Diss. ETHNo. 15413
MOLECULAR INSIGHT INTO TRANSITION METAL
TRANSPORT IN THE GREEN MICROALGA
CHLAMYDOMONAS REINHARDTII
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH
for the
degree
of
Doctor of Natural Sciences
presented by
ALEXANDRA ROSAKIS
Dipl. Biol., University
of Konstanz
born 23. 08. 1976
citizen of
accepted
on
Germany
the recommendation of
Prof. Dr. Alexander J. B.
PD Dr.
Zehnder, examiner
Wolfgang Köster, co-examiner
Buckhout, co-examiner
Prof. Dr. Thomas
Zurich,
2004
Chapters
Chapter
submitted for
2
publication:
(submitted): Rosakis,
microalga Chlamydomonas
A. and
Köster, W. Divalent metal transport in the green
reinhardtii is mediated
by
a
protein
similar to
prokaryotic Nramp
homologues.
Chapter
5
(in press): Rosakis,
microalga Chlamydomonas
A. and
reinhardtii
-
Köster, W. Transition metal transport in the green
Genomic sequence
Abbreviations:
Cd: cadmium
CDF: cation diffusion facilitator
Cu: copper
DMT1: divalent metal transporter
diamine tetra acetic acid
EDTA:
ethylene
EGTA:
ethylene glycol-bis(2-aminoethyl)-tetra acetic
acid
Fe: iron
Mn: manganese
Nramp: natural resistance associated macrophage protein
SOD:
superoxide
dismutase
TM: transmembrane domain
ZIP:
Zrt, Irt-like protein
Zn: zinc
analysis.
Res in Micro
Table of contents
Summary
1
Zusammenfassung
3
1 General Introduction
5
metals"? A short
"heavy
insight
into
confusing terminology
5
1.1
What
1.2
Functions of metals
1.3
Transition metal
1.4
Transition metal transport
11
Transport systems
Transport protein families
11
1.4.1
1.4.2
are
a
7
toxicity
and
availability
9
13
CPx-type ATPases
The CDF family
The ZIP family
The Nramp family
Mode of transport
Nramp regulation
Functional analysis
I)
II)
III)
IV)
The
13
14
15
17
21
21
26
and transition metal
1.5
Algae
1.6
Chlamydomonas
1.7
Motivation and aim of the thesis
2 Divalent metal
29
transport
reinhardtii
-
a
short introduction to the
"green yeast"
32
34
transport in the green microalga Chlamydomonas
reinhardtii is mediated
by
protein
a
similar to
prokaryotic Nramp
35
homologues
2.1
Introduction
35
2.2
Materials and methods
37
2.2.1
Strains
2.2.2
Culture conditions
38
2.2.3
39
2.2.4
Cloning
DMTl expression
2.2.5
GenBank accession numbers
40
2.3
andplasmids
37
39
41
Results
Identification and
2.3.2
2.3.3
Deduced amino acid sequence analysis
Heterologous expression of C. reinhardtii DMTl in S.
2.3.4
DMTl restores
cloning
growth
of the
reinhardtii DMTl gene
2.3.1
of a S.
Chlamydomonas
cerevisiae
smfl
43
49
cerevisiae
mutant under manganese
limiting
conditions
2.3.5
2.3.6
41
50
Overexpression of DMTl in S. cerevisiae mutant smfl increases sensitivity to
manganese, cadmium, and copper, but not to zinc
DMTl does not complement EDTA sensitivity of S. cerevisiae zrtl/zrt2 double
52
mutant..
54
2.3.7
2.4
DMTl function in iron
Discussion
transport
55
56
3
Supplementary
3.1
chapter
2
Further verification that DMTl mediates
limiting
4
material to
60
growth improvement
60
of DMTl mutants
3.2
Study
3.3
Role of DMTl in Zn-mediated reduction of Mn
Experiments
4.1
under metal
conditions
on
the
61
regulation
toxicity
of DMTl
Materials and methods
64
66
66
4.1.1
RNA isolation
66
4.1.2
Culture conditions
67
4.1.3
In-vitro-transcription
RNA gel and Northern
Hybridization
4.1.4
4.1.5
4.2
67
blot
68
68
69
Results
4.2.1
Detection of RbcS2 and DMTl mRNA
4.2.2
Comparison
4.3
of electrophoretic and transfer conditions
Discussion
5 Transition metal
reinhardtii
-
69
69
70
transport in the green microalga Chlamydomonas
Genomic sequence
analysis
71
5.1 Introduction
71
5.2 Materials and methods
74
5.3 Results
75
5.3.1 The
5.3.2 The
5.3.3 The
5.3.4 The
irt-like
ZIP-family (zrt,
protein)
CDF-family (cation diffusion facilitator)
family of CPx-type ATPases
Nramp-family (natural resistance associated macrophage proteins)
5.3.5 Further genes involved in metal transport
5.4 Discussion
6 Conclusions and
References
77
80
83
85
86
88
perspectives
89
91
Summary
Summary
Trace amounts of certain transition metals often referred to
cofactors for
others with
essential function
concentrations.
cell
of
are
involved in vital cellular processes. The very
proteins
no
as
be harmful to the cell if
can
mechanisms
Consequently,
preserving
In this context,
outstanding importance.
enable the passage of these micronutrients
with the necessary amounts of metals is
a
exceed
decisive role is
in
is not
algae
as
Chlamydomonas
by algae
some
to
membranes.
function. The molecular
studied
extensively
reinhardtii
as a
as
model
in other
background
in order to
sequence data available and it is established
(expressed
sequence
the amino acid level showed
(natural
resistance associated
tags)
database I
high similarity
transport of divalent metal cations. They
remarkable sequence
similarity
the wide distribution and the
importance
to
cell survival.
and
occur
displaying
high degree
in
a
To
study
After
growth
a
a
microalga
which
study
identify
are
used
since there
are
In the C
sequence which
Nramp family. Nramps
group of proteins
acting
in the
great number of organisms sharing
We reasoned that
specificity.
of conservation of these
Therefore, the main part of this work
DMTl
function,
I
proteins
underscore their
concentrates on
Nramp homologue, which
performed yeast complementation
transforming
rates
the strains with
expression
of the
a
the
cloning
named
was
The DMTl
{smfl),
zinc
assays with three
{zrtl/zrtl)
gene in
transformed with the empty
mutant
vector)
strains
1
or
EDTA
were
in terms of
iron
growth experiments.
of each yeast mutant strain in minimal medium
expressing
or
uptake
plasmid moderately expressing DMTl,
foreign
minimal medium with the metal chelators EGTA
compared.
able to
a
by
present study.
monitored the effect of
maximal
be achieved
laboratory organism.
broad substrate
different yeast mutant strains deficient in manganese
(fet3/fet4).
can
identify systems
constitute
and functional characterization of the C reinhardtii
DMTl in the
was
a
systems which
or
of these transport processes
members of the
to
macrophage proteins)
as
which
of the cell
We chose the green
organisms.
organism
as
physiological
Supply
guaranteed by uptake systems
facilitate metal transport. C reinhardtii is suitable for such
reinhardtii EST
on
reverse
well
as
played by systems
release metal stores from intracellular compartments, and metal resistance
systems serving the
as
stable metal environment within the
a
biological
across
metals
same
they
metals" act
"heavy
(metal
compared
change
in
to
deficient
For
(control)
growth
rate
this,
and in
conditions)
the control strain
I
were
(mutant
under the different
Summary
conditions. In the
case
(verified by
DMTl
compared
to
of the manganese and iron
uptake
deficient
blotting) improved growth
western
the control cells. These results
clearly
strains, stable expression of
under metal limitation conditions
demonstrated that DMTl is
capable
of
metal transport.
To further
experiments
limits
-
in
investigate
a reverse
of the
conditions
increased
showed
that DMTl
DMTl.
for the C reinhardtii
conditions did not
sensitivity
zinc
can
of the
elevated metal concentrations. I tested
rendered
expression
In the
homologue.
the
smfl
mutant
when DMTl
was
sensitive
to
cadmium, suggesting that these metals
of
case
of DMTl
expression
Thus, the broad specificity described for Nramps
change
smfl
to
performed growth
I
Nramp knockout, under different culture
an
manganese, copper and the non-essential metal
transported by
protein,
and checked whether
-
sensitivity
which is
smfl yeast strain,
and
of the transport
specificity
fashion than above
growth by conferring
growth
the
zinc, the growth
rate
was
under metal
strain to increased manganese concentrations. This
on
also confirmed
limiting
addition of zinc reduced
expressed. However,
compete with manganese for binding sites
are
the DMTl
might
protein
indicate that
even
if it is not
transported.
In further
experiments,
function, gaining information
I tested mutants of the DMTl
on
the
essentiality
of the three mutated
protein
although
conserved amino acid
an
absolutely
Recently,
Genome
conducted
a
Institute,
a
versions
California,
thorough
was
a
Only
to
genomic
alga
substituted in this
was
protein.
made available
sequence which allowed
possesses.
different
By this search
protein
in the genome.
2
families
one
yeast complementation assay
(Doe
http://genome.jgi-psf.org/chlrel/chlrel.home.html
search in the
systems belonging
homologues
functional in
and
expression
on
of individual amino acid residues.
first draft of the C reinhardtii genome
into the metal transport systems the
transport
was
protein
a more
a
total
[52]).
I
general insight
I identified several
and
Joint
of five
putative
Nramp
Zusammenfassung
Zusammenfassung
Bestimmte
Elemente, die
"Schwermetalle" bezeichnet
haben.
der
Gruppe
die
kann das
die
Mechanismen,
für
Metallkonzentrationen innerhalb der Zelle sorgen,
Rolle dabei
Membranen
biologische
Eine
sorgen.
intrazellulären
Systemen
erreicht
dieser
Hintergrund
in anderen
als einen
Kompartimenten
Transportprozesse
als
Alge
sequence
um
in
ist
Algen
tag)
eine
Substratspezifität.
hohe
Systeme
zu
Metallionen durch
Systeme,
geeignet,
weil
an
die Metallvorräte
Metallresistenz
kann
mit
molekulare
Der
einzellige Alge Chlamydomonas
in
identifizieren, die
reinhardtii
Algen
dem
Metalltransport
Sequenzierdaten
zum
Teil vorhanden
schon etabliert ist. In der C reinhardtii EST
Proteinen der
die auf Aminosäure-
Sequenz,
Nramp Familie aufweist. Proteine die
Sequenzähnlichkeit
Die weite
Proteine unterstreicht ihre
reinhardtii
von
Eine
Bedeutung.
umfassend untersucht worden als
Datenbank identifizierte ich eine
zu
der
Gleichgewicht
zu
der
Nramps (natural resistance associated macrophage proteins) gehören, agieren als
zeigen
Hauptteil
stellen.
weniger
Transporter für divalente Metallionen. Sie kommen
vor,
oder durch
Verfügung
zur
Labororganismus
Ebene eine hohe Ähnlichkeit
der
stabiles
ausserordentlicher
von
gewährleistet
dienen. C reinhardtii ist für diese Studie
Gruppe
in
für die Zelle
Versorgung der Zelle mit der nötigen Menge
Wir wählten die grüne
Modellorganismus,
(expressed
ein
werden, die die entgegengesetzte Funktion erfüllen.
Organismen.
sind und weil die
negative Auswirkungen
die für den Durchtritt
spielen Systeme,
Metallen wird durch Aufnahme système
aus
und oft als
werden, spielen eine wichtige Rolle als Protein-Kofaktoren
physiologische Grenze,
sind
Folglich
ausschlaggebende
Übergangsmetalle gehören
der
zellulären Prozessen. Überschreitet die intrazelluläre Konzentration dieser
lebensnotwendigen
Übergangsmetalle
zu
dieser Arbeit
Verbreitung
Bedeutung
auf die
Nramp-Homologs,
in einer grossen Anzahl
untereinander
und
und der hohe Grad der
von
besitzen
Organismen
eine
breite
Konservierung
dieser
für die Zelle. Aus diesem Grund konzentrierte sich der
und
Klonierung
welches in der
funktionelle
vorliegenden
Charakterisierung
des
C
Arbeit als DMTl bezeichnet
wurde.
Um die Funktion
von
DMTl
zu
untersuchen, benutzte ich drei verschiedene Hefe-
Mutantenstämme, bei denen die Transportaktivität für Mangan {smfl), Zink {zrtl/zrtS) und
Eisen
(fet3/fet4)
transformiert
Auswirkung
stark
beeinträchtigt
ist.
Nachdem ich
hatte, das eine moderate Expression
der
Expression
des
von
die
Stämme
DMTl
erlaubte, verfolgte ich die
mittels Wachstumstests.
Fremdgens
3
mit einem Plasmid
Dafür wurden die
Zusammenfassung
maximalen Wachstumsraten
Hefestammes in Minimalmedium
jedes
Minimalmedium mit den Metallchelatoren EGTA oder EDTA
Mutanten, die
mit leerem
exprimierten,
DMTl
Veränderung
Stabile
Kultivierungsbedingungen verglichen.
verbesserte
blots)
western
das
im
Metallmangel-Bedingungen,
(Metallmangel) verglichen.
wurden mit dem Kontrollstamm
hinsichtlich einer
Vektor)
Vergleich
der
DMTl
von
und
smfl
fet3/fet4
Kontrollkultur.
zur
(Mutante
in
Die
transformiert
der Wachstumsrate unter verschiedenen
Expression
Wachstum
und
(Kontrolle)
Diese
mittels
(bestätigt
Mutanten
unter
Ergebnisse zeigten
deutlich, dassDMTl Metallionen transportierte.
die
Um
Frage der Spezifität des Transporters weiter
Wachstumstests
Sensitivität
Zellen
gegenüber
beeinträchtigen
Knockout
Kupferandere
durch, und untersuchte ob
ist,
unter
Expression
kann. Ich testete das Wachstum der
verschiedenen
Metallbedingungen
transportieren
betrifft, bewirkte die
unter
Metallmangelbedingungen.
eines
smfl
sein, dass
kann, auch
wenn
gegenüber
Zink mit
um
zeigte,
reinhardtii
Expression
erhöhte
keine
welche ein
dass DMTl
Spezifität,
Veränderung
Zugabe
von
Nramp
Mangan-,
Homolog bestätigt.
Mangankonzentrationen.
die für
Was die
im Wachstum
Zink die Sensitivität
Das könnte ein Hinweis
die Bindestellen auf dem DMTl Protein konkurrieren
transportiert
wird.
Ferner untersuchte ich Mutationen im DMTl
Bedeutung
und
Jedoch verminderte die
Mangan
Zink nicht
DMTl
erhöhten
smfl Mutante,
kann. Somit wurde die breite
Nramps beschrieben wurde, auch für das C
Stammes
eine
DMTl
von
hohen Metallkonzentrationen bewirken und somit das Wachstum der
und Cadmium-Ionen
zrtl/zrt3 Mutante
darauf
die
untersuchen, führte ich
zu
einzelner Aminosäuren für die
bezüglich
der
und die Funktion des Proteins
zu
Protein,
Expression
bekommen. Nur eines der drei mutierten Proteinderivate
um
war
Informationen
funktionell in einem Hefe-
obwohl in diesem Protein eine absolut konservierte Aminosäure
Komplementierungssystem,
ersetzt war.
Unlängst
reinhardtii
wurde im Internet eine erste Version der
aufgeschaltet
(Doe
Joint
psf.org/chlrel/chlrel.home.html, [52]).
Sequenz
erlaubte
Einsicht
in
Eine
verschiedene
identifizierte mehrere mutmassliche
gehören,
Genome
Institute,
gründliche
California,
Recherche
Metallsysteme,
Transportsysteme, die
sowie fünf Nramp-Homologe im Genom der
4
genomischen Sequenz
Alge.
zu
die
in
die
von
C
http://genome.jgider
Alge
genomischen
besitzt.
Ich
verschiedenen Proteinfamilien
General Introduction
1
Chapter
General Introduction
Transition metals
as
structural
are
involved in
functional cofactors for
or
a
broad range of cellular functions
proteins.
acting especially
Several mechanisms have evolved in order to
guarantee the supply and also the homeostasis of these micronutrients in the cell. In this
is attached to systems which transport metals into and out of the
regard great importance
cytoplasm
and
or
the cellular
general transport principles,
At the end of the
1.1
Metals
What
are
chapter
are
defined
malleable and
the
"heavy
This introduction
organelles.
followed
organism
studied in this
metals"? A short
insight
a
metal function
on
of selected metal transport systems.
project
into
overview
an
will be
shortly
introduced.
confusing terminology
electricity,
have
a
metallic
luster,
ductile, form cations, and have basic oxides" [6]. Following this definition
table
metal"
species
in which the element may exist
are
used
periodic
"heavy
compounds
referring
have the
which does not
correspond
is very
defined
loosely
properties, depending
synonym to toxic
or
identical. In order to
metallic elements
to
on
reactivity.
not
same
on
to
the
in
[35]).
This
and
In addition to this
atomic
weight,
periodic
was
table may be
grouped according
to
possible
more
that
the term
or
"heavy
In this classification the metallic elements
other chemical
a
a
are
chemical classification of
In the
configuration
are
as
"toxicity"
periodic
and thus
divided into
block, d-block-transition and f-block elements (Fig. 1.1). Elements referred
5
metal"
and is often misused
appropriate [35].
valence electron
chemical
toxicological properties,
that "heaviness" and
suggested
over
that the pure metal
atomic number
respective author)
species, assuming
confusion it
discrepancy,
are
the terms "metal" and
implies
physicochemical, biological
the definition of the
guard against
(Fig. 1.1). Usually,
the pure element but also to all
(reviewed
density,
harmful metal
are
metals
are
only
reality.
(based
following
table the elements
chemical
description
a
"elements which conduct
as
3/4 of the elements in the
and all its
by
gives
to
s-block,
as
by
p-
"heavy
General Introduction
metals"
metals
filled.
found in the class of d-block
are
characterized
are
do not
They
and 3d electrons
In my PhD
metals", referring
"metal"
the
or
energetically
close
(e.g.
are
use
3d electron
a
thesis, the
to
name
(Fe3+)).
iron
gives
Thus, transition metals
can
take up different
the
of
term
"transition metals" will be
relevant elements. For
biologically
an
preferred
over
practical
the ionic form will be
specified (e.g.
"metal
transport",
ferrous ion
or
Fe2+).
d-block/transition elements
p -block elements
3
5
4
6
8
7
3
10
11
12
13
14
15
18
17
18
He
B
Be
'NaJ (Mg
Sc
Ti
V
I
Sr
Y
Cs
Ba
Im
Hf
Ta
Fr
Ra
Ac
Th
Pa
Bulk
Nb
1 o
biological
K-,...y elements
Cr
Mil
I
(t)^Ui{0)
At
Si
'"p)i
Cj©
/AS
Se
Fe
Co
Ni
'Cu
Zn
Ga
Pd
Ag
Cd
In
Sn! Sb
Hg
TI
Pb
F
Ne
sV'cf
'
Ar
Br
Kr
Te
I
/Y©
Po
At
Rn
i
Rb
Mo
Tc
Ru
Rh
jw
Re
Os
ir
Pt
Bi
metals
to be essential for
1.1 Periodic table of the elements
or
(adapted
!
bacteria»
animals
from
u
non-me
Possibly essential
Trace elements believed
plants
Figure
"heavy
the word
reasons,
element will be used in the context of "metal
appropriate
the term
{ H
^
(Fe2+),
sometimes its two 4s electrons
very versatile for biochemical reactions.
elements
Li
already
number of electrons in chemical reactions since the 4s
s-block
£*
4s orbital is
same
tolerance" etc, and where
1
orbitals, while the
the
and sometimes adds
oxidation states and
filled 3d
by incompletely
always
are
elements, also called transition metals. Transition
trace elements
some
for
species
[44]). f-block elements (lanfhanides and actinides)
not shown. The group of the d-block/transition elements is
highlighted by
a
square. Most "trace elements"
are
are
(boxed). Bulk essential elements are circled. The black line is the border between
left) and non-metals (on the right). This border is not strict, since the elements near it
do not possess features strictly metallic or non-metallic, respectively. It is noteworthy that the elements essential
for life are the lighter elements, which are also most abundant on earth [44].
found within this group
metallic elements (on the
6
General Introduction
Functions of metals
1.2
large
A
number of
analyses
of several
and tissues made clear that of all the
for life
atoms
of all
constituted
are
living organisms),
organic
structures.
and carbon and
Seven additional
required by
chromium,
but not all
most
elements
are
chlorine, comprising
copper,
body
high
organs
essential
99% of the
water content
the basic elements of the
are
sodium, potassium, calcium,
The
0.9%.
and in
biological systems,
iron, cobalt, nickel,
manganese,
These
nitrogen (10.8%).
as
absolutely
are
due to the
(88.2%,
well
as
of the human
example
and oxygen
essential
sulfur and
magnesium, phosphorus,
elements
by hydrogen
In the
species,
elements 11
naturally occurring
(bulk biological elements, Fig. 1.1).
present
and animals'
plant
remaining
varying
0.1%
vanadium,
amounts:
zinc, molybdenum, boron, silicon,
selenium, fluorine, iodine, arsenic, bromine, and tin, where for the last three elements it is
entirely
clear whether
few bacteria
they
are
indeed essential. Iron and zinc
belong
However, this does
trace amounts.
reactions these ions
are
not
involved in
in these metals
have
can
to
evolving complex
as a
derogate
their
comprise
in
photosystem
cofactor in MnSOD
copper have the facile
oxidation states.
a
They
ability
are
importance.
major part
for
a
for the
an
of
a
large
transcription
cofactor of enzymes involved in the
specific
production
synthesis
Both
are
A
level.
in the oxygen
superoxide
highly important
essential element in the cell. Iron and
donate and accept electrons and
cofactors of
functionality
organism
II and for its action in the dismutation of the
as
needed in
of the cells metabolism.
(manganese superoxide dismutase).
to
only
are
On the contrary, the cellular
in the oxygen
number of
redox reactions. Zinc is essential for the formation of the
some
species except
consequences at cellular and
severe
cellular reactions which establish manganese
important
in all
the group of transition metals
Manganese is mainly known for its involvement
anion
occur
not
[44].
Essential elements which
deficiency
are
factors in
can
easily
shift between two
metalloproteins
"zinc-finger",
a
which
catalyze
structural element
transcriptional regulation.
It is also
a
of nucleic and amino acids. A short overview of
functions of selected metal cations is
7
presented
in table 1.1.
Table 1.1 Selected enzymes
utilizing
Metal
Protein
Copper
Cytochrome
transition metals
as
cofactors
[44].
Function
oxidase
respiratory
formation)
Cu, Zn-superoxide dismutase (see also zinc)
electron transfer chain in mitochondria
conversion of
superoxide
radical to
(reduction of 02
to
H20 linked
to
hydrogen peroxide (cytosol, mitochondria) 202"
proton gradient
+
2H+ -> H202
o2
Blue copper
electron transfer
proteins
Caeruloplasmin (multicopper oxidase)
Plastocyanin
Manganese
Mn-superoxide dismutase
Oxygen Evolving Complex, Photosystem
Isocitrate dehydrogenase
Mn-catalase
Imidazole
Zinc
glycerol phosphate dehydratase
Galactosyl
transferase
Glutamate
synthetase
Fe2+
to
photosynthetic
(membranes, thylakoid vesicles)
Fe3+ in the presence of molecular
electron transfer chain in
oxygen
(which is reduced
superoxide radical to hydrogen peroxide (mitochondria, chloroplasts, prokaryotes)
dioxygen release (chloroplast)
citric acid cycle (mitochondria)
oxidative stress defense: 2H202 -> 2H20 + 02
histidine synthesis
formation of oligosaccharides of glycoproteins (Golgi)
glutamine synthesis
Zinc
finger proteins
dehydrogenase
Aspartate transcarbamylase
Dehydroquinate synthase
RNA polymerase
Reverse transcriptase
tRNA synthase
transcription factors (binding to promoter sequences)
redox reaction, NAD+ + C2H5OH -> NADH + CH3CHO + H+
nucleic acid synthesis
synthesis of aromatic amino acids phenylalanine, tyrosine, tryptophan
RNA synthesis
DNA synthesis
transfer RNA synthesis
Haemoglobin
transport of 02
Transferrin
glycoprotein, transport vehicle
storage of Fe3+ (cytoplasm)
Fe-superoxide
Cytochromes
dismutase
H20)
conversion of
II
copper is the functional
Ferritin
to
chloroplasts
Cu, Zn-superoxide dismutase (see also
copper)
Alcohol
Iron
converts
conversion of
cofactor, zinc is
for
an
extra
stabilizing factor
Fe3+
superoxide radical to hydrogen peroxide (chloroplasts, prokaryotes)
(respiration, photosynthesis)
electron transfer chain
+
General Introduction
Transition metal
1.3
Despite
needed
physiological
levels.
limit. Redox
exploited by
they
physiological
the cell in concentrations
the cell to generate reactive chemical centers for
purposes, may lead to substantial
by
enter
no
damages.
Reactive oxygen
to
of DNA,
damage
Fe3+
+
02" -» Fe2+
Fe2+
+
H202 -» Fe3+
02"
+
H202 -> 02
Redox-inactive metals like
displacing
Other
toxicity
It is
interesting
and would
to note
play
OH"
physiologically
essential
be
produced
species (ROS)
one
magnesium
and calcium
were
+
or
V
[7] leading
OH' (Fenton reaction)
binding
may induce oxidative stress
sites
include
of the
(toxicity
via release of Fenton
binding
catalysts
indirectly by
superoxide
anion 02" is
from iron-sulfur enzymes,
macromolecules
to
of the
by displacement
enzymes
that the elements essential for life
earth
on
reason
native
altering
their
metal
ion, and
why
subgroups
are
the bulk
preference
more
of the
abundance of the elements their
but
table
can
can
differ
lighter
elements
metals
analogous
markedly
in their
sodium, potassium,
perform
the tasks that other
perform [44].
biological availability
9
the
lithium, rubidium, cesium, beryllium,
abundant and
periodic
they
biological
to
are
Some elements have
(Fig. 1.1, [44]).
biological role,
selected in
strontium, and barium is that they
same
can
OH' (Haber-Weiss reaction)
+
similar
a
Presumably
elements of the
routinely
permeability.
abundance.
general
+
mechanisms
also the most abundant
properties
the tolerance
exceeding
02
OH"
indirectly
interference with membrane
are
+
exceed
lipids.
Cd, Hg, Ni, and Pb
conformation, inactivation of
which
+
or
native metals from cellular
believed to be mediated
[7]).
proteins
are
function has been
the Fenton and Haber-Weiss reaction in the presence of Cu, Fe, Cr
eventually
they
of transition metals like iron and copper, which is
capacity
a
become toxic for the cell when
can
metal ions for which
Similarly,
activity,
essential transition metals
physiological significance,
enormous
be detrimental when
can
availability
and
in trace concentrations and
only
shown
their
toxicity
is of
But besides the
particular importance
for
General Introduction
These two attributes do not
living organisms.
of the
one
comes
to
major players
its
in cellular
necessarily
metabolism, stands
[44]. The
in
pH and
aerobic environment and at neutral
insoluble. However,
are
availability
together.
living organisms
of metals and
sophisticated system
of
is that iron
it is
strikingly iron,
of the most
one
spontaneously
in the form of ferric
efficient in
extremely
are
several orders of
by
uptake, storage
the cells with essential
occurs
able to maintain metal
are
environmental concentrations
reason
Most
the bottom of the ladder when it
at
accessibility by biological systems (Fig. 1.2), although
abundant transition metals in earth crust
an
go
oxidizes
complexes
overcoming
which
limited
concentrations which exceed the
magnitude (Tab.
and detoxification is at
play
1.2
[44]). Obviously,
which not
a
only supplies
metals, but also maintains the desired concentration balance of the
various metal elements.
Principal species present:
:
Cation
Anion
•
Hydroxy complex, oxycation,
a
Carbonate
oxyanîon
or
Chloride
complex
•
Br
•
CI
•
F
complex
«
"Ê
m
»Mg
|K
o
S
S
iNa
m
O
N
B
o
--2
.
«Sr
,
Mo
c
U
iRb
Se
Sb
Au
As
yCcf
iCs
m
o
Ba
W
c
V
so
T3
Th
c
_j
Xi
^8
m
*Y
*o
ö)
Hf
tTi
Ni
Ta4-Cr
Ga
Ge
Sn#Bj
Mn
Sc
-8-
iZn
*Nb
La»Lu
O
ACu
i
Co
AI
Fe
^J
IA
_L
j
I
Group
Figure
access
to
elements,
no
IB
L
HB HIB IVBVBVIBVIIB
periodic table
1.2 Ease of extraction of elements from the earth to the
systems will have
the
Viil
in
J
I
_1
IIA MIA IVAVAVIAVI1A
sea as a
guide to the ease with which biological
[44]. The top of the figure indicates
matter what the absolute abundance
speciation.
10
General Introduction
Table 1.2 Accumulation of metallic elements in
Concentration
algae [134].
of
Metal
Type
3,700
Zn
Green
21,600
Zn
diatoms
Zn, Cu, Cd
Chlorococcus
2,000,000
Cd
Chlorella
<4,000
Cd
Chlorella
various spp.
Chlorella
factor*
ca.
4,000
1,000-100,000
Cd, Pb, Hg
4,000
U
>100,000
Pu, Am, Np
algae
algae
paris
pyrenoidosa
pyrenoidosa
500-30,000
various
various spp.
various spp.
3056
Cu
Chlorella
849
Ni
Chlorella
2327
Cd
Chlorella
570-31,000
Pb
various spp.
2,000-25,000
Pb
16,000-20,000
Pb
620-7,700
Pb, Cd, Zn,
Cladophora glomerata
Cladophora glomerata
Green algae
Cr
*metal in biomass/ metal in solution
1.4
Transition metal transport
As mentioned
environment,
are
in the cell. A
not
above, different elements do
equally bioavailable,
stunning
number of
uptake
not occur at
the
same
and must be maintained at different concentrations
mechanisms for micronutrients such
have evolved and many of them have been characterized in pro- and
principles
are
of
activity
systems
Metal
organisms.
high affinity uptake systems,
active.
are
intracellular
transporter
valid in all
systems
which
and
extrude
depletion
while under
Compartmentalization
transport systems,
concentration in the
into
more
growth
cellular
mechanisms
metals
eukaryotes.
metal ions
The
or
organelles
may
direct
general
medium induces the
favorable conditions low
different
detoxification
unwanted
of the
as
them
also
into
be
affinity
requires
based
on
intracellular
compartments.
1.4.1
Transport systems
The passage
of solutes
protein-dependent transport
energetic
criteria
across
membranes
processes. The latter
[1].
11
can
can
be
occur
via
grouped
protein-independent
into classes
according
or
to
General Introduction
1) Protein-independent
Diffusion is
the solute
and with
transport process which does
a
through
C02, NH3)
diffusion
cross
require
a
protein
the membrane
more or
less
2)
facilitate passage of
weak solute
their concentration
freely following
of metabolic energy. Diffusion is not
conditions which demand fast and efficient solute movement
growth,
to
the membrane. Some small molecules like water, ethanol and gases
consumption
no
not
and cannot guarantee
gradient)
an
(e.g.
adequate
nutrient
(O2,
gradient
process under
limitation, fast
specificity.
Protein-mediated transport
In this group
one or more
proteins
transporters, expanding the possibilities of the
act as
transport process regarding direction, substrates and velocity.
Channels
a)
Although
channel facilitates
a
their concentration
Aquaporins
the cell
such
are
without
proteins
of the
requirement
of energy,
similarly
to
diffusion.
which form channels for the entrance of water into
molecule which is also able to enter
(a
membrane, the solutes still follow
by diffusion).
Carriers
b)
Carriers
and
be
gradient
permeation
are
play
at
when transport has to
i)
to
another energy
Primary
In this
such
as
in
of the
membrane
can
and
uptake
gradient
reaction must
reaction.
be driven
by
ATP
hydrolysis
photosynthesis-coupled
membrane
potential,
(eukaryotic Na+/K+ ATPase,
expense of ATP. ABC
and also
concentration
this
place
bacteria, mitochondria and chloroplasts
respiration-
direction
releasing
a
active
class, transport
synthases
against
energy. In order to take
consequently requires
coupled
occur
into
ATP.
bacterial K+
convert
ABC-type uptake systems
of
a
variety
of H+
in the
in
the
plasma
pump ions at the
are
of substrates to ATP
have been identified in
12
energy,
ATPases
ATPase)
ATP
primary
movement
(ATP binding cassette) transporters
couple transport
synthesis.
or
eukaryotes,
widely spread
hydrolysis.
but
No
ABC-type
General Introduction
efflux systems abound in both
for
Here, the chemical coupling makes
electrochemical
process
insofar
by
a
potential
example
described in 1.4.2.
family
place
special
case
gradient. Examples
as
as
of the free energy
use
of another solute.
the action of
take
can
Another
active
Secondary
maintained
eukaryotes.
active transporters is the CPx-ATPase
primary
ii)
and
prokaryotes
the
more
used
or an
solute
transported
described in
are
symport
a
the
which
are
Pre-existing gradients
primary systems
uniport,
a
produced by
uses
driving
as
The
antiport.
its
own
forces. The
uniport
is
electrochemical
detail in 1.4.2 include the ZIP, CDF and
Nramp family.
iii)
Group
translocators
This group has
the substrate
1.4.2
during
Transport proteins
are
Several classes of
On the
The
organisms
and
membranes
(e.
ATPases
g.
expense
intermediate
ATPases
proteins
into families
have been
I will
according
proposed
concentrate
eukaryotic organisms (but
are
on
to
sequence and functional
demonstrated to
or
four
play
families
major
also found in
essential
active transporters
and
of ATP,
their
defined
as
pump
and
a
variety
are
catalytic
of
compose
charged
the
cycle [135].
CPx-ATPases due to
13
a
metal
subgroup
a
substrates
distinguished by
reaction
expressed
toxic
potentially
Cu2+, Zn2+, Cd2+, Pb2+). They
during
were
primary
are
transporting
P-type ATPases, which
the
of
a
role in
mediating
prokaryotes).
CPx-type ATPases
CPx-type
at
grouped
following
transition metal transport in
phosphorylation
the transport process.
families
metal transport.
of
been described in bacteria and involves
Transport protein
similarity.
I)
only
in
formation
wide range of
cations
of the
across
a
cell
large superfamily
biological
of
across
a
membranes
phosphorylated
The divalent metal cation
conserved intramembraneous
P-type
cysteine-
General Introduction
proline-cysteine/histidine/serine (CPx)
transduction
In
cyanobacteria,
detoxification
the CPx-ATPases PacS and CtaA
by pumping
membrane.
cytoplasmic
localized in
S.
In
the
Ccc2p
to
and
thylakoid
into
transported
from
in
reported
Arabidopsis thaliana,
shown to localize in the
identified
chloroplast
inner
[118]. Another homologue, PAA2, might transport
may
plastid targeting signal. HMA5,
mitochondrial
supply
mitochondrial
putative
was
a
shown to
rescue a
proteins
CPx-ATPases
such
targeting signal
yeast ccc2
copper into the active site of the
ions to toxic levels
and
II)
in S.
a
role in
is
chaperone Atxlp
copper
post-Golgi
by
vesicles. The Cu is then
envelope
third
Cu
across
[65].
In
c
[118].
aureus
[135].
It
was
homology
to CtaA
membrane and
was
plastid envelope
the
thylakoid
discussed
of
ATPase
the
homologue
cytochrome
sequence
mutant
across
(P-type
PAAl
[122].
suggested
membrane
membrane since it
by
the
same
authors,
oxidase with Cu, since it possesses
RANI
Arabidopsis
(responsive
RANI is
to
a
antagonist 1)
required
for
loading
ethylene receptor [64].
be
might
as
a
was
sequence
important
not
only
in
transition metal ions for essential cell functions but also in
(CadA
play
lumen. CtaA is localized in the
higher plants
transport Cu for the Cu/Zn superoxide dismutase
possesses
may also
Cu for
supply
to
multicopper ferroxidase Fet3p [137].
Arabidopsis 1_)
was
function in metal cation
suggested
were
Cu bound to the
cerevisiae,
The first CPx-ATPase
PAAl
to
thylakoid membranes,
Cu into the
excess
transferred to the CPx-ATPase
on
thought
[119] (Fig. 5.1).
photosynthesis [128]. PacS,
loaded
motif which is
shown for
prokaryotic
obtaining
preventing
sufficient
of
amounts
accumulation of these
CPx-ATPases that
they
mediate efflux
exports cadmium [95], and ZntA in E. coli catalyzes efflux of Zn, Cd, Pb,
Hg [106]).
The CDF
CDF
identified in
family
(cation
diffusion
potential. Many
in the transport of zinc in
organellar compartments.
pro- and
are
a
bacteria, archaea and eukaryotes (reviewed
electrochemical H+
II and III.
facilitator) proteins
Subfamily
a
I includes
are
divided based
mostly
are
in
family
direction away from the
CDFs
eukaryotic proteins
members of this
family
on
[46]). Transport
have been
cytoplasm,
equally.
14
is
coupled
to
the
implicated specifically
i.e. out of the cell
or
into
sequence similarities into subfamilies
eubacterial and archaeal
distributed
of metal cation transporters
proteins,
I,
while in II and III
Both the C- and the N-terminus of
a
General Introduction
typical
protein
CDF
between them
containing
(Fig. 5.1).
expressed but
no
longer bound
by
zinc
vacuolar membrane and
frequently
This
G).
or
[11]. Proteins lacking the loop
(TM)
loop
was
stably
were
zinc.
homologues
seem
are
to
was
shown to act in cobalt transport
compartmentalization [24, 25].
thought
which is maintained
membrane
n=3-6 and X often S
(HX)n (where
[69]. Another homologue, Cotlp
zinc detoxification
CDF
is found between TM IV and V,
in A. thaliana ZATl
binding
with 6 transmembrane domains
cytoplasmic
yeast S. cerevisiae the CDF homologue Zrclp is involved in zinc and cadmium
In the
potential
be
to
long loop region
A
histidine-rich motif
a
shown to act in metal
resistance
predicted
are
by
play
and exports zinc
a
to
Both
proteins
are
[25] and
expressed
in
the
on
sequestrate zinc into the vacuole, driven by the proton
the V-ATPase
(vacuolar ATPase) [80].
role in zinc detoxification. Zntl is
[99], while
expressed
zinc into
Znt2 transports
mammals,
In
the
on
two
plasma
endosomal/lysosomal
vesicles, thereby protecting the cell against toxic zinc concentrations [98].
The A. thaliana CDF
zinc
resistance.
in
Overexpression
accumulation in roots,
the root cells
homologue
showing
[129]. When
ZATl is
leads
plants
that zinc must be
ZATl
was
Ill)
specific
The ZIP
for zinc
an
at 1%
al
m
Arabidopsis
[56] divided the
nematodes),
review two
more
[138],
thaliana
ZIP
proteins
Irtl
proteoliposomes,
zinc
it did not
showing
that
well
of unknown
proteins
like the
as
ZIP
from eubacteria and archaea
subfamily
by uptake
Liv-1 is
family [46].
cerevisiae. For
measurements
expressed highly
functions
includes members from
family
I
(fungi
and
uptake.
The
ZupT from
plants)
and II
as
an
plants,
[56]. Guerinot
(mammals
and
Gaither and Eide add in their
gufA subfamily
function, like the GufA of Myxococcus
Zrt3p of S.
(zinc regulated transporter 1)
Zrtl
(iron regulated transporter 1)
[37]. The
as
into
groups to the ZIP
proteins
proteins.
and
compartment (vacuole) in
of the zinc transport rate,
protein.
with many members involved in zinc
demonstrated
a
resistance
family
protozoa, insects, mammals and fungi,
et
into
zinc
[11].
S. cerevisiae zinc transporter
iron transporter
enhanced
to
transported
The abbreviation ZIP stands for Zrt, Irt-like
is
in all organs and is involved in
and reconstituted in
purified
transport cobalt, and transported cadmium only
ZATl is
expressed
xanthus
E. coli
a
includes
prokaryotic
[90], and also eukaryotic
function in Zn transport
was
[51]. The Liv-1 subfamily includes only eukaryotic
in certain metastatic breast
15
cancer
tissues
[89] but its
General Introduction
function is unknown. This group is
proteins
but
none
Most ZIP
amino- and
5.1).
of the Liv-1
proteins
based
only
members
subgroup
predicted
were
ends located
carboxyterminal
conserved
[56]),
for
binding,
this
loop
between TM II and III, which is
Residues
residues often with
aqueous
source
cavity
plasma
because its
a
membrane
sequence is
adjacent polar
in the transporter
charged
or
through
proteins.
the
to
notably
not
are
often
Residues
loop region
and may be the initial site of substrate
were
in TMs IV and
V, and
conserved and contain histidine
highly
amino acids.
mapped
Thus, they
are
predicted
which the cationic substrate passes
line
to
[38]. The
symport mechanism
was
an
energy
characterized, but these systems probably function
Zn2+/HC03_
active carriers. A
are
mapped
were
[56] (Fig.
between transmembrane
site of the
binding
of IRTl
specificity
TMs IV and V
[110].
metal
potential
for function of IRTl
for transport has not been
secondary
transmembrane domains with the
eight
region",
the other ZIP
to
tested to date.
long cytoplasmic loop
a
extracytoplasmic
important
include three histidines
is
substrate
determining
binding [110].
functionally
similarity
which accommodates many histidine residues. Since histidines
involved in metal
important
was
sequence
the outside of the
on
the "variable
(called
high
to possess
A remarkable feature of many ZIPs is
domains III and IV
on a
as
for the human
proposed
hZip2 [45].
In
while
yeast, Zrtlp and Zrt2p
are
responsible
Zrt3p probably mobilizes the vacuolar zinc
the CDF members
for the
store
subfamilies except
ever
increasing
to
limited
subfamily
identified
II. IRTl
Zn2+, Mn2+
plants,
Cd2+ [74].
and
seems
to
lie
mainly
transported
iron-limited
higher
as
the
thaliana, ZIP1-4,
accumulated
major
may
play
cerevisiae increased zinc
expression
or
iron
a
[37], but
by
identified, spread
zinc
uptake,
the action of
a
also shown
by
expressed only
Zn2+, Mn2+
and
metal
iron,
not
fact, it
mislocalization.
16
mutant
uptake
assays
excluding
was
that
shown that
Cd2+ [23, 74, 133].
[53]. Overexpression of
the yeast. ZIP 4 failed to
the
in the roots of iron
from the soil. Four other ZIP
role in zinc transport
was
yeast iron transport
in the accumulation of
levels of
among all ZIP
from A. thaliana
under these conditions. In
uptake system
uptake by
was
Since IRTl is
other metal cations may also be
considered
were
[37]. The protein complemented
its function
plants
affinity
is maintained
(iron regulated transporter 1)
the iron content of the yeast cells
transport
(which
and low
Zrclp and Cotlp) [87, 138, 139].
In the A. thaliana genome 18 ZIP members
first ZIP
high
IRTl is
proteins
in A.
ZIP 1-3 in S.
complement perhaps
due to poor
General Introduction
IV)
The
Nramp family
Nramps (natural resistance associated macrophage proteins) form
group of divalent
a
metal cation transporters which contribute to cell metal homeostasis and have been
conserved
throughout
the pro- and
In the
evolution.
domains
eukaryotic
They
in many different
expressed
are
and share
(Tab. 1.3),
high similarity
the cell
surface, but it
expressed
at
to copper
and cadmium accumulation
from intracellular vesicles and to
was
Smf3p, expressed
at
[84]. Smf2p
thus mobilize iron stores
high affinity
iron
analogous
to
the low
[86].
[103]. Although
two
and
multiple pathways
other
to
the amino acid level.
at
for
suggested
to
are
the
AtNramp3
was
recently
characterized in
microscopy
and GFP fusion
proteins,
metal ions from the vacuole into the
sensitivity
in
probable
AtNramp3
that
AtNramp3 might
This would
A.
to
[84].
Fet5p, homologous
to
mobilize vacuolar iron
following
Smf3p
may be
which takes up
assays
a
[30, 127].
functional model
shown
as was
explain
thaliana, which
by
was
fluorescent
the
previously
not
was
observed
accompanied by
overexpressed.
FR02, which is regulated by iron [27, 107],
moved
Since total Fe content did not
iron from the vacuole
also influence accumulation of Zn and Mn
shown to transport Mn and Zn under iron deficient
In mice with defects in nutritional iron
mice),
direct iron
in iron
uptake by peripheral
injection
in
[127]. Similarly, expression of the primary iron uptake transporter
was
AtNramp3
detail and the
cytoplasm.
IRTl and of the root ferric chelate reductase
altered when
(MnSOD)
and acts in the mobilization of metal stores, i.e. exports
AtNramp3-overexpressing
increase in total Cd content
and
by yeast complementation
more
stores
3 and 4 contribute to manganese,
localizes to the vacuolar membrane
suggested [126]. AtNramp3
dismutase
plasma membrane,
Arabidopsis thaliana, AtNrampl,
shown
Mn2+
mobilize
iron out of the vacuole.
In the thale
was
and contribute
export iron from the vacuole and
suggested
cations, including Fe2+ [33].
as
to
superoxide
of divalent metal
cress
Fe2+ uptake [21]
proteins (Fthlp
moving
on
high affinity Mn2+ transporter
also shown to transport cobalt
was
Fet3p)
affinity Fet4p transporter
iron and cadmium transport
was
It
membrane, is believed
uptake proteins Ftrlp
stores, there may exist
was
the manganese
supply
the vacuolar
a
also shown to mediate
the mitochondria with these essential ions
Cd
organisms belonging
yeast Saccharomyces cerevisiae three Nramp homologues have been identified and
characterized, SMF1, SMF2 and SMF3 [103, 121]. Smflp is
variety
highly
did not correct the clinical
tissues
[124].
It
was
17
into the
change,
it
was
seems
cytosol. Furthermore,
by downregulating
IRTl
(IRTl
conditions, [74]).
absorption (microcytic
picture [8], suggesting
a
anemia in mk
second block
demonstrated that in mk mice the
Nramp2
General Introduction
gene is mutated
[42].
In
parallel, Nramp2 (DMTl, DCT1)
in the duodenum of rats and
that this
suggestion
thorough study
on
markers for late
marker for
[124].
may be
the localization of human
light
and
of these results it
of iron in non-intestinal cells
Three
iron
pathways
absorption
as
transferrin-dependent
the ways in which
iron
as
in
that
Nramp2 is thought
Nramp2
of iron from
to
cells
nutritional
Fe2+ (reduced by DcytB) by utilizing
the
a
side of enterocytes
apical
the proton
gradient
(Tf).
Fe3+-loaded
Fe3+
at
the cell surface of
erythrocytes
by Iregl.
is released from transferrin
by
The
or
reduced, probably by
still obscure but
probably
and/or
can
iii)
oxidase
the
through
hephaestin,
a
take up
the action
by
of iron to transferrin
transferrin binds to
blood,
requires
and efflux out of
transferrin receptor
other cells it is internalized
(TfR)
by endocytosis.
acidification of the endosome via the action of vacuolar
H+-ATPase. The apo-Tf-TfR complex is recycled
endosome is
loading
multicopper
When
the enterocytes
expressed
the
nutritional
1.3 visualizes
maintained
transferrin
Fe3+, probably by
i)
Nramp2
bound to the
to
role:
in iron distribution from the intestine to
Iron is distributed to other cells
Fe2+
a
in the distribution
[124]. Figure
exchanger.
oxidation of
which is
(TfR,
phagocytosed erythrocytes,
of Na+/K+ ATPase and Na+/H+
plasma protein
a
(see below).
play
may
and other cells
act
peripheral
at
the
to
in non-intestinal cells
Nramp2 is involved
of intestinal iron
in which
erythrocytes
[88, 124]: Expressed
ions, leading
absorption [57]. Recently,
plasma membrane)
absorption
proposed
in
expressed
gene
Nramp2 showed that the protein colocalizes with
suggested
ii) recycling
uptake
as a
but not with the transferrin receptor
was
well
have been
in enterocytes,
for intestinal iron
endosomes and the
recycling
divalent metal
transporting
responsible
endosomes/lysosomes
sorting
In the
protein
of
capable
identified
was
an
oxidoreductase,
involves recruitment of
Fe3+
the cell surface. The free
to
to
Fe2+.
Nramp2
to
in the
Maturation of the endosome is
the endosomal membrane. The
transporter exports Fe2+ ions into the cytoplasm by using the proton gradient established by
the vacuolar H+-ATPase. In
similar way,
a
phagocytosed erythrocytes, by releasing
proposed
for murine
phagosome
be
expressed
in the
membrane upon
intracellular
contribute to
the metal from the
lysosomal
phagocytosis
was
of iron from
recycling
phagosome.
A similar model
was
demonstrated
reported
vesicles of
of
a
macrophages
mouse
that mutation of
pathogens [132]. According
to
was
and to be recruited to the
pathogens [55]. However,
knockout
was
in the
[54].
Nramp 1, the first identified member of the Nramp family
role in iron distribution since
[124]. Instead, it
can
Nramp2 for which co-localization with transferrin and expression
and the endosomes
Mammalian
Nramp2
it is not believed to
phagosomal
play
does not show defects in iron
Nramp 1
causes
susceptibility
to
shown to
a
major
absorption
infections
by
the direction of transport which is not yet settled
18
General Introduction
Mode of
(see
or
to
transport), Nramp 1
concentrate
deprived
is
them within it.
thought
the
Thereby,
from metal ions essential for the
either extrude metal ions from the
to
production
the increase of the metal concentration in the
oxygen radicals to combat the
Some
the
case
with
pathogens
of the
Gram-negative
suggested
bacterium Salmonella
in
and Escherichia coli
Since
that
a
Mn2+
phagosome
the
phagosome.
are
few enzymes
typhimurium
The MntH
-,
which
proteins
are
known to need
Mn2+
in these
play
a
protective
role
independent
peroxide [72]. Additionally, Mn2+
or
of
shown for
Lactobacillus
species
which lack
19
a
cytoplasmic
might compete
from Salmonella
a
Mn¬
sensitivity,
it
perhaps by
direct
superoxide
Mn-SOD
MntH in
organisms (e.g.
of any enzyme
may scavenge the
-
Cd may also be
knock-out mutant of mntH resulted in increased H2O2
may
production
Nramp homologues
specific Mn2+ transporters (but
dismutation of
some
proteins (e.g. Fe-SOD),
would support the
have also been described to express
substrate) although only
SOD) [72].
of defense
would either be
pathogens.
Nramp 1 for metal ions
typhimurium
phagocytosed pathogen
phagosome
[3].
was
radical
as
General Introduction
Intestinal lu
H*
•
24
•
Fe
fc2t
°0
ft3+
•
Tl*. empty transferrin
m
Fe;Tf. lrim>fcrrin with
X
Empty transferrin
Fe34
receptor
O
Nranipl
Na/KL'-ATPase
and Na
/H'-exchanger
Nramp2 (adapted from [88, 124]). Nutritional Fe3+ is reduced
Fe2+ which is taken up by Nramp2 together with H+ in a 1:1
stoichiometry. The proton gradient required for the Fe2+ uptake is maintained by the Na+/K+-ATPase and the
Na+/H+ exchanger. Fe2+ in the cytoplasm is converted to Fe3+ and stored bound to ferritin. For distribution via the
blood, iron may be exported by the transporter Iregl in parallel with the oxidation of Fe2+ to Fe3+ by the
ferroxidase hephaestin. In the blood, Fe3+ is bound to transferrin (Tf). Iron-loaded transferrin can bind to the
Figure
1.3 Model of the fonctions of mammalian
at the surface of an
enterocyte by DcytB
to
(TfR) and the complex can be internalized by erythrocytes or other cells via endocytosis. In
endosome, Fe3+ is released from Tf (acidification of the endosome via H+-ATPase favors this process) and is
transferrin receptor
the
reduced to
Fe2+ by
the endosome to
an
oxidoreductase. The empty Tf-TfR
complex
is
recycled
to the cell surface. Maturation of
late endosome is not well understood but may involve recruitment of Nramp2 to the
endosome membrane. Nramp2 exports Fe2+ to the cytoplasm as an Fe2+/H+ symporter. The H+ gradient is
a
H+-ATPase. DctyB: Ferric reductase; Hephaestin: Multicopper ferroxidase; Iregl (ironregulated transporter 1, ferroportin, metal transporter protein-1 MTP1): Protein for iron efflux.
maintained
by
the
20
General Introduction
Mode of
transport
It is
generally
Studies with
potential.
dependent
on
overexpressing
believed that transport via
their native
mi croelectrode
as
Nramp transporters
divalent metal
a
voltage clamp analysis
showed that metal transport
Xenopus oocytes
pH [21], and Mn2+ influx
extracellular
Nramp2 occurred
in
Smflp expressed
Nramps is driven by the electrochemical
was
in E.
coli
also sensitive to
as
was
Nrampl ([13-15],
being imported
must in
this
also
see
metal
act as a
electrophysiological
transport process is dependent
on
the
on
high degree
of sequence
mice extruded
Nrampl-expressing
the
pH-gradient
in the
inhibited
Mn2+
mechanism. In
symporting
orientation
same
identity.
proton gradient, again
as
in
pH
way which
a
Mn2+
the
by
deprive
the
pathogen
twosome
regulation
acidic compartment,
supported by
which showed that the
speaks
for
an
Nrampl
antiporter
Nrampl
mechanism
phagosomes
Nrampl-deficient mice,
[68]. This would speak for
a
and
metal
despite
isolated from
dissipation
of
cation/proton
addition, HA-tagged Nrampl inserted into the plasma membrane
Nramp2 and mediated uptake of
as seen
Fe2+, Mn2+
and
Co2+
down the
forNramp2 [43].
seem
possible
when the role of
phagosome
would
Nrampl is taken into
catalyze production
the Fenton/Haber-Weiss reaction in the microenvironment
On the other
pathogen [140].
an
shown that
was
faster than
consideration. Metal cation movement into the
radicals
the
on
may have different modes of transport
In contrast, it
extrusion
Both transport directions
hydroxyl
by
there is
This model is also
Xenopus oocytes,
[50]. This suggests that Nramp2 and Nrampl
the
is
phagosome
cation/proton antiporter.
measurements
shown
Nramp regulation) support the model of metal cations
into the endosome. Since the
case
typhimurium
Xenopus oocytes [57]. However,
controversy regarding direction and mode of transport of Nramp 1. Studies
of murine
S.
pH [72]. Transport of rat
cation/proton symporter
in
and
was
of toxic
surrounding
hand, extrusion of metal cations from the phagosome would
from factors essential for defense
(e.g.
metals
as
cofactor for SOD,
[68]).
Nramp regulation
Regulation
homologue
deficiency)
increases
to
of
Nramps is discussed
homologue.
Inactivation
suppresses oxidative
sensitivity
damage
toward Cd and Cu
by
at
several
mutation
in yeast
lacking
toxicity [81].
21
levels
of the
and
seems
to
vary
from
gene
BSD2
(bypass
SOD
the Cu/Zn
superoxide
Western blots in
a
dismutase and
yeast bsd2
mutant
General Introduction
showed that
Bsd2p
controls
the half life of
Smflp
was
of
Smflp
via
degradation
for
ubiquitination
Smflp
the bulk of
directed to the vacuole.
this time
plasma membrane,
the
endoplasmic reticulum,
depending strictly
not
on
inducing
metal
synthesis [83].
manganese is
thus
on
allowing transcription
the
in
be
complemented
on
an
(TfR).
shown for
Smf2p
regulation
Smflp effectively
a
switch for
rapid
plasma
protein
new
membrane when
in
way similar to
a
an
iron
binds to this sequence and confers
of TfR and accumulation of the
possible
seems
the level of mRNA
binding
and
for rat
in
mouse.
In
Irtl)
is
synthesis
sites for APT
of A. thaliana
are
-
responsive
stability
in the
protein
Nramp2 since there has been
low
to
the
plasma
an
IRE
synthase (NAS)
the metal chelator nicotianamine
role in metal transport
similar
thought
to
a
the chloronerva mutant (nas
was
mutant)
regulation. [NAS
found in all
undetectable
Lelrtl and
22
-
and the
was
plants
irrespective
Both genes
functional FER
lie in the internal transport of iron and other
LeNrampl expression
factor
also
[9]. LeNrampl and
regulatory pattern.
required
for proper
(NA)
dependent regulatory
{Lycopersicon esculentum), LeNrampl
a
a
for yeast SMF3. The promoter
[103]. Transcriptional regulation
tomato
showed
proposed
iron
an
under iron deficient conditions and
factor and nicotianamine
role of NA is
cobalt did
with the difference that
The mRNA of TfR contains
smfl yeast mutant, suggesting
(a homologue
Lelrtl and
provides
pathway,
transcript [57].
tomato
up-regulated
localizes at
or
copper
starvation, without the need for
was
fact,
conditions, but is
Bsd2p
Because
post-transcriptionally regulated
is induced when iron levels
transcription
produces
to
binding protein
An IRE
of the gene contains two
Lelrtl
of toxic metals and also
In
SMF1 is distributed to the
control of
post-translational
regulatory pathway
supply.
is decided in the secretory
Smflp protein
levels. This
of the transferrin receptor
Regulation
studied
limiting,
concentration, since addition of zinc,
proposed
was
membrane. This type of
postulated
membrane under favorable
Bsd2p -independent pathway.
a
or
limiting [103].
(IRE).
transcript,
plasma
protein
to
Instead, Bsd2p probably targets
under conditions of sufficient manganese
under conditions of metal
same
Nramp2
regulation
element
The
of the transporter
endocytosis
in the proteasome.
the fate of the
state
[83]. The pathway leading
is distributed to intracellular vesicles instead of the
protein
Rat
by
hyperaccumulation
uptake
deleted
when manganese becomes
manganese
change Smflp steady
minimizes the
the
degradation
Only
was
did not involve
Bsd2p
is not localized at the
Smflp
Nramp homologue Smflp protein and that
increased when BSD2
subsequent degradation
the vacuole for
to
of the yeast
stability
transcription
is the enzyme which
tested to date
metals.]
of iron
were
In the fer mutant,
supply
LeNrampl expression
[125]. The
was
conditions. In
higher
in iron
General Introduction
sufficient and lower in iron deficient conditions
double mutant gave the
LeNrampl
was
hardly
chloronerva mutant
picture
same
the
as
detectable. Since the
allele, the
the
to
fer single mutant,
fer
mutant
fer
wildtype.
i.e.
an
act in
chloronerva/fer
of Lelrtl and
epistatic
the
signaling pathway
gene
A
expression
allele had
products probably
two gene
authors suggest that NA acts in the
compared
as
same
the
effect
the
on
pathway.
The
sensor
for iron
more
into the
availability [9].
Another group
of the involved promoter
structure
non-canonical E-boxes
but lacks
Spl site,
bHLH
a
transcription
suggested by
Max, is bound
of
with the
not
of
factor
a
that
core
promoter of Nrampl contains multiple
Myc-Max binding sites,
Co-transfection with
role for the
transcription
the authors is that in
and
Nrampl regulation going
loops
initiator elements
factor Miz-1 in
proliferating
cells
the
(Inr)
activity.
represses promoter
cells
and
an
heterodimer
Further
Nrampl activation. The
c-Myc, possibly
interact with Inr-bound Miz-1 for
to
cells
two
Nrampl promoter-constructs and the
c-Myc showed that c-Myc
proliferating
finding
or
murine
on
[13-15]. The
non-proliferating
In
Nrampl.
complexes
at E6
(E1-E6)
TATA box.
experiments suggested
model
concentrated
as a
dimer with
transcriptional repression
Mad-Max
replaces Myc-Max
This is consistent
thereby allowing Nrampl transcription.
Nrampl, which is repressed by c-Myc, is expressed only
in mature
(but
immature) macrophages.
c-Myc
was
shown to
the labile iron
respectively,
positively
or
negatively regulate
pool (cytosolic
iron
pool).
genes that increase
cytoplasm. Thus,
in contrast to
mode of transport would be
Nramp2 which
was
as a
shown to be
a
decrease,
The authors argue that since
Nrampl, the function of the transporter should lie
represses
or
in
depletion
divalent cation / proton
symporter ([57],
see
c-Myc
of iron from the
antiporter [13-15],
also
chapter
Mode of
transport).
In S.
regulated
on
typhimurium
the
the
Nramp homologue
transcriptional
level
by
bind to the promoter in the presence of
activator
OxyR
which activated
Mn2+-MntR regulation
was
the
Mn2+
MntH is involved in H2O2 resistance and is
transcriptional
or
transcription
Fe2+, respectively,
and
[100].
the
by
in the presence of H2O2
also shown for E. coli MntH
23
repressors MntR and
Fur, which
transcriptional
[72]. Fe2+-Fur and
Table 1.3 Overview of selected
Name
SMF1
Nramp homologues
Organism
Localization
Specificity
Function
Saccharomyces
Plasma
Mn^TCiPVCd2"
~7Z*~
Mn
cerevisiae
membrane
[84]
uptake [23],
Xenopus oocytes [21]
transport
in
SMF3
AtNrampI
Posttranslational
vacuole via
is sufficient
(baker's yeast)
SMF2
S. cerevisiae
Intracellular
(baker's yeast)
vesicles
S. cerevisiae
Vacuolar
(baker's yeast)
membrane
Arabidopsis.
thaliana (thale
cress)
probably
intracellular^
[30]
Mn'+, Co'+ [84]
Fe
7T~
References
Regulation
Fe2+
Mobilization of Mn stores, which
are
[86], Fe2+
Xenopus oocytes [21]
vacuole via
transport in
is sufficient
[103]
by
targeted
Bsd2p when Mn supply
[83]
to
[121]
Posttranslational
delivered to Mn-SOD
Mobilization of Fe stores
Mn:
by Mn: targeted to
Bsd2p when Mn supply
[103]
Transcriptional by iron: two potential
binding sites (iron regulatory
factors) in the promoter [102]
Induced by iron starvation
Aft
Divalent metal
cations
Fez+, Mn^+, Cd^+ transport
in
yeast
complementation assays (S.
cerevisiae smfl, fet3/fet4) and in A.
[30, 127]
thaliana
overexpression
leads to increased
resistance to toxic iron concentrations
AtNramp3
AtNramp4
A. thaliana
Vacuolar
Divalent metal
Mobilization of metal stores from
(thale cress)
membrane
cations
vacuole
A. thaliana
Divalent metal
(thale cress)
cations
Fe^+, Mn^+, Cd^+ transport in yeast
complementation assays (S.
cerevisiae smfl, fet3/fet4) and in A.
Induced
by
iron starvation
[126]
Induced
by
iron starvation
[127]
thaliana
LeNrampl
Lycopersicon
roots
esculentum
Divalent metal
Complementation
cations
smfl
of S. cerevisiae
(tomato)
LeNramp3
Lycopersicon
roots, leaves
esculentum
Divalent metal
Complementation
cations
smfl
Fez
Pathogen
of S. cerevisiae
Via FER
(TF) and NAS
(nicotianamine synthase),
by iron deficiency
Independent
[9]
induced
of iron conditions
[9]
(tomato)
Nrampl
Homo
sapiens
Phagosome
membrane of
macrophages
resistance
[55]
[17]
Name
Organism
Localization
Nramp2
(DMT1)
H.
Late
sapiens
Specificity
Fez+
endosomes,
lysosomes
(HEp-2 cells)
Nrampl
Nramp2
(DCT1,
DMT1)
Slc11a1
DMT1
NrampC
Function
Phagosome
Divalent metal
Metal cation /
(mouse)
membrane of
cations
[68]
Rattus
macrophages
Enterocytes
Dog
(Rottweiler)
Repression by c-Myc,
Miz-1 [13-15]
proton symporter,
independent iron uptake
Putative IRE element
Divalent metal
Metal cation /
transferrin
Danio rerio
Red blood
(zebrafish)
cells
by
[57]
[2]
Fez+
Fez+ uptake in human kidney
overexpressed
[34]
cells
when
Expression induced after induction
macrophages and after LPS
MntH
MntH
Escherichia coli
S.
typhimurium
Bacillus subtilis
[20]
of
treatment offish
Mature
Mn2+, Fez+
Involved in the
taste
neurons,
signal
perception
transduction of
[97, 109]
macrophages
MntH
[131]
Sequence variants associated with
susceptibility (Leishmaniasis)
Ictalurus
Drosophila
melanogaster
induction
disease
punctatus
(channel
catfish)
Mvl
[124]
proton co-transporter
cations
Macrophage
in
absorption
enterocytes; recycling of iron from
phagocytosed erythrocytes;
transferrin-dependent iron uptake in
erythrocytes and other cells
Mus musculus
norvegicus (rat)
References
Regulation
Nutritional iron
Cell membrane
Cell membrane
Specific for Mn2+
[72] and Fe2+ [72,
100]
Specific for Mn2+
and Fe2+ [72]
Mn2+ and Cd2+
Mn-transporter;
involved in response
to reactive oxygen
Mn-transporter;
[72]
involved in response
to reactive oxygen
[72]
Repressed by
[71, 100]
Fe-Furand Mn-MntR
Transcription activated by hydrogen
peroxide via the transcriptional
activator OxyR. Repressed by FeFurand Mn-MntR [71, 100]
Transcription repressed under Mn
excess
conditions via the
metalloregulator
MntR
[104]
General Introduction
Functional
For
analysis
of S. cerevisiae
Smflp
functional
a
analysis
several mutated versions of the
protein [82] (Tab. 1.4).
transmembrane domain 4
and G424A in the
resulted in
In
a
loss of function
addition, those
variants
respond
by
were
shown
as
by
immunofluorescence
the other two functional mutants
essential for
targeting
to
depending
or
and
(Q408A
the vacuole
proteins
were
G418A). Apparently,
by Bsd2p
deplete
(Q419A
was
conditions.
and
not
the
functional
a
metal
on
E423A)
redistributed to the
not
This
microscopy.
assays.
showed that these
microscopy
functional. However, two of them
did not allow response to decreased metal levels and the
membrane
complementation
redistributed to the cell membrane under metal
Four other mutations in the CTM
plasma
functional
immunofluorescence
G190A in
transport motif (CTM), both
regulation by Bsd2p
to
of
by generation
point mutations,
Two
consensus
shown
as was
experiments by
not
were
phenotype
did not
mutants
conditions. Localization
protein
(TM4)
conducted
was
for
case
Smflp
is
but is not sufficient for redistribution to the
cell membrane.
An extensive functional
amino
acids
replaced
were
complementation
analysis
and
was
function
assays in yeast and
by
Table 1.4
labeling.
mutations in the
gives
a
by
fluorescent metal transport assays in CHO cells
[79,
in yeast
expression
mutated
was
on
(Q384E,
G394V
proteins
checked
by
the cell surface
summary of the mutational
CTM
Nramp2
of the
Nramp2. Several
checked
101]. Expression of the mutated proteins
membrane fractions and in CHO cells
also conducted for murine
was
western
was
blotting
tested
phenotypes. Interestingly,
biotin
by
point
two
[101]) prevented complementation
of the
yeast mutant, while the corresponding mutated variants of Smflp (Q408A, G418A [82])
functional
(see above). Perhaps
for correct localization
G185R is
iron
deficiency
the yeast
a
or
this
reflects the need for
finding
function of the mammalian
naturally occurring point
and
smfl/smß
microcytic
mutant
protein, missing
[42].
[79]. This might indicate that the mutated protein
was
targeted
compartment in the yeast, suggesting that the mutation affected
already
seen
occurring
disrupts
above for
Nramp2
G169D mutation in
function of
mutants
Nrampl
Nrampl and leads
-
to
in the yeast system.
Nramp2G185R was
significant transport activity
were
interaction partners
mutation in TM4 and is associated with
anemia in mice
but showed
specific
of
in
unable to
a
to a
severe
complement
metal transport assay
transport-incompetent
correct
localization
[79],
as
Q384E and G394V. Interestingly, the naturally
a
residue
adjacent
susceptibility
26
to
to Gl85
pathogen
in
Nramp2
-
totally
infections in mice. This
General Introduction
mutation
probably
Nrampl protein
two
in
causes a
structural alteration which prevents accumulation of the mature
macrophages [132].
evolutionary absolutely
Two further mutations
rescued
by lowering
of H267/H272 is
required
residues would
shift the
protonation
for the
of other groups
Table 1.4 Mutational
analysis
of
inserted in
conserved histidine residues H267 and H272
and found to be essential for function of
mutants were
were
the
Nramp2 [79]. Strikingly,
pH from
protein
6 to 5. The authors
to assume a
side chains to create the
Smflp
and
were
substituted
of the histidine
some
suggested
that
protonation
functional conformation. Loss of these
pH for maximal transport
or
Nramp2. The
to
same
a
more
acidic
value, requiring
conformational state
[79].
Nramp2.
A: The effect of individual mutations is shown. Protein
expression was stable for all mutants and all proteins
expression in the yeast membrane fraction. For transport experiments with CHO cells Nramp2
expression/localization on the cell surface was determined by biotin labeling. Smflp localization of G190A,
Q419A E423A and G424A was checked by immunofluorescence microscopy. Corresponding residues of
Nramp2 and Smflp are in the same column. Bold residues are evolutionary absolutely-highly conserved, nd: not
were
checked for
determined
Graphical representation of the Smflp and Nramp2 amino acid chain (not drawn to scale). The black bars
symbolize the transmembrane regions of the proteins (numbered 1 through 12) and mutated amino acid residues
are indicated above or below the corresponding TM. CTM: consensus transport motif.
B:
27
Table 1.4 Mutational
analysis
of
Smflp
and
Nramp2.
A
SMF1
G190A
Q408A
G418A
Q419A
E423A
+
+
+
+
nd
nd
+
+
+
+
[82]
[82]
[82]
Q384E
G394V
Q395E
complementation
-
of smfl
regulation by
Bsd2p
regulation by
metals
reference
[82]
Nramp2
complementation
D86A
-
R119A
R146A
+
+
E154A
nd
nd
not
[79]
expressed
[79]
D161A
G185R
+
-
D192A
-
-
+/-
+/-
E225A
E299A
+
-
-
G424A
[82]
[82]
R416A
+/-
-
-
-
of smf1/smf2
transport in CHO
-
cells
reference
[79]
Nramp2
complementation
[79]
H267A
H272A
+
-
of smf1/smf2
transport in CHO
cells
rescue
by
+/-
(very low
expression)
y
y
[79]
[79]
+
[79]
[79]
H267A/H272A
[79]
H267C
-
nd
-
[79]
H272C
nd
-
[79]
[101]
H267C/H272C
+
nd
-
[101]
H267R
nd
not
[101]
expressed
[79]
H272R
H267R/H272R
-
-
-
-
-
-
(very low
expression)
+/-
y
y
y
nd
n
n
n
[79]
[79]
[79]
[79]
[79]
[79]
[79]
-
-
-
not
expressed
low
pH
reference
Û40§â
B
mmk
(MfSA
SMF1
E423A
G190 A
GM24A
General Introduction
and transition metal
Algae
1.5
Molecular data
assimilation
transition metal transport in
on
in the green
pathway
[61, 78] (Fig. 1.4). Based
mediated
by
a
reduced
to
Fe2+ by
similar mechanism
the iron permease
to
Fe2+ by
Fe3+.
as a
as
II
an
iron
was
identified and characterized
high affinity
iron
uptake
cerevisiae. In the
Fet3p oxidizes Fe2+
In A thaliana
plants)
in C reinhardtii is
yeast, Fe3+ is firstly
Frelp and Fre2p.
[107] but it is taken
(Strategy
Only recently
scarce.
C reinhardtii
reductases
ferroxidase
Grasses
is
algae
Saccharomyces
Ftrlp into the cell.
up
utilize
a
Fe3+,
to
(Strategy
the
by
I
the
Subsequently,
which is then
plant), Fe3+is
Fe2+ transporter
imported
also reduced
IRTl without
different mechanism and take up iron
Fe3+-phytosiderophore complex.
Similar to yeast,
(FRTl [78])
were
a
multicopper
respectively.
The
obligatory
an
directly demonstrated,
conditions and
Fe2+
Fe3+
to
mechanism is
proposed
reinhardtii involves
depleted
ferroxidase
inhibited
was
and
the
by
in cell
activity
multicopper
a
cellular copper status.
Thus,
copper is not
required
and
biosynthesis
a
of FOX1/FLP
in
[78],
a
similar way
With the reoxidation of iron to
assimilation system not found in
FRT1
system,
deficiency
higher plants
further iron assimilation
a
did not lead to
ferroxidase accumulation
secondary
was
Fe3+
as was
for
so
iron-responsive regulation
together
to
on
of the
ATX1 copper
supply
Cu for the
[137].
C reinhardtii possesses
must
be present in the
deficiency by influencing
reduced. This
dependent
was
of
an
iron
far. However, in addition to the FOX1/FLP-
pathway
iron
was
deficiency, independently
shown for yeast
uptake,
in C
[62]. The transcripts
protein, however,
ATPase may work
putative Cu-transporting
uptake
ion,
increased under iron
suspensions
ferroxidase, but for the accumulation of the protein itself [78]. The identified
chaperone
which
[36]. Activity of FOX1/FLP
oxidase inhibitor
for the
proteins
that iron
finding
for the ferroxidase and the iron permease accumulated under iron
copper concentration. Accumulation of the ferroxidase
iron permease
of the trivalent
subsequent uptake
supported by
an
C reinhardtii, two
alga
reduction of Fe3+-chelates
but ferroxidase
and
(FOX1/FLP [61, 78])
identified and characterized in the
may account for the oxidation of
not
in
Fe3+-chelate
the
the reductase FR02
reoxidation to
microalga
these studies
on
metalloenzyme multicopper
by
transport
backup system
copper nutritional status in order to guarantee sufficient iron
must
supply
alga
iron
be
since copper
uptake, although
independent
under copper
of the
deficiency
[78] (see also chapter 6).
Another
gene for the
protein
previously
with
a
possible
role in iron assimilation in C reinhardtii is H43. The
described C02-inducible
29
polypeptide
H43
[73]
was
recently
isolated
General Introduction
H43 is not related to any other
[112].
marine
Chlorococcum
alga
in the database except for HCR1 from the
protein
littorale, which is induced by high CO2 and iron deficiency [117].
Induction of H43 mRNA under conditions of cadmium
demonstrated. Based
in the cell
deficiency
on
their
and iron
toxicity
results, the authors suggest that cadmium
by saturating
the iron transporters,
inhibiting
or
response which includes induction of H43
leading
deficiency
was
causes
iron
to an
an
iron stress
propose that H43 and HCR1 may
[112]. They
represent novel, algal-specific proteins that may facilitate iron transfer from the ferric
reductase to the iron transporters
littorale incubation under
needing
more
[112] (Fig. 1.4). Induction by CO2 might be indirect.
conditions resulted in increased
high CO2
iron for the
of
biosynthesis
proteins
involved in
In C
photosynthesis, possibly
photosynthesis (cytochromes,
ferredoxin) [117].
In contrast to the iron
uptake system,
identified yet in C reinhardtii. Also for
Only recently
a
family
of
putative
plants
in
observed
tomato
for this
responsible
plant
deficient in copper
mutant
uptake
copper
In C
a
whether the
sole enzyme
enzyme(s)
Fe3+
and
uptake
Cu
a
[66] and
Only
roots
pea
one
activity
cupric
-
as
reduction to
Ctr3p [130].
radioisotopic
Fe2+ (Frelp
In C
copper
uptake
The
affinity
transport system
expression
was
in yeast
Cu2+
and
a
assay
more
is
a
activity
has been
[133] the
enzyme
a
Cu reductase in
family
of
relation to copper
measured
was
copper
is then taken up
uptake system
by
higher
copper-deficient
unaffected
in
of the system is induced in
by
the
of the
identity
same
two
reductases
as
Ctrlp
has not been identified yet, but
than in
a
high affinity
copper-sufficient
copper-deficient cells, indicating
30
displayed by
copper conditions but the
copper-deficiency.
[61]. The
the Cu transporters
demonstrated the existence of
active in
are
because the
performed by
together with
conditions
deplete
unexplored yet,
reduction to Cu+ is
constant was
20 times
-
Fre2p). Cu+
reinhardtii,
transport pathway which is
(Fig. 1.4).
under iron and/or copper
case
is unknown. In yeast,
in
[26].
reductase and the ferric reductase activities
is the
(COPT family),
member of the FRO
no
scarce.
overexpressed
role for
possible
a
when
Cu reductase
reinhardtii, induced cupric chelate reductase activity
chelate reductase
question
by
vesicles
has not been established yet.
homeostasis has been demonstrated
or
high affinity
identified
was
is
uptake
copper
ferric chelate reductases has been characterized to date but
putative
ferric
proteins
has not been identified and
activity
regarding
uptake [70, 116]. Although
membrane
plasma
information
copper transporter
with COPT1 and COPT2 shown to mediate
yeast
copper assimilation mechanism hasn't been
a
In the
same
that the
study cupric
copper
cells
activity
a
[63]
of the
activity
or
reductase
General Introduction
and copper
activity
are
uptake
were
regulated coordinately [63].
repressed by equal
It is therefore
amounts
possible
may resemble that in yeast, which includes reduction and
of copper
that copper
suggesting
uptake
subsequent uptake
that
they
in C reinhardtii
of Cu.
>H43
uptake
system? DMTl?
copper
1.4 Scheme of copper and iron homeostasis in C. reinhardtii. FOX1: multicopper ferroxidase, FTR1:
iron permease, ATX1: copper chaperone, H43: putative iron chaperone, DMTl: divalent metal transporter 1.
When copper is limiting (-Cu / +Fe) cupric chelate reductase activity [61] and high affinity copper uptake is
Figure
induced
[63]. Under iron limitation (-Fe / +Cu) ferric chelate reductase activity and iron uptake via the high
affinity FOX1/FTR1 system is induced [61, 78]. Fe2+ may be delivered from the reductase to FOX1 by H43
[112]. ATX1 and Cu-transporting ATPase are also induced and may contribute to FOX1 biosynthesis by copper
delivery to the secretory system [78]. Under copper and iron limitation (-Cu / -Fe) an alternative uptake pathway
for iron must exist since FOX1 cannot be
conditions
chapter
[61]. The role of
DMTl in
synthesized [78]. Both
uptake of iron or copper
6.
31
reductase activities
as
indicated in the
are
induced under all three
figure
will be discussed in
General Introduction
Chlamydomonas
1.6
reinhardtii
-
a
short introduction to the
"green yeast"
Taxonomy
Superkingdom:
Eukaryota
Kingdom:
Viridiplantae (green plants)
no
rank:
Chlorophyta (green algae)
Class:
Chlorophyceae
Order:
Volvocales
Family:
Chlamydomonadaceae
Genus:
Chlamydomonas
Species:
Chlamydomonas reinhardtii
Chlamydomonas
different
species,
glycoprotein
chloroplast,
formed
is
found in
genus of unicellular green
a
equal flagella.
single large pyrenoid,
photosynthetic products.
excretory function,
are
sensitive, allows the cell
located
to
algae, including
soil, fresh and marine water, and
wall and two anterior
which contains
from
a
near
the
swim towards
Two
a
proteinaceous
contractile
and
a
light (Fig. 1.5).
Figure 1.5 The morphology of Chlamydomonas reinhardtii [31].
mitochondrion, N: nucleus, P: pyrenoid, V: vacuole
32
snow
C:
red
than 500
[59]. The cells have
The nucleus is enclosed in
small
flagella
in
more
structure
a
cup-shaped
where starch is
vacuoles, which have
pigment spot
which is
The genome counts 100
chloroplast,
a
ES: eye spot, F:
an
light-
Mbp.
flagellum,
M:
General Introduction
The most
species
are
widely
about lOum
but
inorganic salts,
provided
also
in
cells of
the
system for research
be handled
which is
a
life
vegetative
a
suitable
A
life
phenotypes
cycle
major
nuclear genome
and is often
replacement
is
and
are
occurs
at a
frequencies
low
the
releasing
a
hard outer
four
especially
It is
used
as
haploid
assays allowed
a
time
(6 hours)
is
(2
of
phenotype
forms colonies
of C reinhardtii
was
Doe Joint Genome
a
of the gene of interest is
and
short,
a
wide
University,
the
during
genome
a
efficient system for directed
out
of 3
an
on
recombinants, [94]).
particularly important.
project, precise
by
function
In the
was
"green yeast"
and has two
mating types,
February
2003
of
a
unknown and it seemed
the
state. On
case
gene
hardly applicable. Concerning
of the transport function of the
as
At such low
in order to detect
alternative method. Yeast functional
plates,
disruption
transformants, [94])
good screening system
is unknown this method is
diploid
released
an
recombinants out of 2000
frequency (3
thorough investigation
haploid
the
but
recombination between introduced DNA and the
C reinhardtii is sometimes referred to
quickly,
since
model
a
eukaryotic organism,
a
Genetics Center, Duke
immediately expressed
availability
fruitful to start characterization
on
zygospore with
generation
Chlamydomonas
deletion events
studied in this
protein,
a
The
techniques.
lacking. Homologous
mutants
can
(e.g. nitrogen deprivation)
meiosis
and is
flagellar motility:
drawback of C reinhardtii is that
where the mutant
between
but
cycle
haploid.
accompanied by
recombination
grows
darkness if acetate is
great advantage for research purposes. Cultivation is simple and cheap, and
of nuclear genes is
DMTl
medium of
simple
a
fast mitotic life
diploid
a
laboratory organism
standard microbial
by
Mutant
vegetative
on
cycle.
range of mutant stocks is available at the
Durham.
a
diploid zygote undergoes
photosynthesis
on
fuse to form
can
reinhardtii. Cells of this
in total
heterotrophically
C reinhardtii has
source.
the
improve,
C reinhardtii is
can
photosynthetically
grow
also grow
can
opposite mating types
resume
can
Chlamydomonas
controlled sexual way. Under adverse conditions
a
wall. When conditions
cells that
long. They
they
is
laboratory species
alternative carbon
as an
reproduce
haploid
used
a
the
more
complementation
protein.
because it is
unicellular,
which allows it to switch
first draft of the
complete
genome
(version 1.0, http://genome.jgi-psf.org/chlrel/chlrel.home.html,
Institute, California, [52]).
http://www.biology.duke.edu/chlamy/
More information
and in
33
[59].
on
C reinhardtii is available
General Introduction
Motivation and aim of the thesis
1.7
Transition metals deserve
micronutrients and
as
aspects of acquisition
families
protein
potentially
or
lot of attention because of their dual action
a
toxic substances. A lot of work concentrates
extrusion of metallic ions
in
involved
characterized to date. In this
transition
the basis of the food
more
chain, but also
than 90% of the worlds'
of oxygen.
source
and
splitting,
a
significant
in the overall
from
been
identified
in
of the
aquatic ecosystems
planet, contributing
photosynthesis
on
a
manganese for water
requires
process
important
especially dependent
are
and
molecular mechanisms
only
ecology
molecular
and several
which makes them the most
photosynthetic
resulting
stress
have
status not
on
organelles,
analogous
Algae, being photosynthetic organisms,
oxidative
cellular
or
homeostasis
photosynthetic activity,
of metal balance. The
precise regulation
metal
cells
little is known about
regard,
present in algae. Yet, these organisms have
being
by
essential
as
be
must
antagonized by
metalloenzymes.
The aim of this work
comes
to
metal transport,
the existence
green
of,
to
was
on a
identify
partial
and to
reinhardtii.
sequence database
to
proceed
with molecular
The
various
cloning
of the
assays in several yeast mutants
approach was
was
to
successful and the results
families
the
presented
and,
in
to
the database of the
perform
this
alga.
a
The
recently
thorough investigation
insights
of this
study
released
on
are
genomic
to
explore
chapters
a
C
yet available) for
if such
a
transporter
was
sequence. A detailed
of yeast
means
on
the
complementation
specificity.
This
2 and 3.
Further, elucidation of the regulation of the identified transporter
Finally,
not
was
follow, together with studies
are
was
search in
first to
was
complete coding
by
have when it
metal transport system in the
sequence
protein
functional characterization of the transporter in vivo
a
strategy
(the genomic
to
algae
this, the chosen approach
investigate functionally
potential transport systems belonging
identified,
the alternatives green
on
molecular level. For
microalga Chlamydomonas
reinhardtii
gain insight
to
was
attempted (chapter 4).
sequence of C reinhardtii
was
utilized
the presence of different metal transport systems in
presented
34
in
chapter
5.
The
Chapter
Divalent metal transport in the green
is mediated
by
a
protein
2
microalga Chlamydomonas reinhardtii
prokaryotic Nramp homologues
similar to
Information about the molecular mechanisms of metal transport in
the
despite
we
significant
describe the
status
cloning
DMTl)
in the green
of the
cloned DMTl
transmembrane
these
organisms
have in
aquatic ecosystems.
and functional characterization of
domains.
The
encodes
a
protein
protein belongs
to
of 513
the
transporters and shows surprisingly higher homology
polypeptides. Especially
considered in this
prokaryotic
impaired
acting
the
-
characteristics. Functional
uniquely
among
longest
amino
open
(named
reading
acids with
frame
putative
11
Nramp family of divalent metal
to
than to
eukaryotic
every other
homologue
prokaryotic
eukaryotic Nramps
complementation experiments
in the transport of several divalent metals
present study,
divalent metal transporter
N-terminus, which is longer than of
study, displays
is scarce,
algae
In the
exclusively
-
in yeast strains with
metal transport systems, revealed that C reinhardtii DMTl has
excluding
a
broad
(manganese, iron, cadmium,
and
specificity,
copper),
but
zinc.
Introduction
2.1
Metal ions
several
are
sites in the
indispensable
concentration in the
for cell survival since
This
mainly
reality
fulfil
environment,
[44]. Different metal elements do
are
not
equally bioavailable,
different concentrations in the cell. Iron for
occurs
they
important
functions at
cell, including stabilisation of cellular structures, enabling of redox
reactions and activation of enzymes
but
a
reinhardtii. The
microalga Chlamydomonas
cDNA
reinhardtii
Nramp homologue ofC
instance, is needed
in the form of insoluble and thus not
forces the cell to express
a
variety
readily
not occur at
35
required
same
and have to be maintained at
in greater amounts than copper,
bioavailable ferric
of transport systems in order to
range of these essential micronutrients in the
the
complexes.
acquire
a
broad
concentrations. A number of metal
The
reinhardtii
Nramp homologue ofC
transport proteins have been characterized in bacteria, yeast, mammals and plants,
them
classification
a
similar
displaying
see
[114]).
molecular level.
involved in
78]
was
features
Less is known
belonging
regarding
the
to
family (for
same
overall
an
divalent metal transporters in green
algae
iron transport in the green
microalga Chlamydomonas reinhardtii [61,
published.
Nramps (natural resistance associated macrophage proteins) form
group of divalent
a
metal cation transporters which contribute to cell metal homeostasis and have been
conserved
evolution.
throughout
the pro- and
They
cerevisiae three
are
expressed
and share
eukaryotic domains,
Saccharomyces
in many different
high similarity
Mn2+ ions,
SMF2 is
but it
[86]. SMF2
dismutase
level
posttranslational
contribute
to
by
the
iron
Nrampl
the
expressed
phagosomal
manganese
depletion
and
cadmium
in the
of
phagocytosis
ions out of the
broad
displays
endosomal/lysosomal
decreased
Nramp homologues
typhimurium
MntH
-
proteins
which
pathogenicity.
-
MntH
in
as
[103].
space
pathogens
Some
of the
case
with
to
was
important
3
and 4
by
yeast
as
as
a
the
pH-
shown to transport
mediating
a
defence
metal
cofactor for
mechanism,
have also been described to express
Gram-negative
and Escherichia coli
reactive oxygen
bacterium
in the
were
Salmonella
phagosome.
The
shown to be
Mn2+
[72]. Several other organisms
Nramp homologues [20, 34, 39, 109] and putative
36
the
and is recruited to
[68]. Since Mn2+ is
an
at
The mammalian
that its role lies in
Nrampl for metal ions
typhimurium
transporters involved in the response
It
ions
In the thale
shown
was
the
vacuole, is
AtNrampl,
macrophages
pathogens [55].
phagosomal
the
might compete
from Salmonella
described to express
found.
Mn2+
regulated
in the
specificity [57].
vesicles of
phagosome, suggesting
of divalent metals from the
to
were
transport
MnSOD, its lack would deprive the pathogen from
resulting
are
for
[84].
supply
to
iron transport system in the intestine. It functions
membrane upon
(Mn2+)
suggested
iron
down-regulated by
Nramp homologues
divalent cation transporter and
is
specific
[30, 127]. Mammalian Nramp2 (DMTl, DCT1) is described
tests
major transferrin-independend
dependent
was
protein Bsd2p [83, 84]. SMF3, expressed
thaliana several
manganese,
complementation
the cell surface and is
[84]. SMFl and SMF2
believed to mobilize vacuolar iron stores and is
Arabidopsis
at
in the mitochondria with the essential
(MnSOD)
also shown to transport cobalt
was
to
the amino acid level. In the yeast
in the membrane of intracellular vesicles and
superoxide
manganese
organisms belonging
also shown to contribute to copper and cadmium accumulation
was
expressed
at
highly
Nramp homologues have been identified and characterized,
SMFl, SMF2 and SMF3 [103, 121]. SMFl is expressed
cress
at
the molecular identification and characterization of genes
Only recently,
high affinity
and
of
some
genes
were
encoding
The
members of the
sequence
Nramp family have been identified
a
in many other
organisms
based
on
homology.
The present
of
reinhardtii
Nramp homologue ofC
study
describes the
identification, cloning and functional characterization
divalent metal transporter from the green
protein designated
members of the
DMTl
shows
highest
microalga Chlamydomonas
amino acid sequence
Nramp family. Functional complementation
homology
manganese,
2.2
Materials and methods
2.2.1
Strains and
copper and iron but
cerevisiae strain Y16272
ura3A0; smfl::kanMX4) is
obtained
a
yeast
mutant in
Dr. D.
protein displays
broad
zinc.
from
(BY4742; MATa; his3Al; leu2A0; lys2A0;
which the SMFl gene has been
Frankfurt
EUROSCARF,
The
frankfurt.de/fbl5/mikro/euroscarf/index.html).
provided by
no
impaired
plasmids
Saccharomyces
was
cadmium,
prokaryotic
to
in yeast strains with
manganese, zinc and iron transport systems revealed that the DMTl
specificity, transporting
reinhardtii. The
following
two
disrupted
(http://www.unistrains
were
Eide, University of Missouri-Columbia and have disruptions
zinc and iron transport,
respectively:
S. cerevisiae strain ZHY3
his3; leu2; trpl; ura3; zrtl::LEU2; zrt2::HIS3) [139] and strain
leu2; his3; canl; fet3::HIS3; fet4::LEU2) [32].
C reinhardtii strain
For
a
listing
cwl5arg7 (kindly provided by
and
kindly
in genes for
(MATa; ade6; canl-100oc;
(MAT
DDY4
of yeast strains
E. H. Harris
[59])
see
was
a;
ura3; trpl;
also table 2.1.
used for RNA
isolation for RACE and RT-PCR.
To express the C reinhardtii DMTl in yeast the
cloned into the yeast
University
of
vector
pUG35 (kindly provided by
Düsseldorf, Germany; Guidener, U., and Hegemann,
preparation). pUG35
MET-25
expression
corresponding full-length
has
a
CEN/ARS
origin
of
replication (1-2 copies
promoter for moderate expression of heterologous genes.
37
J.
per
J.
H.
H.
cDNA
was
Hegemann,
manuscript
cell)
and
uses
in
the
The
Table 2.1 List of S.
cerevisiae
strains used in this
Name
Deletion in
Genotype
Y16272
SMFl
MATa;
reinhardtii
Nramp homologue ofC
study
Obtained from
his3Al;
leu2A0;
lys2A0;
EUROSCARF, Frankfurt
http://www.uni-frankfurt.de/
ura3A0;smfl::kanMX4
fb 15/mikro/euroscarf/index.html
ZHY3
ZRT1/2
MATa; ade6; canl-100oc; his3;
leu2;
ura3;
zrtl::LEU2;
trpl;
DDY4
FET3/4
MATa; ura3; trpl; leu2; his3; canl;
fet3::HIS3;fet4::LEU2
zrt2::HIS3
Eide,
Department of Nutritional Sciences,
University of Missouri Columbia
Dr. D.
Dr. D. Eide
Culture conditions
2.2.2
S.
cerevisiae: For selection of transformants cells
minimal medium
containing
sulphate, DIFCO),
agar. For
0.5% ammonium
above
as
supplemented
chelators EGTA
(Ethylene
medium in
acid).
and incubated with
a
250
ml
2%
cells
(without
glucose,
were
grown
grown in
tests 5 ml
growth
shaking (200 rpm)
flask
Erlenmeyer
at 30°C
acetic
medium
for
a
density (OD6oo)
of 0.1-0.3 at 600nm and divided into several flasks
and without stress
30°C for the
course
chelators
(metal
of the
The
overnight.
or
metals).
growth experiment.
overnight
These
Growth
1-2 hour intervals for 7-10 hours. The maximal
without any stress conditions
was
defined
as
same
cells in different concentrations of stress
were
performed
several
at
experiments
least in
triplicate
is shown in the
was
a
culture
were
was
growth
100%)
and either
constant
illumination and
was
expressed
an
and
optical
medium with
then incubated with
rate
growth
diluted to
a
growth
preculture
containing
monitored
shaking
at
by measuring OD6oo
at
of each strain in the medium
of the strain and
growth
in relation to it. All
representative experiment
or
of the
growth
the
tests
mean
of
corresponding figures.
C reinhardtii: For RNA isolation C reinhardtii strain
liquid Tris-acetate-phosphate
inoculated with
few hours. 45 ml of
shaking
or
and EDTA
acid)
were
same
of metals
(see results)
incubated with
at 30°C
medium with the
liquid
inoculated with this yeast
were
synthetic
mixture of amino acids and 1.8%
a
with various concentrations
For
solid
on
amino acids and ammonium
(Ethylene glycol-bis(2-aminoethyl)-tetra
diamine tetra acetic
single colony
base
nitrogen
sulphate,
complementation experiments
composition
metal
0.17% yeast
were
medium TAP
cwl5arg7
was
grown at 25°C in
[59] supplemented with 50mg/l arginine under
agitation.
38
The
2.2.3
Research
Institute, Japan identified
AV389837)
with
identified
amplified by
pUG35.
high homology
by
nested PCR
Total
and RACE
sequence
transcription step
full-length
(EcoRI)
was
cDNA
reinhardtii
instructions.
followed
by
by RT-PCR,
haemagglutinin tag (HA-tag)
was
named
and ends 129
HA-tag
a
transformed with
pARl
and
as
of c_DNA
accession
(GenBank
ends). Full-length
were
restriction sites
the
by
use
of
partial
one
EST-
reverse
(Xbal)
and 3'
appropriate primers.
A
after the start codon and the
contains the start
codon, followed by
downstream of the native stop codon. Yeast mutants
pUG35
then
was
steps. For cloning of the
inserted at the 5'
immediately
pARl
to
[58]. Briefly,
two PCR
were
second PCR step
The insert of
by
cDNA
Reagent (GibcoBRL)
designed specific
described elsewhere
reaction and
cDNA
no.
and cloned into the yeast vector
extracted with Trizol
fused to the 5'end
pARl.
of Kazusa DNA
tag) library
sequence
transcription
was
tailing
two
was
bp
EST
RACE-primers
performed
was
sequence
Nramps. The 5'end of the corresponding
reverse
end of the cDNA in the
resulting plasmid
partial
a
known
to
following
of C
RNA
(expressed
(rapid amplification
5' RACE
according to supplier's
the
reinhardtii
Cloning
A search in the C reinhardtii EST
was
Nramp homologue ofC
were
with the YEASTMAKER yeast transformation system
(Clontech).
2.2.4
DMTl
expression
Heterologous expression
according
to
standard
method described in
protocols [115]
Anti-HA
and
of DMTl in S. cerevisiae
protocols [115].
[96].
proteins
protein
tested
gel electrophoresis
were
blotted onto nitrocellulose membrane
(peroxidase conjugate, Sigma)
as
was
the
used
as
the
was
39
extracted
to
by
the
standard
(Schleicher&Schuell).
primary antibody
detection
blotting
western
performed according
secondary. Subsequent
(Amersham Biotech).
by
content was
SDS
High Affinity (Roche)
the ECL kit
Total yeast
was
and
was
Anti-Rat-IgG
performed
with
The
Nramp homologue ofC
reinhardtii
GenBank accession numbers
2.2.5
Cloned C reinhardtii DMTl: AP515631
EST clone from Kazusa institute: AV389837
Nramp homologues
in
2.3: Escherichia coli MntH
figure
CAD07646;
Deinococcus radiodurans
Nostoc
PCC
sp.
Pseudomonas
Oryza
sativa
MntH
aeruginosa
MntH2
Homo
thaliana
aureus
Nramph AAP83825;
Nramph
NP3 74223;
Nrampl AAF36535; Saccharomyces
O.
Nramp2 AAB61961;
sativa
sapiens Nrampl AAG15405, Nramp2
Nramp homologues
MntH
Q9RPF2; Staphylococcus
9a5c
AP515631;
CAA64547, Smf2 AAB68900, Smf3 NP_013134; A. thaliana Nramp2
AF141204, Nramp4 AAP13279,
CAC27822;
enterica MntH
AAP 11265; C reinhardtii DMTl
BAB77244; Xylella fastidiosa
Nrampl S62667; Arabidopsis
Smfl
cerevisiae
7120
Nramph
P77145; Salmonella
in
thaliana
Nramp5
P49281.
2.5: Salmonella enterica MntH
figure
A.
CAD07646; Escherichia coli
P77145; Yersinia pestis MntH CAC92226; Chlor obium tepidum Nramph NP661903;
Wiggle sw or thia
brevipalpis
CAC99502; Nostoc
Nramph
sp. PCC 7120 MntH
ZP_00063650;
Enterococcus faecalis
NP_689011;
Lactococcus
ZP_00069508;
MntH2
lactis
BAB77244;
Listeria
monocytogenes
Leuconostoc mesenteroides
Oenococcus
NP_267238;
loti
Nramph BAB49617;
Nramph NP_522081; Staphylococcus
aureus
Nramph AAF11265; Chlamydomonas
Brucella
Nrampl
aries
NP_038640;
AF153279;
Rattus
Ictalurus punctatus
melanogaster
thaliana
Mvl
Nrampl P49280;
Homo
Nramph NP_374223;
reinhardtii DMTl
Homo
Nramph
P49281;
Nramph
Nramph
aeruginosa
melitensis
Ralstonia
MntH
solanacearum
Deinococcus radiodurans
AT515631;
Bos taurus
sapiens Nrampl AAG15405,
sapiens Nramp2
9a5c
Pseudomonas
NP_539486; Agrobacterium tumefaciens Nramph NP_354720;
NP_777077; Ovis
oeni
Nramph BAB47552; Xylella fastidiosa
axonopodis Nramph NP642362;
Q9RPF2; Mesorhizobium
Nramph
Nramph NP_815583; Streptococcus agalactiae Nramph
Nramph
Lactobacillus brevis
Xanthomonas
AAP83825;
NP_714977;
Macaca
Nramp
Mus musculus
fascicularis Nramp2
norvegicus Nramp2 054902; Takifugu rubripes Nramph CAD43053;
Gallus
Nramph AAM73759;
S56140;
Caenorhabditis
gallus Nrampl P51027; Drosophila
elegans Nramph NP_509132; Arabidopsis
Nrampl AAP36535, Nramp3 Q9SNV9, Nramp4 AAP13279, Nramp2 AP141204,
Nramp5 CAC27822; Oryza
T38466; S
cerevisiae Smfl
sativa
Nrampl S62667; Schizosaccharomyces pombe Nramph
CAA64547, Smf2 AAB68900, Smf3 NP_013134.
40
The
2.3
Results
2.3.1
Identification and
A search for
cloning
of the
Nramp homologous
Chlamydomonas
reinhardtii DMTl gene
sequences in the C reinhardtii EST database of the
Kazusa DNA Research Institute led to the identification of
similarity
to genes
accession
no.
AV389837)
complete only
at
named DMTl
(divalent
and the
untranslated
region
13
C
was
(GenBank
metal transporter
The
longest
open
pairs (bp)
the
[108]. The
accession
Recently, sequencing
reading frame,
long
as
has
G
a
or
since
coding
scaffold_489, contig 3,
sequence
showed
homology
(ATG)
February
2003.
at
pairs
by
was
(Fig. 2.1).
41
position
Nramps,
64. The 3'
typical
codon usage of
position.
accomplished
available
Homology
30332:34793.
us
to
1936
been described for other C
sequence of the cloned DMTl
base
{DCT1,
putative polyadenylation signal
a
already
is
to DMTl
RT-PCR reached
by
C at the third
of the C reinhardtii genome
revealed that the
and 12 introns
which
was
The cloned gene is
analogy
DMTl GC content of 62.9% reflects the
sequence to the cDNA sequence determined
exons
obtained
and contained
polyadenylation site,
psf.org/chlrel/chlrel.home.html
sequence
in
with the translation start
Institute, California [52] and the
(version 1.0)
GenBank
of 5'RACE and cloned the
help
study,
product
reinhardtii, which favors codons containing
Genome
(CM051d09,
AP515631).
no.
in this
1)
cDNA
330 nucleotides
bp upstream
reinhardtii genes
of the clone
displaying high
obtained from the Kazusa Institute revealed that the cDNA
longest
consisted of 1542 base
EST sequence
an
the 3'end. We identified the 5'end with the
in mammals.
nucleotides
(TGTAA)
Nramp family. Sequencing
cDNA via RT-PCR
full-length
Nramp2)
of the
reinhardtii
Nramp homologue ofC
in
at
the Doe Joint
http://genome.jgi-
search in this database
corresponds
Comparison
to
of the
genomic
genomic
showed that the DMTl gene consists of 13
The
Nramp homologue ofC
reinhardtii
chromosomal DNA
548
650
1466
1553
2138
\
Ï
64
385
914
962
1743
2242
2837
/
1798
2913
3510
3629
4203
4295
4859
mz±2485
2600
3126
3281
3864
4008
4405
4529
mRNA
64 Translation start
1
{_/
5' UTR
coding
3' DTR
sequence
Organisation of the DMTl gene. Total length of the genomic DMTl DNA region is 4859 base pairs,
poly-A tail (cDNA) 1936 nucleotides and of the coding region 1542. Exons and the
are
coding region
represented by black, introns by white and the 3 and 5 untranslated regions by grey boxes.
Number 1 indicates the first nucleotide of the sequence which was obtained by 5 'RACE and the other numbers
mark the position of start and end of exons (genomic DNA) or of the coding sequence (mRNA). Boxes are
drawn to scale in order to give an impression of the organization of exons and introns in the genomic sequence.
Figure
2.1
of mRNA without the
'
The introns
are
rather small with the
largest
one
However, total length of intronic sequence is
only
1542
bp.
described to
introns
total
equal
splice
comparative
reaching
2923
bp,
504
bp
while the
and the smallest 110
coding
bp.
sequence reaches
This is rather unusual for C reinhardtii genes where total intronic sequence is
(Tab. 2.2)
reinhardtii
'
coding
sequence
showed that
sites
sequence
[108].
[108]. Analysis of the boundaries between
they correspond
well to the
At least three further
analysis [111]
in the
recently
42
consensus
Nramp homologues
and
exons
sequence for C
were
identified
released genome of C reinhardtii
[52].
by
The
exon
int r
g
IHM
SJCOIÏ
-3
ensesstis
W/
JTm
splica
1 §11
3 splice site
sitä
positions
eson
3
__Ä
5
reinhardtii
Nramp homologue ofC
xntron
posi.fci.eas
1 il vîTOil
-2
-1
/
+1
+2
+3
+4
+5
A/C
S
/
G
T
G
&/C
S
positions
-3
-4
G/A
-2
-1
exon
/
CAS/
positions
+1
G/A
UltEOB 1
A
t
G
G
m
a
g
G
G
C
A
G
2
C
A
G
G
T
a
ft
t
A
C
A
G
G
t
3
C
il
13
G
T
G
2>
G
t
t
A
G
G
4
9
Ä
G
G
T
8
g
\3
A
C
A
G
c
5
C
Ä
G
G
A
X3
«
&
G*
C-
A
t?
G
S
L
C
G
G
T
G
G
G
c
G
A
G
G
1
C
Ä
G
G
T
d
c|
G
\2
L
Ä
o
G
8
C
Ä
G
G
T
G
C
&
G
C
A
G
G
3
t
C
a
G
1
3
J-l
G
G
C
A
G
L
10
C
Ä
G
G
T
G
C
G
A
C
A
G
A
11
C
Ä
G
G
T
G
cj
G
G
C
A
G
C
12
G
f
G
G
T
ts
A
G
A
a
A
G
G
Splice sites in the genomic sequence of DMTl Shown are the sequences at the splice sites of each
(1-12) of DMTl On the left part of the table the 5 splice sites are listed and on the right the 3 splice sites
The positions of the nucleotides relative to the splice site are indicated by numbers on the top Underneath, the
consensus sequence for C reinhardtii splice sites (according to [108]) is typed in bold Nucleotides identical to
the consensus sequence appear in capital letters, variant nucleotides in small
Table 2.2
intron
2.3.2
Deduced amino acid sequence
analysis
Translation of the DMTl cDNA
which contains
comprise
key
structural features of the
11 transmembrane domains
terminus in the
cytoplasm
a
513 amino acids
Nramp family (Fig
2
2)
and the C-terminus
extracellularly Comparison
the
loops linking
amino acids
positive charged
cytoplasmic
side than at the extracellular side of the membrane
arginine)
[76]),
sequence
predicted
to
with the N-
of the amino acid
transmembrane domains
showed that
(lysine
and
putative
protein
DMTl is
the programme TMHMM 2 0,
(by
of the intra- and extracellular
content
rise to
gives
are
found
more
The CTM
often at the
(consensus
transport motif) sequence motif described for other Nramp homologues [16] is also present in
DMTl
(Fig
2
predicted only
Brunak,
S
2)
Putative
with low
manuscript
N-glycosylation
probability by
in
sites
as
described for other
the programme
preparation, http
NetNGlyc
//www cbs dtu
43
1 0
Nramps [57, 132]
(Gupta, R, Jung,
dk/services/NetNGlyc)
E
were
,
and
The
Nramp homologue ofC
reinhardtii
1
MVLGRLFARSESAAPALADYNNEG^GSCTIDTSVVAVEVEASGSPSSATVR
51
GDSTLNEPLLEGFKDRGELPSLPESJMASJLSFPEESAPWWRKFFAFMGLGF
101
LIS¥GYMDPGNWATDLAAGSSFGYILLF¥¥LCSSLCAMFLQYLSLKLGIA
^
>-
.
2
1
151
SDRDLAQACRDAYHPR¥NKLLW¥¥AEAAXAATDLAE¥¥GCAIAFNLLLGI
>-
•
-<
•
3
201
PLWAG¥LITAAD¥IILL¥AEARSFRFLEXL¥GALTALISACFIYEL¥KAQ
»>
•
4
5
251
PDMGK¥MRGYtPSKEÏITNKDMLYLATGÏLGAI¥MPHNLYLHS5¥XQTRA
301
YPRTTAGRT1AXKLGL¥DSILSLL¥AF¥INSSILI¥AAAAFHYAIPPLED
1
3 51
IADITDAYELLASSLGSKAASILFG¥ALLAAGQteÄTGTIjSSQlWIEGF
4 01
LSFKMRPWLRRIITRGAAI¥PAAT¥AA¥MGREG¥SÜLLVLSQ¥ILSLTLP
8
^-
-*
9
10
451
FA¥FPL¥HFTSSRKYLGNFANRWYAS¥IAWLLFLF1SALN¥NL¥¥QSA1S
501
GSFGGLGHRRAÄY*
11
Figure 2.2 Deduced amino acid sequence of DMTl. Putative transmembrane domains (predicted by
TMHMM2.0) are indicated by black arrows under the corresponding sequences and numbered 1 through 11. The
arrowheads point to the outside of the cell. Predicted extracytoplasmic loops are shaded in grey, predicted
intracellular loops are not shaded. The consensus transport motif (CTM) appears in bold types and putative Nglycosylation sites are boxed.
Interestingly, comparison
reinhardtii
similarity
the
to
the
prokaryotic
core
an
(as
of DMTl to the
(58.06% identities)
2.4 for
Nramp homologues from various species
Nramp showed that the predicted protein
hydrophobic
similarity
of
than to the
defined in
sequence
of DMTl
[16])
of the
proteins
in
figure
Nramp homologue of the cyanobacterium
and lowest
similarity
Pairwise
eukaryotic Nramps (Fig. 2.3).
to
Oryza
alignment).
44
sativa
2.3
to
the C
shares
more
alignment
of
reveals the best
Nostoc sp. PCC 7120
Nramp2 (32.51% identities,
see
Fig.
The
Ec MntH
Nramp homologue ofC
reinhardtii
96%
54%
Se MntH
H
Dr
Nramph
a
m
Cr DMTl
O
Nsp MntH
O
58%
56%
38%
Xf Nramph
69%
50%
Pa MntH2
Sa
Nramph
Os
Nramp 1
At
Nrampl
43%
68%
4l°A
Se SMFl
55%
36%
Sc SMF2
S
o
a
s
58%
Sc SMF3
At
Nramp2
At
Nramp4
Os
Nramp2
At
NrampS
Hs
Nramp 1
Hs
Nramp2
m
80%
77%
74%
55%
73%
Figure 2.3 Homology tree of selected members of the Nramp family. The homology tree for the hydrophobic
core (as defined by [16]) of 19 Nramp homologues was constructed by the program DNAMAN. The first two
letters correspond to the initials of the organism (e.g. Ec for E. coli) with the designation of the protein following.
Nramph stands for Nramp homologue. Prokaryotic and eukaryotic groups of Nramp homologues are boxed.
Accession numbers for the used protein sequences in order of appearance in the tree can be found in the
materials and methods section.
45
The
Nramp homologue ofC
reinhardtii
NöStöC
DMTl
Oryza
consensus
NöStOC
DMTl
Oryza
consensus
NostOC
DMTl
Oryza
wx-ji £
o
L£
o
A.
O
LJ.
O
lui
consensus
241
o
\^3 i 1
4^
A
it ^
is? it it
A
Nostoc
DMTl
jKIDINKKSjjp
Oryza
wx-ji £
O
fe* 11
")t
it ^
")t 5fr" it 'Sfc"
5fr"
it 'Sfc" ")t
d
NostOC
DMTl
Oryza
Nostoc
Jg—1
DMTl
DTiDPTlDIpIEAL
Oryza
w-x-ji £
O
\^3 11
O
LJ.
-^ kJ A
O
Nostoc
DMTl
Oryza
Figure
hydrophobic core of Nostoc sp. PCC 7120 MntH, Oryza sativa Nramp2 and C.
hydrophobic core was defined after [16]. Identical amino acid residues are marked by a
similar residues by a dot in the consensus sequence.
2.4
Alignment
of the
reinhardtii DMTl. The
star and
Another
compared
interesting
feature of DMTl involves the N-terminus of the
the N-termini of selected
that the N-terminus faces the inside of the cell
domain with
example play
amino
acids
occurrence
a
a
analyzed homologues (Fig. 2.5).
as
predicted,
it
might
function distinct from the actual transport function of the
role in the
with
regulation
ScanProsite
We
Nramp homologues and found that the length of the C
reinhardtii DMTl N-terminus exceeds that of the other
Presuming
protein.
of
(e.g. phosphorylation sites)
protein
protein stability. However, analysis
[48] produced only patterns with
but
no
a
an
extra
and for
of the first 80
high probability
apparent protein motif known
46
form
to
play
a
of
role in
The
regulatory posttranslational
Zrtlp [49], data
In
a
not
further
modification
(e.g. ubiquitination,
investigation
of the DMTl N-terminus
in respect of the N-terminal
hydrophobic
core.
overall
variability
eukaryotic
or
which
reinhardtii
shown for yeast
was
shown).
homologues
For this
amino acids of the
Nramp homologue ofC
core
alignment
we
up to the first
sequence,
we
compared
which is not
used the N-terminal sequence
completely
of the sequence there
are
prokaryotic homologues (Fig. 2.5).
47
conserved residue
as
selected
conserved
overlapping
Nramp
as
the
the first 9
(glycine). Despite
distinct conserved residues shared
the
only by
I-
S"
Kilt K
£P
Mn t H
UL
i
J-ICipJX
JL
i>l
tlsp
Kr.tH
ü
s.
*
EttxO
1\
^u
.***
MUCDKTEPTKCSWPfcAQNAj
ll%ITipt"t
J11L
^
lj
ii
—
o
O
Li
Nramph
xa
Nr-juph
J?C*]fJ.
t
^Z.
i*»
I
s,
-J.
Ois.1
1
—
—
—
—
—
—.
—
—
—
—
—
—
_—
—
s,
„
-
_—
.
_
—
—
—
_
_
_
_—
—.
—.
—.
—
—
—
—.
—.
là
J.
J
_—
—.
—.
—
—
—
—
p 1 hf
uf
I
-~J. J
Cr
dmtI
if ci
^
|^ xSJI
i*.
1
^
I
î?l P
<Z*
P
*
i\
m
L3 k.
m
L| ff
1U 1U f^ C.
X X 4L 4 L f
J
Q
î
1U llllllll h 11«
PT H r¥ :"
i I
|J £
Mil MRM IC NC i N 3 I3PÏKA JKKDK P¥ i < ; WPP
MG|DI
„
pli
KV IiGPPI1 Ah SI",S
PWM AI
ïHPPGIJGSTI 1
/VA"Kv'KAri
1
hî'l'ÇDhf.HC'RI.SC
l*i
Nrampl
Kr.
Nramp2
$«i
*«
Ti
Uramph
M.V—U t't LK
ä
"^
?
:
rf rf ""* Ci :"
I
s
?
x
Ci ja
Pï 1^1
^
i
.
~—
——
~^|
^W
AS Fi S P
ïj
~~"—
^ Hil
f DUC AJ GiiJH(i C d AS_G. Al N HA 1
-
_
^
SFSSA"rVlAC;i)SÏLlJl«,Pl
——
——
^
-
J
^*5C s FiAJ*fi\ ^ I s R ht s I iAÇ
'
$1 Jr^
^
^i""^
X^ X^^
j^
j
J[
^
S1K
^
ä
W
w
TY
SEK
PÄPO-t^ Pt~ K-P T Y
SEK
J? Pi PQQÄ
fj
^w D f
S $ J. f -H $ l'C
SN
KRRMKDStlC*/MEL3lrL.HNC3''/lOÏ 1x331
H Si
^ fc^xL* x^^
I«,i:rt<PJs;
I
/CClS--Sft'~S PO POQA i1 PCG T Ï
PS
MVLCjLEQKM^iDDSV^ûDHGE^iACLO
Util
^
j^îj yi^x j SC tV3 C-A.1R1HV* T h IxIG WF F R FtG JblA.SMS
,-_,-_.-_..
Mj:
—'i^ji ^\ j£*^£ ^^
*w
*
Ut
—
_jif ^,-xï : a HÄIR AIV :ï 11111H R fïK £ï S ^>f :*, :*, y1
TR
.0
_—
KLiL>KÜHOKKR__YhTUC-J<
Nr%mph
Afu
M
—
isiraaipli
*:f
s.
—
Nrampl".
Satj
X"
—.
MIJIi'jMlPh A">ISPI h «'AHl
e
z
—.
nnm
-
1
P P KK
G iT iT
US EM
# Ls
Pt
1» lTii
NEK
^
LEK
TT V
Si'-SKKQSNISl'ï
SIiNlAGQÏl aSh*1
'
-
H"
PHH hP\« HKA'jTY
I-Ei,
Kvl
s
£*
w
Art
Nrampl
Ai 11
jjl IL
At h
Nmrnp?
At h
tfïrimpb
Ji 1 S3 Sr.f.flb St SUS t-1.1 KOSDSNOi
l§SEKfc
JE
LU
Oi,
w7
L^
1\ X
^
J II L
Sltlfl
^
^
f"^
KKNDv'hhllU.hh*, i)P 1
1
^Ffi 1°"°
lj
ii
-
AY PETE.
PPPPrS'jSLPSÏPSESPA/1
Pt,
,r HDPPPEÄD
j'NE
ri
jflJlJLJl<GU3PKfc"5
i« psppi'pr l'i-Pi'i
ppPS
KTG STV .-RQENS W\R FMDSUGfc t'FPLL" P'.
Mi imp 1
Se
^
M SE'i'D P E P. PGGSSEMP.
^iîTip *i
MG\"1" KAfcl AV Mit K\'\' D D1E AgÄD
y_,:, s Q S Y YI'IMD L DDIjR S L li .= 3Ï
^j
^
I
i.
\X ^-L^'
J&
^
I. JL X^XLä X^.
^
I Èf È*li
L^|Â
I
lj
.&.
r\
X\ tH* X
i^j
^j^j^EXI^^JJ i\^
^
L
WESti/-
-MV tJV G PS HAAV AVDA3 LAKKRN i S L LV i "E1.P.D KKC 3 T.' ¥ I EG -EJU^V RT FT S S S StJH E kJEDT YB
hit
.i,
Qé
ï«,ï
L E
xj
„ï x JNIJNIL
^„
/ Uxl
n
x i—^iJ
¥
«
I
i,
—
11s* xjI\JKJK J.
~~
~~
~~
~~
L
~~
--------
——
—
—
—
—
—
^
—
—
—
&\
^
«1^.4^\^X
XL
3 KHO^/Î^RE JL Ig
£X^
—
N-termlnys
^ u^-
————MtL "i%ÎQi
wor©
The
Nramp homologue ofC.
reinhardtii
Figure 2.5 Alignment of the N-terminal sequence of selected Nramp homologues and of C. reinhardtii DMTl.
The alignment was performed with the N-terminal sequence of selected Nramp homologues including the first 9
amino acids of the core region, which display high similarity among the organisms (rightmost in the alignment).
The alignment is split in two blocks. The upper block is composed of prokaryotic Nramps and CrDMTl (bold),
the lower block of eukaryotic Nramps. White letters shaded in black or dark grey represent residues conserved in
both blocks. Letters shaded in green represent residues conserved only in the prokaryotic block and CrDMTl,
and letters shaded in yellow represent residues conserved only in the eukaryotic block. The cross, the circle and
the star indicate residues which are conserved in both blocks, but are somewhat shifted (because of additional
amino acid stretches in the eukaryotic block). The first two letters correspond to the initials of the organism (e.g.
Se for Salmonella enterica) with the designation of the protein following. Nramph stands for Nramp homologue.
Accession numbers for the used sequences (in order of appearance in the alignment) can be found in the
materials and methods section.
Some
prokaryotic Nramps (e.g. Salmonella,
include these conserved residues
also
C
eukaryotic homologues
them to
a
lesser extend.
the two
major
they
shorter. On the other
are
Clearly,
the C reinhardtii DMTl takes its
This is in agreement with the
the sequences of the
among the
core are
analyzed Nramps
Heterologous expression
2.3.3
For functional
S.
cerevisiae
homologous
was
was
by
or
membrane
again,
of the C reinhardtii DMTl
was
blot
in
untransformed
possess
the pro- and
among the
prokaryotic
features
prokaryotic
DMTl takes
protein
pARl,
proteins [10, 75] heating
As
(but
pUG35 resulting
(Tab. 2.1). Expression
analysis (Fig. 2.6).
cells.
an
a
on
the N-
cluster when
unique position
characteristics.
approach utilizing yeast
chosen, since targeted nuclear
cloned into the yeast vector
detected in cells transformed with
pUG35
of DMTl in the
Once
or
of C. reinhardtii DMTl in S. cerevisiae
mutants
western
place
to
eukaryotic homologue displaying prokaryotic
transformed into various yeast mutants
confirmed
correspond
eukaryotic
no
recombination in C reinhardtii is not established
cDNA of DMTl
was
analysis
transport
placement
compared (Fig. 2.3).
as a
are
matching pattern. Seemingly,
a
the N-terminal sequence
Nramp homologues since it only displays prokaryotic and
terminus.
hand, there
conserved amino acids
typical eukaryotic
regarding
not
ranks, namely the plants, yeasts, Drosophila and
Nevertheless, the vertebrates display
groups formed
domains.
coli, Yersinia, Chlorobium, Ralstonia) do
because
which break the
which do not possess the
elegans,
eukaryotic
mainly
E.
A
specific
see
in
gene
disruption by
[94]).
The
complete
plasmid pARl,
which
of the DMTl gene in yeast
immunoreactive band
was
but not in cells transformed with the control vector
was
already
observed
of the cell extract in SDS
49
with
sample
highly hydrophobic
buffer at 95°C before
The
loading
on
the
lead to
gel
separation gel (Fig. 2.6,
heating,
the
the DMTl
predicted
protein
57 kDa
studied mutants
lane
(data
was
(Fig. 2.6,
not
of the DMTl
aggregation
4).
When the cell extract
identified
lane
Nramp homologue ofC.
as a
which
protein
was
loaded
on
the
rather diffuse band in the
1). Expression
of the DMTl gene
barely
gel
reinhardtii
entered the
without
previous
gel corresponding
to
confirmed in all
was
shown).
Heterologous expression of DMTl in S. cerevisiae strain Y16272 (smfl). Yeast strain Y16272 was
pARl or with the control vector pUG35 and whole cell extracts were analyzed for DMTl
expression by western blotting. The molecular weight in kDa is indicated on the right and arrows point to the
DMTl protein band. Samples 1-3 were loaded on an SDS PAGE gel without previous boiling, samples 4-6 were
boiled. Lanes 1 and 4: mutant smfl with DMTl, lanes 2 and 5: mutant smfl with control plasmid, lanes 3 and 6:
mutant smfl.
Figure
2.6
transformed with
2.3.4
DMTl restores
growth
of
S. cerevisiae
a
smfl
mutant under manganese
limiting
conditions
The
belonging
Smflp protein
to
the
of S. cerevisiae has been identified
Nramp family [121] and shows
Yeast strains with
SMFl gene fail to grow
disrupted
divalent cation chelator EGTA. As
complement
with
a
was
yeast overexpression
minimal medium
vector
containing
{smfl)
tested. For
plasmid pARl containing
to
C reinhardtii DMTl.
synthetic
with respect to
this, yeast strain
C reinhardtii
pUG35
similarity
on
manganese transporter
medium
containing
first step to elucidate the function of DMTl, its
the yeast strain Y16272
concentrations of EGTA
38%
a
as
as
a
growth
Y16272
restoration in
{smfl)
Nramp homologue DMTl,
control. Growth tests
0-10 mM EGTA and the maximal
50
were
growth
was
or
ability
to
inhibiting
transformed
with the empty
performed
rate
the
in
liquid
of each strain in
The
medium with
strains
were
no
EGTA
growth
smfl/pARl
defined
as
100%
growth
expressing
of strain
DMTl
smflVpUG3 5
remained at 100%).
was
Higher
(Fig. 2.7).
for manganese
ions,
At
an
EGTA concentration of 5
EGTA concentrations
in contrast to strain
higher sensitivity
reduced to 78%, whereas relative
both strains. These results indicate that the strain
reinhardtii
of the strain. Growth rates of the two
different and the strain with the control vector showed
EGTA than the strain
relative
was
Nramp homologue ofC.
smfl/pARl
growth
to
mM,
of strain
eventually impaired growth
of
is able to compete with EGTA
smflVpUG35, indicating
that DMTl acted
as a
metal
transporter. At EGTA concentrations below 5 mM, growth of strain smfl/pARl exceeded
100%). This
might suggest
that the
overexpressed
protein imported
DMTl
too many
ions into the cell in medium without the metal chelator and thus lead to
a
metal
slight growth
decline.
140
3**
120
100
O
j:
x
80
m
60
20
0
u
S
10
EGTA
[mM]
smfl/pARl was tested for altered EGTA tolerance. The yeast manganese transport
(smfl) was transformed with the DMTl containing plasmid pARl (filled squares) or with
the control expression vector pUG35 (open squares) and growth of the two strains was compared in medium
containing increasing concentrations of the metal chelator EGTA. The maximal growth rate of each strain in the
medium without EGTA was defined as 100% and growth of the same cells in different concentrations of EGTA
was expressed in relation to it. The graph is derived from two independent experiments.
Figure
2.7 Yeast strain
1o
mutant strain Y16272
51
The
of DMTl in S. cerevisiae mutant
Overexpression
2.3.5
cadmium, and copper, but
manganese,
The fact that metals
levels in the cell
particular
was
smfl
increases
utilised to
metal ion is mediated
study
growth
under
increasing
the
by DMTl,
specificity
the strain
which
were
grown in
of the DMTl
protein.
the DMTl
expressing
smfl/pARl)
(Fig.
was
growth
growth
of the strain and
expressed
containing 0, 10,
rate
of the
growth
in relation to it. Relative
reduced to 89%. and 83%>,
entered
the
leading
In order to
to
Mn2+,
to
uM
(Cd2+),
DMTl but
only
or
mutant
a
decreased for
clear
of 1 mM CuS04
growth
of strain
For
previous
this, cells
MnCl2. Addition of
expressing
DMTl
was
more
growth
no
MnCl2
was
defined
as
cells in different concentrations of MnCl2
of strain
at 1000 uM
smfl/p\JG35
These results indicate that
DMTl
was
still 100%> at
a
MnCl2. Growth in strain smfl/pARl
transporter
where
Mn2+
they
and zinc
(Zn2+)
smfl expressing DMTl
to
an
average
transported by
ions must have
reached
toxic
on
growth
only
a
growth
the
smfl7pUG35
potential growth
Addition of
plasmid pARl.
reduction of 18%> in the strain
(Fig. 2.8B).
reduction of 52%. whereas relative
At 10 uM
growth
of
9%>. These results led to the conclusion that DMTl is able to
difference between the two strains
(Fig. 2.8C).
DMTl in addition
tested for
were
transport cadmium ions into the yeast cells. Addition of CuSÛ4
effect and
1000 uM
2% in the strain with the control vector
CdCl2 strain smfl/pARl showed
smflVpUG35
the
(Cu2+)
copper
CdCl2 into the medium led
expressing
100
the spectrum of metal cations
inhibition of the yeast mutant strain
1
following experiments
growth impairment.
investigate
cadmium
via
same
growth
respectively.
cells
smfl/pARl
concentrations
a
should be
protein
(Mn2+) uptake.
of each strain in medium with
MnCl2 concentration of 100 uM and 99%.
was
of
concentrations of MnCl2 than the control strain with the empty vector
2.8 A). The maximal
100%o
uptake
tested for differences in
were
role of DMTl in manganese
to a
minimal medium
increasing
If
manganese, in order to confirm the result of the
was
MnCl2 showed the expected effect. That is, the yeast
sensitive to
physiological
metal concentrations.
pointed
liquid
and
{smfl/p\JG35
The first element tested
experiment,
to
not to zinc
less tolerant to that metal than the strain with the control vector. In the
the two strains used above
sensitivity
become toxic when their concentration exceeds
can
reinhardtii
Nramp homologue ofC.
Strain
was
smfl/pARl
reduced
by
showed
a
to
was
the medium had
observed at
growth
similar
concentration
reduction of 73%, while
48%>. The fourth metal tested
52
a
a
was
zinc. In this
The
case
there
was no
Nramp homologue ofC.
difference in the response of the two strains after addition of up to 10 mM
ZnCl2 into the growth medium. Both strains showed growth inhibition in the
concentrations
reinhardtii
exceeding
DMTl did not have any
1 mM
same
degree
at
ZnCl2 (Fig. 2.8D), showing that the yeast strain expressing
growth disadvantage
the strain with the empty vector in toxic
over
zinc concentrations.
B
120
120
100->-
100
80
£
"S
80
*c
I
60
m
=
60
Ol
40
40
~
20
0
0
0
10
100
1000
10
1
MnCi2 [pM]
100
CdCI2 [MM]
n
120
80
2
01
40
20
0
0
2
4
6
10
ZnCt2 [mM]
sensitivity to divalent metal ions in yeast strain smfl/pARl. The yeast manganese transport
(smfl) was transformed with the DMTl containing plasmid pARl (filled squares) or with
the control expression vector pUG35 (open squares) and growth of the two strains was compared in minimal
medium containing increasing concentrations of a metal solution. The maximal growth rate of each strain in
medium without additional metal was defined as 100% and growth of the same cells in different concentrations
of the metal solution was expressed in relation to it. A: MnCl2, B: CdCl2, C: CuS04, D: ZnCl2. The graphs for
MnCl2 and CdCl2 are derived from three independent experiments and for CuS04 and ZnCl2 a representative
experiment is shown.
Figure
2.8 Altered
mutant strain Y16272
53
The
DMTl does not
2.3.6
EDTA
complement
Nramp homologue ofC.
sensitivity
reinhardtii
of S. cerevisiae zrtl/zrt2 double
mutant
ZHY3 is
a
yeast
mutant in
ZRT1 and ZRT2 have been
which the
and
disrupted
medium with the metal chelator EDTA
containing plasmid pARl
expression
liquid
would alter the
minimal medium
each strain under zinc
growth
was
it
can
[139]. The
growth
of the
(EDTA
same
1
ZnCl2, which showed
no
performed
ZnCl2. Maximal growth
mM, ZnCl2
1
mM)
was
defined
degree
and
no
clear difference
was
overexpressed protein
was
not
sensitivity
to
able to
smfl
zinc when
in
of
100%>
was
growth
of
observed between
the C reinhardtii DMTl did not show any
enhanced
as
rate
cells in lower concentrations of ZnCl2
the cell. This is in agreement with the results obtained for the
with
were
At lower zinc concentrations in the medium
same
be assumed that the
in order to test whether DMTl
1 mM EDTA and 0-1 mM
(Fig. 2.9).
expressing
pUG35
in
transformed with the DMTl
mutant was
behaviour of the strain. Growth tests
conditions
reduced to the
them. Since the strain
growth,
replete
in relation to it
both strains
growth
zinc transporter genes
affinity
which, consequently, shows reduced growth
the control vector
containing
of the strain and
expressed
or
and low
high
import
advantage
in
zinc ions into
mutant grown in
medium
expressing DMTl.
120
100
/
»sJP
CT*
JE
%
80
o
OJ
»
t-
80
40
20
0
0
0.2
0.4
0.6
0.8
ZnCfe [mM] (EDTA 1mM)
limiting conditions. The yeast zinc transport mutant
containing plasmid pARl (filled squares) or with the control
expression vector pUG35 (open squares) and growth of the two strains was compared in minimal medium
containing 1 mM EDTA and increasing concentrations of ZnCl2. The maximal growth rate of each strain under
zinc replete conditions (EDTA 1 mM, ZnCl2 1 mM) was defined as 100% and growth of the same cells in
limiting concentrations of ZnCl2 was expressed in relation to it. Shown is a representative experiment.
Figure
2.9 Growth of yeast strain
strain ZHY3
[139]
was
ZHY3/pARl
under zinc
transformed with the DMTl
54
The
we
investigated
in which
high
improvement
the
same
liquid
reinhardtii
whether DMTl acts in iron transport. Yeast mutant DDY4
(fet3lfet4),
DMTl function in iron
2.3.7
Next,
Nramp homologue ofC.
and low
affinity
containing
excess
of the strain and
(EDTA
of the
in relation to it. At lower FeCb
(Fig. 2.11). However,
the strain
strain with the control vector
pUG35.
same
tested for
and
were
growth
compared
to
performed
in
FeCb. Maximal growth
mM, FeCl31.25 mM)
was
defined
as
DMTl had
reduction of
clearly
a
approximately
FeCb concentration of 0.5 mM). This result suggests
a
was
growth advantage
40%
as
opposed
rate
100%>
cells in lower concentrations of FeCb
concentrations, growth of all strains
expressing
(growth
1
was
Growth tests
1 mM EDTA and 0-1.25 mM
conditions
growth
impaired [32],
containing plasmid pARl,
strain transformed with the control vector
of each strain in iron
expressed
iron transport is
after transformation with the DMTl
minimal medium
growth
transport
was
impaired
over
the
to 70% at a
role of DMTl in iron transport.
120
100
,
/
80
'
,
O
60
OÏ
m
__
.
^
^z'
40
0
0
0.25
0,5
0,75
1
1.25
FeCIa [mM] {EDTA 1mM)
limiting conditions. The yeast iron transport mutant
[32]
containing plasmid pARl (filled squares) or with the control
expression vector pUG35 (open squares) and growth of the two strains was compared in minimal medium
containing 1 mM EDTA and increasing concentrations of FeCl3. The maximal growth rate of each strain under
iron excess conditions (EDTA 1 mM, FeCl3 1.25 mM) was defined as 100% and growth of the same cells in
limiting concentrations of FeCl3 was expressed in relation to it. Shown is a representative experiment.
Figure
2.11 Growth of yeast strain
strain DDY4
was
DDY4/pARl
under iron
transformed with the DMTl
55
The
Discussion
2.4
We have cloned
reinhardtii, named
characteristics
Nramp
reinhardtii, that is
13 base
was
released
recently
showed that the DMTl gene consists of 13
than double the
length
reinhardtii, where intronic
the
pairs upstream
of the
sequence
coding
equals
Joint Genome
and 12
the
coding
Analysis
and
a
of the
Institute, California, [52]),
introns, with the introns reaching
This is rather
sequence.
displays
60%>
over
site.
polyadenylation
(Doe
exons
of
content
C
microalga
The cloned cDNA
1.
GC
a
in the green
family
gene
for divalent metal transporter
of C
typical
sequence which
genomic
member of the
a
DMTl
polyadenylation signal
more
reinhardtii
Nramp homologue ofC.
uncommon
for C
length [108].
sequence
The deduced amino acid sequence reached 513 amino acids and contained structural features
for the
common
Nramp family. Firstly, the
transmembrane domains
(TMs).
In
terminus
For other
[28, 29].
to
agreement
that the E. coli MntH forms 11 TMs, with
protein
DMTl
a
that it
was
a
predicted
shown
to
comprise
11
by topological analysis
N-terminus and
cytoplasmic
Nramp homologues
was
number of 10-12 TMs
a
periplasmic
C-
predicted [18,
was
57, 105]. Secondly, the highly conserved "consensus transport motif (CTM) found in every
in DMTl within the
Nramp homologue is also present
This motif
was
similar to the
prokaryotic
suggested
be involved in the transport mechanism of
transporters)
hydrophobic
ATPase subunit
[12, 85, 93].
coupled
[131] for the
to
and to the pore
For the
by
which is
thought
predicted
in
in
a
to
indicate
might
similarity
be
cytoplasmic loop,
TM8 and
TM9).
Nramps, since it is
of
an
unlikely,
important
since
consensus
an
for ion
seems
the pore
selectivity [60].
unlikely
region
is
ATPase subunit in
of cations
was
sequence described in
function in the
to
EAA
interacts with the
uptake
in bacterial ABC transporters.
Nramps is restricted
it
likely
interaction with
an
seems
[16]. The
region
Nramp protein but
Regarding voltage
sequence
Since the CTM of
[16, 136],
Nramps is
that the motif should have
a
similar
K+ channels.
as
Putative
N-glycosylation sites,
(by
to
region
responsible
function
DMTl
hydrolysis
ATP
distinct to the function of the EAA
the
Nramp proteins
H+ import [57]. Nevertheless, the
CTM motif
gated K+-channels,
of K+ channels
region
subunits of the ABC transporters and most
order to facilitate transport
shown to be
region (between
prokaryotic binding-protein-dependent transport signature (EAA region
ABC
found in the
to
same
the programme
preparation), yet
a
third feature shared
by Nramp proteins,
NetNGlyc 1.0; Gupta, R., Jung, E.,
with rather low
probability,
at
56
the N-terminal
and
were
for
manuscript
in
before the first TM
(2
Brunak,
region
predicted
S.
The
and between TM8 and TM9
sites)
intracellularly
in contrast to other
Nrampl is extensively glycosylated, with
protein targeting
and
the
Strikingly,
than to
stability
eukaryotic
algal
as
in the membrane
members of the
for
regulation
clustered
[19].
in
exclusively
group of
group and
one
prokaryotic homologues.
prokaryotic
of such events the
spite
In
The DMTl
eukaryotic homologues,
that it is
explanation [91].
possesses
only
the
phylogenetic
-
of the
protein, although
seems
possible
no
that it
familiar
functional
terminus of
some
Nrampl
Nramps (e.
or
investigate
plays
higher homology
be
an
exception
as
to
prokaryotic
to
globins
Nramps
proposed by
was
in that
study
each other than to
it clusters within the
shown for the
was
in
globin protein
from ciliates and Nostoc than to
were
a common
considered
-
a
as
out
an
that it
eukaryotic homologues
"prokaryotic" protein
in
prove useful for further studies
-
DMTl and tested for
was
a
on
coli)
in
a
is
motif
regulation
or
be present. Tabuchi et al.
[123]
showed that the native N-terminus is essential
an
extremely
protein.
a
interesting result, considering
was
57
was
A deletion mutant for SMFl
as
a
-
utilized
the yeast
expression plasmid containing
conditions. Growth
partially
role for DMTl
that the N-
short.
moderate level
depleted
with the divalent metal chelator EGTA
a
to
seems
role in the function
manganese transport mutant of S. cerevisiae
under metal
DMTl, suggesting
important
considered
homologue
Nrampl and Nramp2 by exchanging the N-termini of the
transformed with
growth
than that of any other
some
protein
the function of the DMTl
Nramp homologue
reinhardtii
prokaryotes
in contrast to other
Nramp2. This is
g. E.
Heterologous expression
to
longer
proteins. Complementation experiments
fully
and
of C reinhardtii DMTl
studied the function of H. sapiens
a
higher similarity
evolution of Nramps.
study,
for
eu-
Nramp homologues might
among
in this
two
for proper
of function.
Chlamydomonas
prokaryotic Nramps,
Since the N-terminus of DMTl is
it
and
Tetrahymena
unique position
eukaryotic organism
important
addition, regarding only the N-terminus of DMTl, it stands
features of
This
(Fig. 2.5).
In
to
similar to
more
be
and gene transfer events between the Nostoc ancestor and
Paramecium,
to
seems
Nramps (Fig. 2.3). Likewise, it
Chlamydomonas eugametos
ancestor
protein
can
mouse
sites between TM6
investigated eukaryotic Nramps
all showed
they
localized
A scenario for the evolution of
Nramp family.
which includes horizontal gene transfer events between
Cellier et al.
are
[132] showed experimentally that
sequence showed
protein
DMTl
reinhardtii
[57] predicted N-glycosylation sites
N-glycosylation,
or
sites
putative
possible N-glycosylation
two
modification, such
al.
et
between TM7 and 8 for rat DCT1, and Vidal et al.
and 7. Posttranslational
All three
(1 site, Fig. 2.2).
Nramps. Gunshin
Nramp homologue ofC.
impairment
restored in cultures
in medium
expressing
C
divalent metal ion transporter. This
The
finding
affirmed
was
the fact that the strain
way
Addition of CdCl2 to the
with the
displaying greater sensitivity against
Mn2+ uptake by
showed that
inhibited
Nramp transporter
the
in
weakly by Cu2+,
sensitivity
an
of the
element which
smfl yeast
with the control vector.
same
mutant
transported by
strain
Apparently,
this
behaviour after addition of
growth
whether zinc
was
indeed not
expressing
was
not true
DMTl
the
the DMTl
{smfl/pARl,
for
also described to pass the
[127] and Kehres
et
similar
a
[72]
shown
by
was
the ABC
specificity
inhibited
was
only
the increased
algal Nramp, compared
were
al.
typhimurium
Mn2+ uptake by
as was
for ZnCl2
we
concentrations, that
In contrast, MntH
increasing ZnCl2
transported by
lower
was
inhibited also the
ions,
growth impairment
transporter which showed
a
above
as
MntH of E. coli and S.
typhimurium [71].
was
to
thaliana
Arabidopsis
Nramp homologue
the MntH of E. coli and S.
responded
DMTl
strongly by Cd2+. Interestingly, Cd2+
as
These results
plasmid.
yeast strains
the metal. Cadmium
transporter SitABCD in Salmonella enterica,
profile
same
medium resulted in
growth
strains, but the strain expressing
cell membrane via the
less tolerant to
was
of DMTl to transport other divalent metal
ability
performed growth impairment experiments
both yeast
DMTl
of DMTl to transport manganese ions into the yeast cell.
ability
In order to check the
smfl/p\JG35).
expressing
concentrations than the strain with the control
increasing MnCl2
demonstrate the
by
reinhardtii
Nramp homologue ofC.
to
the strain
both strains showed the
concentrations. In order to confirm
protein,
DMTl
was
expressed
in the
yeast strain ZHY3 {zrtllzrt2) which shows defects in zinc transport. The phenotype of ZHY3,
namely
DMTl
reduced
growth
in medium with the metal chelator
expression. Thus,
both
experiments
-
the zinc
the
complementation
not
able to transport zinc ions into the yeast cells.
test
obtained for its transport
not
inhibited
with the zrtllzrt2 mutant
zinc in manganese
mention inhibition of Smfl manganese
iron and manganese
Sacher et al.
bind at the
uptake
[113] could
same
site
on
not
via the
import by
sensitivity
the transporter
protein
complemented by
with the
Concerning zinc,
uptake [72],
zinc. In rat, it
65Zn2+
as
test
not
smfl
different results
whereas
was
DCT1
via DCT1
typhimurium
Supek
and
mutant
lead to the conclusion that DMTl
Nramp homologue
show transport of
was
MntH of E. coli and S.
by Nramp homologues.
considerably by
-
EDTA,
et
al.
was
were
were
[121]
shown that zinc inhibited
but
(DMTl, Nramp2) [57],
or
Smflp. Thus, Zn2+ might
the other metal ions but without
entering
the
cell.
For
a
further
characterization,
(fet3lfet4)
in which the
phenotype
of
by expression
high
and low
growth impairment
of DMTl,
DMTl
affinity
under iron
indicating
a
was
expressed
iron transport systems
limiting
conditions
role for the DMTl
58
in the yeast
was
protein
are
strain DDY4
impaired.
The
partially complemented
in iron transport. This is
The
consistent with the
in iron transport
similar to the S.
reinhardtii
are
several
same
finding
that
Nrampl, Nramp3 and Nramp4
[30, 127]. Recently, components of
cerevisiae
Frtlp/Fet3p
[61, 78]. This does
Nramp homologue ofC.
not
in the
uptake systems present
with
our
pathway
Accordingly,
be shut down and
DMTl
might play
a
a
were
described in C
cell, mediating high and low affinity transport of the
mostly important
element is essential for many vital processes. However, under iron
can
also involved
results, considering that generally there
substrate. An efficient transport system for iron is
affinity system
are
iron transport system
high affinity
a
iron assimilation
disagree
in A thaliana
reinhardtii
low
affinity system
role in low
affinity
can
excess
take
on
for the cell
as
this
conditions, the high
the
uptake
iron assimilation when the
of iron.
high affinity
system is down regulated.
In
transporter in
an
conclusion,
alga
-
we
have
the DMTl
identified, cloned and characterized
of C
prokaryotic Nramp homologues regarding
the function of the
showed that the
reinhardtii
sequence
-
which must be
similarity. Furthermore,
protein by complementation experiments
specificity
of the transporter
was
a
divalent metal
placed
we
among
elucidated
in several yeast mutants and
quite broad, including
manganese,
cadmium,
copper and iron but not zinc transport.
Acknowledgements
We would like to thank Dr. J. H.
Hegemann for the pUG35 vector,
Dr. D. Eide for yeast
strains and the Kazusa DNA Research Institute for the C reinhardtii EST clone. We
indebted to Dr. A. J. B. Zehnder for support,
helpful
project.
59
are
discussions and constant interest in the
Supplementary
Chapter
Supplementary
3.1
verification that DMTl
Further
limiting
by
chapter
2
3
material to
mediates
2
chapter
growth improvement
under
metal
conditions
The DMTl
(see
material to
coding
sequence
Materials and methods
was
chapter 2).
cloned in the
pUG35
vector
for moderate
The MET-25 promoter of the vector
expression
be
can
repressed
addition of ImM methionine into the culture medium. In order to further confirm that the
effects observed
on
complementation
the
growth
assay
minimal medium agar
was
plates
rate
of the
performed
smfl yeast
mutant
where the promoter
with EGTA and/or methionine
yeast strains smfllp\JG35 (control) and smfl/pARl
negative
strain
effect
(Fig.
on
the
growth
3.1 A). The DMTl
when methionine
was
of the
added. In
showed that the DMTl
strains, whereas
expressing
protein
addition,
is not
arise from DMTl
switched off. For
was
were
(DMTl).
spotted
EGTA reduced
western
expressed
blot
(see
with dilutions of
growth
on
EGTA but not
chapter 2)
in the presence of ImM methionine
was
no
of the control
Materials and methods
Thus, the complementation observed for strain smflIpABA (chapter 2)
a
this,
Methionine itself had
strain grew better than the control
a
expression,
(IB).
due to DMTl
expression.
B
OmM EGTA
OmM EGTA, 1 mM Met
smf1/pUG35
smf1/pAR1
T
to
CO
ç—
(Z
O
a:
<
=>
<
Q.
Q.
+
Q.
smf1/pUG35
.Jjllllllllgjl
DMT1
smf1/pAR1
Figure 3.1
A) Complementation experiment (spot assay). smfl/pUG35: yeast mutant smfl transformed with the empty
pUG35 vector; smfl/pARl: yeast mutant smfl transformed with the DMTl containing plasmid pARl; Met:
methionine.
increasing dilutions were spotted from left to right.
B)
protein extracts with anti-HA antibody. pARl : yeast mutant smfl transformed with the
DMTl expression plasmid pARl; pUG35: yeast mutant smfl transformed with the empty pUG35 vector;
pARl+met: ImM methionine was added to the culture medium before protein extraction.
10|al
culture of
Western blot of yeast
60
Supplementary
Study
3.2
The
chapter
2
of DMTl mutants
During
mutations
material to
the
were
cloning procedure
of DMTl
(see
materials and methods
introduced into the amino acid sequence
plasmid harboring
acid substitutions
were
the correct sequence
named
was
pARlmut, pNl
and
resulting
in four different
designated pARl,
pN2HA (Fig.
chapter 2) point
the
plasmids
3.2 and Tab.
plasmids.
with amino
3.1).
1
M¥LGRLFARSESAAPALADYNNEGNGST1DT5V¥A¥E¥EASGSPSSAT¥R
51
GDSTLNEPLLEGFKDRGELPSLPESNASLSFPEESAPWWRKFFÄFfÄGF
101
LISVGYMDPGNWATDLAAGSSFGYTLLFVVLCSSLCaMFLQYLSLKLGIA
151
SDRDLAQACRDAYHPB¥MKLLW¥¥AEAAÏAATDLAE¥¥GCAÏAFNLLLGI
201
PLWAG¥L I TAAD¥I I LL¥AE Ai&RFLE I L¥GALTAL ISACFI
_*
Y^^KAQ
'Tel
251
PDMGK¥MRGYLtfStEllTNKDMLYLATGlLGAT¥MPHNLYLHSS¥lQTRA
301
YPRTTAGRTIAIKLGL¥DSTLSLL¥AF¥INSSILI¥AAAAJfHYATPPLED
351
IAD ETDAYELLAS5LGSKÄÄS JLFG¥ALLAAGQNSIITGILSGGjI¥MEGF
401
LSFKMRPWLRRIITRGAAI¥PAAT¥AAVMGREG¥SQLL¥LSQ¥ILSLTLP
451
FA¥FPL¥HFTSSRKY1G ^FANEWYAS¥IAWLLFLFISALN¥NL¥¥QSA1S
S
\Ry
501
GSFGGLGHRRAA¥*
pNl (G97S, D353G, W473R)
pN2HA (A137T, G467S)
pARlmut (S223G, L246Q, S263F, F341L)
3.2 Point mutations in DMTl.
Regular amino acids correspond to plasmid pNl, bold to pN2HA, and
pARlmut. Residues absolutely conserved throughout evolution are marked by a grey box with a black
line. Residues highly conserved are in a grey box without a black line and residues partially conserved are in a
white box with black line. Residues not at all conserved are circled. *This amino acid is conserved as a glycine
in all organisms except DMTl where it is replaced by a serine.
Figure
italics to
61
material to
Supplementary
levels of the mutated
Expression
Materials and methods
while
and
showed weaker
protein
detected
no
staining
of
In
yeast
a
was
gel
ran
in
mutant on
achieved in the
and
not
smfl yeast
mutant
the mutant
whereas the mutations in
S in
with other
pNl
protein
in
by
western
blotting (see
almost similar to the
were
stability
was
content was
was
wildtype,
affected in
severely
checked
able to
not
(Fig. 3.3B).
pN2HA
coomassie blue
by
comparison
phenotype
on
in
to
the
no
and
pNl allowed the expression of
was
This
be attributed to
can
pNl
was
functional
(Fig. 3.3B)
affected
functional
a
smfl
which
In contrast,
pN2HA
an
was
medium with 15mM EGTA
pARlmut
of
complementation
pARlmut plasmid
(Fig. 3.3C).
wildtype.
growth
rescue
Also
strain transformed with the
concentrations of EGTA
varying
checked
were
protein stability,
protein. Sequence
Nramp homologues (not shown) revealed that G97, which is substituted
pNl, is conserved
in all
Nramps (Tab. 3.1). Whether this is
a
functional conservation
has to be tested with further amino acid substitutions. It is notable that evolution insisted
using glycine
at
this
position although
substitution with serine in DMTl
function. However, this amino acid could also be involved in
which would not be detected in this
protein,
Smflp
and
Nramp2
in
chapter
regulation
or
did not affect
specificity
correspond
to
the
ones
1.4.2.
B
CD
in
co
,-
CD
ce
3
<
CL
CL
iHlltllk
uBaaîlÉÉ«*
—
DMT1
pUG35
3
E
IT)
C)
<
X
CD
ce
ce
C\l
_)
<
<
z
CL
Cl
Cl
Cl
5
-DMT1
on
of the
experimental setup.
The substituted amino acid residues described here do not
for
2
shown).
Thus, the introduced mutations
by
total
observation, pN2HA
protein stability
complemented
alignment
Protein
medium with 15mM EGTA
tested in medium with
the decreased
this
expression.
(Fig. 3.3A,
parallel,
to
agreement
Protein levels of
chapter 2).
pARlmut
proteins
chapter
pN2HA
EGTA
62
10
[mM]
described
Supplementary
material to
chapter
2
Figure 3.3
A) Western blot of smfl yeast strain protein extract. pUG35: empty vector; pARl: plasmid with correct DMTl;
pNl, pARlmut and pN2HA: plasmids with mutated DMTl
B) Spot assay. Cultures of smfl yeast mutant transformed with empty vector (pUG35), and plasmids with
mutated DMTl (pNl, pN2HA) were spotted on minimal medium agar plates with and without the metal chelator
EGTA.
C) Growth
smfl yeast mutant with empty vector (pUG35, squares), correct DMTl (pARl, circles) and
(pARlmut, triangles) in increasing concentrations of the metal chelator EGTA. Maximal growth
rate of each strain in minimal medium without EGTA was defined as 100% and growth of the same cells in
different concentrations of EGTA was expressed in relation to it.
curve
of
mutated DMTl
Table 3.1
A: Overview of the DMTl mutants
pNl
expressed
and functional
pN2HA
expressed
not
substitution
G97S
D353G
W473R
A137T
G467S
conservation*
absolute
partial
no
high
partial
refers to the
B:
the
degree
of conservation of this
particular
pARlmut
expression, not functional
S223G
L246Q S263F F341L
low
absolute
amino acid residue
as
G
throughout
partial
no
partial
evolution
Graphical representation of the DMTl protein. Black bars symbolize the predicted transmembrane regions of
protein. Mutated amino acids are indicated below the corresponding TM. CTM: consensus transport motif
63
Supplementary
Role of DMTl in Zn-mediated reduction of Mn
3.3
further
strain
investigate
in
0).
figure
2.8A. When the medium
the
inhibitory
was
effect of
a
10%
was
defined
addition of Zn had
a
protective
of the yeast
(Fig. 3.4,
as was
with 1000 uM ZnCl2
almost
effect
100%
as
growth reduction,
supplemented
Mn2+
was
already
(MnCl2
entirely suppressed. Already
ZnCl2 ratio of 1:1 (100 uM MnCl2, 100 uM ZnCl2) led
indicating that
growth
DMTl in medium with various manganese to zinc concentration ratios
Growth in medium without additional metal
1:10),
2
DMTl. In order to
transported by
interaction between DMTl and Zn I tested
Addition of 100 uM MnCl2 resulted in
ratio of
to
possible
smfl expressing
(Fig. 3.4).
Zn
a
chapter
toxicity
2.3.5 and 2.3.6 I showed that Zn is not
chapters
In
material to
to
a
slightly
better
Mn
0,
shown
to
ZnCl2
a
MnCl2
growth,
against Mn toxicity.
120
100
80
OS
60
*5
40
20
Mn
100p.M
Zn 0
MrtlOOjiM
MntuOuM
Zn
Zn
1ûûnM
1000fiM
Figure 3.4 Competition between zinc and manganese for DMTl. The yeast manganese transport mutant strain
smfl (Y16272) was transformed with the DMTl containing plasmid pARl and growth under different conditions
was tested. Growth in minimal medium without any additional metal was defined as 100% and growth of the
same cells under different metal concentrations was expressed in relation to it. Mn: MnCl2, Zn: ZnCl2. The graph
is derived from two independent experiments.
64
Supplementary
Three
possible explanations
for this effect
are
imaginable, a)
material to
chapter
2
Zn enters the cell via other
transporters than DMTl (e.g. by the Zrtlp), accumulates intracellularly and protects the cell
against
Mn
toxicity
from
inside,
e.g.
by competing
with Mn for
protein
active sites,
b)
Zn
prevents Mn from entering the cell by blocking the binding sites of the DMTl transport
protein, c)
Zinc prevents Mn from
the cell
entering
by
other
some
unspecific
To
way.
differentiate between the first and the other two alternatives the metal content of the yeast
cells
can
be measured
by inductively coupled plasma
concentration in the yeast cells grown in
excess
yeast cells grown in Mn and Zn, then Zn does
protective
Mn is
not
culture, then
the DMTl transporter
or
in the medium
{smfl/p\JG35)
one can
metals which
(b),
Mn
indirectly by
(c). Distinguishing
import
is either inhibited
binding
to
interactions of the two metals at the cell surface
or
between b and
c
may be
directly by
Zn
accomplished by comparing
mutant
in terms of adsorbed metal concentration.
only
carrying only
Again, using
the
the empty vector
ICP-MS
as a
measuring
adsorbed to the cell surface. The adsorbed amount of metals is calculated
the intracellular concentration
metal chelator EDTA and
thereby removing
concentration. If the adsorbed Zn reaches
control, then
toxicity
In contrast, if less Mn
(a).
differentiate between intracellular metal concentration and the concentration of
by subtracting
entering
the Mn concentration in the
to
prevent Mn from entering the cell and the
yeast strain expressing DMTl {smflfDMTl) with the
in the
equal
mechanism of Zn may be localized inside the cell
accumulates in the second
tool,
spectrometry (ICP-MS). If the Mn
mass
Zn
probably
(determined
all metals at the cell
higher
after Zn addition may not be
an
equal
effect
protein
for both
specific
65
washing
surface)
concentrations in the
binds to the DMTl
the cell. If the concentration is
after
and
the cells with the
from the total metal
smflfDMTl
thereby
strain than
hinders Mn from
strains, then the reduction of
of DMTl
expression.
Mn
DMTl
Chapter
Experiments
Most characterized
Nramps
are
SMFl and SMF2
homologues
protein (see
Nramp regulation).
1.4.2
sequences similar to BSD2
revealed
putative
are
the
on
regulated
produced
BSD2 sequences
or
depend
investigating
on
DMTl
4.1.1
RNA isolation
Total RNA of C
(GibcoBRL) according
(3345
x
g,
to
in
only
reinhardtii
supplier's
removed
was
dichloromethane
were
centrifugation (12000
tube. 500 ul
x
isopropanol
incubation at RT
this
cwl5arg7 cells
(10 min)
were
added.
aqueous
was
10
g,
(upper) phase
x
removed. The
may be restricted to
We
decided to start
were
min, 4°C).
hand for 15
was
g,
10
Reagent
centrifuged
(proteins,
ul
200
sec.
transferred to
a
of
After
fresh
min, 4°C) followed
pellet was
ethanol 75%, dried at RT and dissolved in 50 ul RNase free water.
66
and
under different conditions.
vigorously by
Centrifugation (12000
and the supernatant
data base for
in 1 ml Trizol. Insoluble material
x
Bsd2p
Magnaporthe grisea
25 ml of culture
by centrifugation (12000
min, 4°C) the
the
extracted with Trizol
was
the yeast
redundant data base
non
organisms.
synthesis
Briefly,
Only
genomic
regulatory pathway
the mRNA
instructions.
level.
protein stability by
crassa,
in other
Bsd2p
added and the tube shaken
g, 15
transcriptional
Neurospora
4min, 4°C) and the pellet resuspended
polysaccharides)
the
hits. BLAST search in the
no
regulation by studying
Materials and methods
at
A search in the C reinhardtii
factors other than
4.1
of DMTl
the level of
at
Schizosaccharomyces pombe (not shown). Thus,
fungi
4
regulation
regulated
Regulation
washed with 1 ml
Regulation
DMTl
4.1.2
Culture conditions
1200 ml TAP medium
mg/1 arginine
overnight
(see
inoculated with 50 ml
were
and at time
flask, control
4.1.3
was
left
density
9 and 23
total RNA
agitation,
was
cells/ml. In order to induce
Samples
(hours
After
were
taken at time
point
0
after addition of ImM EGTA to
untreated).
an
RNA-probe
for
from
with Clal and Xhol. A band of 1 kb
from the agarose
gel
and after
Switzerland) harboring
an
corresponding
purification
EcoRI
carboxylase/oxygenase (RbcS2)
appropriate
pGEM7
restriction
sites).
hybridization in-vitro-transcription
it
was
was
Michel
fragment
After
were
T7-polymerase.
1 ug linearized DNA
blot
was
performed
RNA
The reaction
where
was
ligated
with
the 750
new
purified
was
lx
to vector
pGEM7
reversely
was
digested
a
control
lack
was
of other
ligated
to
with BamHI. Both
T7.
For
transcribed in the presence of lOmM
RNA
of DMTl and RbcS2
67
bisphosphate
transcription by polymerase
labeling
incubated 2h at 37°C. To test
probe, digoxigenin-labeled, Roche).
to
bp long fragment
plasmid
for
(due
EcoRI
digoxigenin-UTP
labeling efficiency
excised
was
Zf+. As
with
digested
was
part of the DMTl sequence
of the gene of subunit of ribulose
purification
DTT, 20u RNasin ribonuclease inhibitor,
20u
to
digested only
linearized with Clal and
in-vitro-transcription
performed
Goldschmidt-Clermont, University of Geneva,
Zf+. In order to check orientation the
plasmids
was
Promega. Plasmid pARl (harboring DMTl)
plasmid pCS2.1 (kindly provided by
(Actin
approximately 5xl06
treated with EGTA.
was
points 1, 2, 3, 6,
Riboprobe System-SP6/T7
new
of
(2 1).
In-vitro-transcription
To generate
the
a
the culture
transcription,
(no treatment)
the
and divided into two flasks
preculture
with 50
chapter 2) supplemented
incubation at 25°C under constant illumination and
isolated when cultures reached
DMTl
Materials and methods
mix
(Roche)
labeling efficiency
was
compared
to a
a
and
dot
control
Regulation
DMTl
RNA
AAA
and Northern blot
gel
Two combinations of different
1)
RNA
to
5 ul
the tube. For
(50%)
2)
denaturated for lh at 50°C
was
glyoxal 6M,
sucrose
RNA
protocols
DMSO,
loading
and
ul
RNase free water ad 19
the 1% agarose
on
bromophenol blue).
denaturated for
was
(2
for RNA
formaldehyde, lxMOPS,
10
contained lxMOPS, 1.85%
lOmM
min
RNA
acetate
(0.05M),
autoclaved
with
electrophoresis
which
formaldehyde
Both
gel types
might
was
Pharmacia Biotech
mixed with 0.1 vol
was
in
(1:2
buffer
was
the
bromophenol blue).
The
sulfonic acid
and stored in the
(3M NaCl,
0.3M
3.5 ul
was
added
loading
buffer
buffer.
formamide,
6%>
1% agarose
gel
was
the
running
(MOPS) (0.2M), 6.8g
dark).
Na-citrate,
tested:
pH7,
running
deionized
50%
RNAse free water ad 1
(0.01M),
yellow)
turns
20xSSC
were run
Blotting
standard
(buffer
buffer lOOmM
and ethidium bromide. lxMOPS
(lOxMOPS: 41.8g 3-Morpholinopropane
were
After 0.5h ethidium bromide
Na-phosphate
buffer
pellets,
ul).
0.05%
formaldehyde
20ml EDTA 0.5M
and northern blots
Na-phosphate
65°C
at
glycerol,
10%>
gel
gels
liter,
The
pH7)
pH7 with
to
Na-
NaOH
washed after
gel
was
in
order
to
remove
inhibit transfer.
at 40V
for 3h.
performed
either with the VacuGeneXL
according
to
supplier's
The
protocols (overnight).
instructions
membrane
used
vacuum
(3h),
was
or
blotting system
from
by capillarity following
positively charged nylon
a
membrane from Roche.
4.1.5
Hybridization
Prehybridization (2h)
at 68°C.
Roche,
Probe concentration
or
stringency
Detection
hybridization (overnight)
was
Ultrahyb Ambion).
approximately
The
was
performed
on
the
with
100
membranes
wash: RT 2xSSC, 0.1%SDS;
according to supplier's
depending
and
of the membranes
ng/ml hybridization
were
high stringency
washed
(Roche).
Films
signal strength.
68
were
performed
solution
(EasyHyb
hybridization (low
wash: 68°C O.lxSSC,
Anti-Digoxigenin-AP (1:10000)
instructions
after
were
exposed
and CSPD
for
varying
as
0.1%SDS).
a
substrate
amounts
of time,
DMTl
4.2
Results
4.2.1
Detection of RbcS2 and DMTl mRNA
DIG-labeled
controlled
by
and blotted
a
for
an
probe.
dot blot
case
of each blot
Hybridization
buffer
(Ambion)
generated
corresponding
see
to
which is
designed
to
labeling efficiency
loaded onto
with
the mRNA of RbcS2
below). However,
transcription. Still,
was
and
performed
was
that the DMTl mRNA is transcribed at
with ImM EGTA to induce
Ultrahyb
were
Total C reinhardtii RNA
transfer.
solution. A band
explanation
In
(Fig. 4.1).
by capillary
hybridization
for RbcS2 and DMTl
probes
no
a
no
signal
was
an
was
increase
was
sensitivity
visible
(Fig. 4.2,
even
of the
gel
probe/ml
detected
detected
agarose
was
ng
100
by
very low rate the cultures
signal
Regulation
the DMTl
were
after
use
treated
of the
hybridization (not
shown).
A
M
Figure
4.1 A:
B
t-
o
h-
i=
5
<=
DMT1
Agarose gel
with DMTl labeled RNA
probe.
B: Dot blot of DMTl
probe compared
to a labeled
control.
4.2.2
Comparison
of
electrophoretic
Four different blots
provide
prepared
in order to test which
experimental
conditions
the best results:
Blot Nr.
Gel
Blotting
1
Na-phosphate
Formaldehyde
Na-phosphate
Formaldehyde
Vacuum
2
3
4
Hybridization
and 4
were
and transfer conditions
were
visualized
method
Vacuum
Capillary
Capillary
and detection conditions
on
the
same
film.
were
identical for all blots. Blots 1 and 3, and 2
Comparison
69
of the blots shows that the
capillary
blot
DMTl
was more
than
efficient than
vacuum
blotting.
Also
Na-phosphate gels
seem
Regulation
give sharper
to
bands
formaldehyde gels.
1
I
1
i^L
lw*
3
Tf
O
ë
2 3 4 56 Î
RbeSZ
RtoS2
1
2
3
4
S
-
G
7
1
2
3
4
S
6
7
^RtaS2
Hbeœ
Figure
4.2
of
Comparison
and transfer conditions. For blots 2 and 4: 1=1.79, 2=4.47, 3=8.93,
electrophoretic
4=17.86, 5=26.79, 6=35.72,
7=44.65 \ig total C reinhardtii RNA. For blots 1 and 3:
4=14.5, 5=21.8, 6=29, 7=36.4 |xg total RNA. Northern blots
hybridized
1=1.45, 2=3.6, 3=7.27,
with the RbcS2
probe.
Discussion
4.3
For murine
by transcriptional
regulation
RbcS2
Nrampl,
activators
of DMTl
differentially
at
probe (the
detected
expression
on
Nrampl and
tomato
was
expression
varying
expression studies).
was
were
shown
(see
Rubisco gene is
probe
Firstly,
was
generated
a
could be that the
transcript
Membrane embedded
to RNA
all the methods
As
under the
were
a
first step to
study
are
below
optimized using
and used
same
as
a
control
conditions but
metal transporter, it is
is induced when the metal conditions
prior
Nramp regulation).
1.4.2
constitutively expressed
the blot. Since DMTl is
added to the medium
typhimurium MntH, regulation
decided to check whether the DMTl gene is transcribed
we
culture conditions.
A DMTl
E. coli and S.
no
quite possible
optimum. Therefore,
extraction. However, the DMTl mRNA
was
for
signal
that its
EGTA
not
an
was
visible. It
is very low abundant and thus below detection limit of this method.
proteins
cannot
be
since this would interfere with membrane
expressed
integrity.
70
to
levels
as
high
as
cytoplasmic proteins,
Transition metal transport in C. reinhardtii
Chapter
5
Transition metal transport in the green
reinhardtii
and export systems
Uptake
this
study
Chlamydomonas
comparative
approach
CDF,
plants
Using
sequence
we were
two
major
and other
able to
on
the four
recently
transition metal transporter
protein
genomic
identification of ESTs
sequence
(expressed
Nramp-family,
families
we
obtained
sequence
identified ESTs will be valuable for the
tags)
our
current
sequences
one
knowledge
which
a
By this
(four ZIPs,
addition, by subsequent
cDNA
and the
performed
we
members of all four transporter families
CPx-ATPases, and five Nramps). These findings advance
of the
of
microalga
released genome of C reinhardtii.
the metal transport processes present in C reinhardtii. In
splicing
major
the ZIP-, CDF- and
organisms:
in the
identify
objective
metal transport mechanisms in the green
the information available for these
analysis
analysis
role in transition metal homeostasis. The
reinhardtii. We concentrated
families found in
CPx-ATPases.
a
Genomic sequence
-
identify potential
to
was
play
microalga Chlamydomonas
of
in silico
led
to
the
in the C reinhardtii EST database. These
cloning
and characterization of several
metal
transporters utilized by the alga.
5.1 Introduction
Transition metal ions
with their broad range of
effects of the
same
homeostasis. A
only supplies
play
a
significant
role in cellular
elements demand mechanisms that guarantee
sophisticated system
the cell with essential
prokaryotic,
available
but also
on
regarding uptake
Along
processes.
action, which makes them indispensable for cell survival, toxic
of
uptake, storage
a
subtly regulated
and detoxification is at
play
metal
that not
metals, but also maintains the desired concentration
balance of the various metallic elements. Research
on
physiological
mammalian and
on
metal transport has concentrated
plant systems.
A vast
and efflux of transition metals in several
71
pool
mainly
of molecular data is
organisms. Interestingly,
Transition metal transport in C. reinhardtii
up to
now
have not been
algae
group is
especially dependent
process
requires
photosynthesis
on
be
a
for
manganese
must
prominent
water
based
data
in
reinhardtii
[112],
In order to shed
we
were
more
have conducted
a
algae, general
[47, 63, 120]. Only recently,
proteins,
suggested
light
on
(multicopper
two
the genome of the model
to
transferrin in Dunaliela salina
be involved in iron
metal homeostasis
Foxl/Flp, [61, 78]
For
this,
we
performed
organism Chlamydomonas
genomic
sequence is available
H43
in C
network in
algae,
[41] and
a
acquisition
to gene
thorough
families known to be
sequence
analysis
reinhardtii. C reinhardtii is the first
(Doe
http://genome.jgi-psf.org/chlrel/chlrel.home.html [52]).
72
A. and Köster W.
uptake.
potential homologues belonging
(Fig. 5.1).
from
resulting
metal transport systems
ferroxidase
the nature of the transition metal
search for
involved in metal transport
for which the entire
In
stress
[78]; divalent metal ion transporter DMTl, Rosakis
Two other
preparation).
oxidative
photosynthetic
[67, 77, 92], but information about metal transport is
have been identified and cloned in C reinhardtii
and iron permease Ftrl,
and
antagonized by metalloenzymes.
physiological
on
splitting,
Yet, like plants, this organism
of metal balance. The
precise regulation
has been studied at the molecular level
mainly
in such research.
Joint Genome
of
alga
Institute, California,
Transition metal transport in C. reinhardtii
ZIP
Nramp
4»***
.'\
,-*-%
,
ni;
•"
'
h
t
t
f?
cytoplasm
GQNSTITGTLSGQIVMEGF
CDF
CPx ATPase
".""'."i
r
ff
Ck
«
5
:
•,
i
,')%
-">'"
"V
•
/\ 4%. Y\
iJX
j,#—•.
i,I
t
(PAA1)
ll<
H
f
•'i«f»:
"^
rttptat binding
meta!
motif
î
| H-t/E-X-H-X-W-X-L-T-XB-H
V
AJtûtif
(Hx||rfotif
!
bînping domain(s)
/
GMfCxxC
1M|G>
domain
-
N
cytoplasm
phosphorylation
domain
DKTGTLT
representation of four transition metal transporter families with approximate positions of
ZIP-family [46] : The histidine rich variable region and the conserved residues essential for
IRTl function are indicated. CDF-family [46] : The (HX)n motif and the metal binding motif are indicated. CPxATPases: A. thaliana PAAl as an example [118]. The metal binding, phosphorylation and ATP binding domain,
and the CPx motif are indicated. Nramp-family: For most Nramps 10 or 12 transmembrane domains are
predicted (dark grey), but C. reinhardtii DMTl is predicted to have 11 (light grey). The conserved transport
motif (CTM) for C. reinhardtii DMTl is indicated.
Figure
5.1 Schematic
conserved motifs.
73
Transition metal transport in C. reinhardtii
5.2 Materials and methods
(expressed
ESTs
the
to
genomic
sequence
sequence,
the untranslated
and
similarity
regions.
search
reinhardtii genome
was
was
which have the
This
directly
with the
BLOSUM62,
were
a
filter for low
For EST search
complexity
ESTs
was
applied
and Lukashin A.
M.
we
used the
sequencing
sequence
possible
identified
to
were
on
the
respective
reconstruct
a
provisionally
were
recently
an
Therefore,
released C
The matrix for
ungapped alignment
reinhardtii
sequence
as
well
silico
and translation.
scaffold. Since the scaffolds
sequence
named
by
by stringing
the
they belong. Only
performed by
ClustalW 1.8
family
them
name
are
was
not
or
together.
non-redundant ESTs
always
genomic
truncation of the
randomly numbered,
followed
are
the
GeneMark.hmm,
Still, it
splicing,
as
splicing
Eukaryotic
mistakes which would lead to false
sequence scaffold to which
Alignments
results.
no
library,
Kazusa DNA Research
original genomic
in
unpublished)
EST
EST clones to selected sequences. This may be due to gaps in the
assign
to
sequence,
not
and
an
searched in the C
were
after
resulting
Borodovsky
genes in
identified in the
firstly
(http://opal.biology.gatech.edu/GeneMark/eukhmm.cgi,
genomic
potential
(http://www.kazusa.or.jp/en/plant/chlamy/EST/blast.html;
sequence
sequence often limited
published
in the C reinhardtii EST database gave
organisms
the cDNA instead of
harboring
(http://genome.jgi-psf.org/chlrel/chlrel.home.html [52]).
Institute, Japan, [4, 5]).
possible
of
identification of
hampered
performed. Subsequently, corresponding
database
advantage
only partially available,
genes from other
homologous
BLAST
are
tags),
by
The
putative
it is
genes
the number of the
given.
(http://searchlauncher.bcm.tmc.edu/) (Tab. 5.1).
74
Transition metal transport in C. reinhardtii
Table S.1 Parameters for ClustalW 1,8
DNA
Sequence
Fast Pairwise
Parameters
Alignment
K-tuple (word)
alignments
Parameters
size: 2
Multiple Alignment
Parameters
Gap opening penalty:
Gap
Scoring method: percentage
Weight transitions:
Yes
Multiple Alignment
Parameters
Number of top
Gap penalty:
Protein
diagonals:
Sequence
5
4
Parameters
Alignment
K-tuple (word)
Parameters
Weight
size: 1
Gap
method: percentage
Number of top
matrix: blosum
Gap opening penalty:
Window size: 5
Gap penalty:
penalty:
5
Fast Pairwise
Scoring
extension
10
Window size: 4
diagonals:
5
3
extension
10
penalty:
0,05
Hydrophile
gaps: On
Hydrophil»
residues: GPSNDQERK
Residue-specific
gap
penalties:
On
5.3 Results
Table 5.2
gives
In addition to
text
a
summary of all results.
our
search
search
in
as
described in the materials and methods
the
website
JGI
with
bin/searchGM2.cgi?db=chlrel&dName=chlre)
ATPase, and
ZIP. For
(Smith-Waterman Hits,
Nramp and
CDF
all available
no
new
databases).
also other hits besides the sequences identified
investigated
further in this
by
study.
75
the
we
performed
a
(http://genome.jgi-psf.org/cgiwords
sequences
For
section,
were
CPx-type
Nramp,
CPx-type
CDF,
identified
by
this search
ATPase and ZIP there
us, but with smaller
scores.
These
were
were
not
Table 5,2 Overview of C reinhardtii clones and
putative
transition metal transport systems
Cloned transport systems
Family
Fox 1/Pip
multicopper ferroxidase ÀF450I37/AYÛ74917
Ftrl
iron permease
AF478411
f78]/[6i]
[78]
DMTl
Nratnp
AF515631
Rosakis and Köster, in
PutatiYe transport systems
Family/Subfamily
Position in genome version 1.0
ESTs*
ZIP77
ZIP/ZIP I
scaffold 77
ZIP735
ZIP/ZIP I
ZIP162
ZIP1381
ZIP/gufA
ZIP/gufA
contig 3
scaffold 735 contig 2
scaffold 162 contig 3
scaffold 1381 contig 1
CDF820
CDF/CDF III
scaffold 820
CDF2650
CDF/CDF I
CPxll84
CPx-ATPases
contig I
scaffold 2650 contig 1
scaffold 1184 contig 2, 3 and
CPx89b
CPx-ATPases
scaffold 89
Nramp579
Nramp3557
Nra»p672
Nramp2132
Nramp
Nfamp
Nraittp
Nramp
scaffold 579
'only
non-redundant ESTs
identical
to
are
or
eniyme
Genebank
Reference
ace. no
contig
-
AV630909
-
AV635915,AV389034
3 and 4
contig 7
scaffold 3557 «»tigs 1
scaffold 672 contig 1
scaffold 2132 contig 1
ÀV395632, but alignment only partial
-
4
ÀV636551
AV387187, BE129393, BE76Ï353, BE761354
BE72433Î, BE337524
and 2
AY62892Û
_
given
ESTs BE761354 and BG844651 identified
by |78]
as a
putative
préparation
copper
transporting
ATPase
Transition metal transport in C. reinhardtii
ZIP-family (zrt,
5.3.1 The
The ZIP
as
well
the
irt-like
protein)
includes members from
family
from eubacteria and archaea
as
thaliana genome
Arabidopsis
transporter 1)
was
plants, protozoa, insects,
mammals and
in
[37, 38, 45, 49, 51, 138] (and reviewed
18 ZIP members
the first ZIP identified
identified. IRTl
were
[37] and is considered
the
as
(TMs)
with
(called
amino- and
extracytoplasmic
feature of many ZIPs is
the "variable
a
predicted
were
to possess
accommodates many histidine residues. Since histidines
this
is
loop
function
We
metal
binding
major
performed similarity
(Ace.
corresponds
sequence after in silico
due to
including
these
For the
used
as an
database
which
seem
than to ZIP1.
and
similar to
ZupT,
a
belongs
[56] (Fig. 1).
A
particular
often involved in metal
which
binding,
IRTl, residues important for
In
ZIPs
are
divided into 4
for
a
subfamily
proteins
of the ZIP
subfamilies,
(LIV-1,
good alignment
The
high degree
genes among the ZIP
were
to ZIP
homologues.
ZupT, especially
at
tree
contig
2. EST
regions
Zrtlp
which do not fit very
of conserved amino acid
well,
residues,
(Hisl97, Serl98, His224, Glu228,
(Ace.
no.
database, but
two
P24198, [51])
hits in the
was
genomic
Scaffold 162 and 1381 harbor sequences
the second half of the amino acid sequence,
possess His224 and Glu228.
Serl98 is
replaced by
Only ZupT
and
Asn.
homologues
for better visualization
77
The sequence
for ZIP735 with S. cerevisiae
from Escherichia coli
8 amino acid sequences of 8 ZIP
similarity
family.
family.
found in the EST
well, but the
I, revealed the
found for ZIP77. Translation of the DNA
Translated ZIP77 contains
splicing.
ZIP
3 and for ZIP735 in scaffold 735
ZIP162 and ZIP1381
as
to
essential for function of IRTl
correspond
aligned
generated
are
putative
ZIP162 possess Hisl97
We
splicing yielded
No similarities
to
are more
contig
gufA subfamily, zupT
input.
proteins.
to ZIP735. No ESTs were
in the
errors
which
possibly coding
(Fig. 5.2).
the four that
[110]), places
transmembrane domains
searches with members of each of the 4 subfamilies
for ZIP77 is located in scaffold 77
possibly
role in zinc
notably conserved, [56]),
are
[110] (Fig. 5.1).
AAC24197),
no.
existence of at least two genes
and A thaliana ZIP1
uptake
II, [46]). Similarity search in the C reinhardtii genome with the A.
ZIP I and ZIP
AV630909
a
iron
similarity [46].
sequence
thaliana ZIP1
site of the
in TMs IV and V
mapped
were
according to
gufA,
potential
a
In
between transmembrane domains III and IV
because its sequence is not
region",
ends
carboxyterminal
long cytoplasmic loop
eight
[46, 56]).
(iron regulated
system from soil. Four other ZIP proteins in A. thaliana, ZIP 1-4, may play
transport [53]. Most ZIP proteins
fungi,
(Fig. 5.3).
or
putative homologues
ZIP735
and ZIP77
are
Transition metal transport in C. reinhardtii
positioned
closer to A. thaliana ZIPl, while ZIP 162 and ZIP1381
similar to E. coli
are more
ZupT.
The nucleic acid sequence of EST AV635915 is identical to part of the
Interestingly,
ZIP1381 DNA sequence.
ZupT
on
the in silico
amino acid sequence level than the EST
sequence is still present in the EST which
was
spliced
ZIP1381 bears
(not shown).
spliced
It is
out in
similarity
more
possible
putative
that
an
to
intronic
silico. Another EST which
matches ZIP1381 is AV389034.
We did not find
cerevisiae YIL023c
T39242)
as an
performed
and
a
(Ace.
input.
homologue
no.
in the LIV-1
NP_012241)
and with
Search for ZIP II members
with Homo sapiens hZIPl
Caenorhabditis
elegans (Ace.
reinhardtii possesses
representatives
when
we
H
also
no
hits. Here,
sapiens hZIP2 (Ace.
T29592) hypothetical protein.
of at least 2 ZIP subfamilies.
78
searched with S.
Schizosaccharomyces pombe (Ace.
produced
(NP055252),
no.
subfamily
a
no.
In
search
no.
was
AAF35832)
summary,
C
Transition metal transport in C. reinhardtii
ZI P7 7
EXP735
î* irt 1
i
msnvttpwwkqwdpsevt|
LFV
-tIveIg AT AC gHaII
D^HAS
E1P77
45
EIP ? 3 b
45
GJIiISMI HS »GM»Ji11sg1»WDHRRRAGTI
ZIPl
46
KATKLK
sJfFTMJ
Ertl
61
consensus
61
ziei i
91
EIP 7 3 b
96
*
*
«
SkikSlrI pIy« yHfBk y
rr^gtsstad^Bs---PEND
llFlxfiKgEaÄ^¥ ILC TC3
iiiiHMDPr
*
v|gds|s|p|kpH|vB
~-~
--—
ZIPl
106
1'xJBgmegeSksdP8ghhyhhoooqhnohnhhehchah
daferlsspclWttIgkfppagMJlsI
Ertl
115
tg^otgmwSlyswcp'hJtslIptfBtp
consensus
121
AFSL
FSMNllBSKOVH
deIkdtvvrnt
IV
EI P 7 7
128
ZIP7 Î5
15b
EIP1
1
IdeBehBghvhih
£Z ft^
T
IsS^DNlJNgTgNGSHDTKNGVEYYg
Ertl
consensus
tsJdvvqsfqaqfyaf:
131
.ifs.
V
ZIP77
139
ZIP735
202
EI PI
214
Ertl
2 3b
consensus
241
VI
VII
ZIP/7
196
|G-
Z1P735
259
El PI
270
HlftjB
jTl |
Ertl
2 92
consensus
301
JAR— IMl) PME V
1s|1glg1kk»ke1
Ulq
*
IpJIr—IvsgIy
jslqlLi^li
RFVPAG
MN
IFÏ PN
*
.
.
Vil
o *j a
EI P7 7
El PI
**
i
31 ?
GEGGAGAGRi
325
rlqsnIwlj
|o)RTKP§REI,SFN¥l
3 4 6
361
Figure
Alignment of S. cerevisiae Zrtlp, A. thaliana ZIPl and two putative ZIP transporters in C. reinhardtii.
approximate position of transmembrane regions predicted for Zrtlp and ZIPl [46]. Residues
be essential for function of IRTl [110] are shaded grey in the consensus sequence.
5.2
Bars indicate the
found to
79
Transition metal transport in C. reinhardtii
A. thaliana 1RT1
43%
26%
A. thaliana ZI P1
S. cerevisiae Zrcl p
22%
ZIP77
51%
15%
ZIP735
ZIP1381
27%
'.aiti ZupT
29%
ZIP162
Figure 5.3 Similarity tree of selected ZIP homologues. The C. reinhardtii putative ZIP homologues identified in
study are boxed. The tree was generated with the full dynamic alignment option of the DNAMAN software
(Lynnon BioSoft). Alignment parameters: protein weight matrix blosum, gap open penalty 10, gap extension
penalty 5; Optimization of group alignment: protein weight matrix GÖNNET, gap open penalty 10, gap
extension penalty 0.2, residue-specific gap penalties, hydrophilic penalties, hydrophilic residues GPSNDQERK.
this
5.3.2 The
CDF-family (cation
CDF
archaea and
function in
proteins
constitute
uptake
thaliana CDF
roots,
[129].
while others
homologue
Overexpression
showing
CDFs
are
mostly
proteins
are
is
plants
[46]).
Certain members of the
efflux. In the yeast
expressed
can
family
Saccharomyces
transported
equally.
or
G).
into
a
proteins,
Both the N-
loop
to
cerevisiae at least
[69]. The
compartment (vacuole) in the
A.
and the
C-terminus
is
thought
[11].
80
a
(Fig. 1).
are
A
metal
cells
to
long loop region
binding
I
eukaryotic
predicted
histidine-rich motif
to act in
root
[46]. Subfamily
while in II and III pro- and
frequently containing
This
thought
in all organs and is also involved in zinc
with 6 transmembrane domains between them
n=3-6 and X often S
bacteria,
lead to enhanced zinc resistance and accumulation in
eubacterial and archaeal
distributed
are
in zinc and cadmium resistance
divided into the 3 groups CDF I, CDF II and CDF III
found between TMs IV and V,
ZATl
in
of metal cation transporters identified in
Zrclp is involved
ZATl
in
facilitator)
family
catalyze
that zinc must be
includes
cytoplasmic
a
eukaryotes (reviewed
5 CDFs have been identified.
resistance.
diffusion
be
is
(HX)n (where
as was
shown for
Transition metal transport in C. reinhardtii
A. thaliana ZATl
belongs
subfamily
to
CDF III and
the C reinhardtii genome database. After in silico
S. cerevisiae
Zrclp (Ace.
no.
NP_013970)
splicing
produced
and
The
putative
metal
and A. thaliana ZATl
terminal tail is also conserved. The
splice junctions
other
In
thaliana
subfamily
(Ace.
protein
region
one
no.
was
CDF
I, scaffold
CAB67634),
has not been
which
a
since the
interpreted
CzcD from Bacillus subtilis
was
loop
is much
AAD11757)
of the CDF
at
family
the C-
longer
assignment
than that of the
CDF820, but with several base
at
alignment
Notably,
a
putative protein
family
of A.
in the review of
between ZATl and the
least 1 of the 3 CDF subfamilies is
a
role
as
a
putative
metal transporter for
CDF2650 did not appear
were
NP834116)
81
to
motif is not conserved in CAB67634. Since
characterized yet,
homologues
no.
similarity
included in the CDF
binding
with caution.
(Ace.
showed
two sequence
functionally
search in the JGI website. No
input. Thus,
proteins
no
(H-D/E-X-H-X-W-X-L-T-X8-H)
identified for
2650
CAB67634 showed that the metal
protein
(Ace.
is also found in CDF820. Errors in the
possible
EST
[46]. However,
CDF2650 must be
as
(HX)n
are
in
only partial alignment (AV395632).
Gaither and Eide
text
this
homologues. Only
mismatches and
this
at
(scaffold 820)
described for CDFs between transmembrane domains
loop
IV and V which contains the motif
of
motif
binding
hit
translation, the alignment with
shows that CDF820 possesses several conserved residues found in
(Fig. 5.4).
one
identified in
subfamily
and ZitB from E. coli
represented
(Ace.
as a
hit in the
CDF II with
no.
Q8X400)
in the C reinhardtii genome.
Transition metal transport in C. reinhardtii
CDF8 20
8
?rcl
12
consensus
61
v^
LJ iL
G-WEATP
RDaATBLHBGgHFEDÄGN;
t-Bkshks
L|1tBdT^HFL^^b1tiByMs1
...
DvBKNBGPD.
....
...
il
O xL LJ
rBI
HMHGHGJNJG^oBppdl
SLPNDNLAlPlPATÏSPGPSi
zrcl
1 90
consensus
241
QipEVLPQSVBNRLSNESQ-——
GÖFlSöflSliS
V^ Li IT
lj* £. LJ
—
---
—
.
—.-
246
ATTREGGHEVPVRWvOOMLAlMIMSMSDHnHKHGOGEGTNGHAGHSSDEEGGHDHHD
212
N
hBdHDHSHES|k|
3 01
ä£ Zj
PïlFA2f1
——
KSKTQVl
240
CPF820
——
O
30 6
*
.
"^
^^
*^
""^
***"
"^
^^
^^
**"
iiii
"^
^^
^*
^^
"^
ii"
"^
^^
^
^^
^^
^^
*^
^^
*""*
""^
llli
"*"
^^
^*"
"^
llli
"^
^
^*
^^
""^
111A
^^
^""
^"
^^
•"
^^
^^
^^
m*,*
œrc
œs,*
tfsss.
^a
bbbtc
«^
.ssrc
mas
tfraa.
aaœrc
^^
œsrc
rasra
HSHEHGAGSCCGGHGQAAAAAGAGAAAAGPSAPDGHGHGHDHGHDHDHKHGGGGDGHGHS
361
V
17
v^
/,
311
L^ i^
o *c°
\J
3 66
revpghshghgeggcghghshghghshgbghghgg
rel
pnn CÏ0.T1 cjiise
4 21
2 81
4 2 6
consensus
481
zatl
338
"fa "# ~i"
^N---«ElKl
Bi^B§KQSQBF
**
.
.
.
.
.
**
L LJ
e
OfcU
4 83
zrcl
31 j
consensus
541
"k û"
^lil
*
*
* *
IB^BJIaBWkvIIacË
HU
HB
iuMpJ^MMÉMg^pfl!
A|J|PG||||JJftuiillFMKÉlÉftiE Sil YHIylffTiïi
*
ifc"
1ST
"%
ifr
.
r
7\
Figure 5.4 Partial alignment of C. reinhardtii CDF820 with A. thaliana ZATl and S. cerevisiae Zrclp. The
putative metal binding motif (H-D/E-X-H-X-W-X-L-T-X8-H) is boxed, with the conserved amino acids
indicated by a star above the sequence. Transmembrane domain V is indicated by a line above the sequence [46].
82
Transition metal transport in C. reinhardtii
5.3.3 The
CPx-type
to
of
family
ATPases
CPx-type
ATPases have been identified in
a
wide range of
transport essential and potentially toxic metal cations (e. g.
belong
to
the class of
P-type ATPases, which
membranes at the expense of ATP
PAAl
[118, 122], PAA2,
inner
chloroplast
copper ions via
were
defined
a
as
envelope
stromal
in
(reviewed
membrane and to
[135]).
Plant CPx-ATPases
supply
metallochaperone [118].
due
CPx-ATPases
are
motif DKTGTLT, found in the
VI and VII. These two motifs
motif GMTCxxC is found in
the
thought
ATP-binding
to
several
to
copies
biological
homologues
are
be localized in the
superoxide
intramembranous
dismutase with
P-type
ATPases
cysteine-proline-
function in metal cation transduction
motif GDGxNDx and the
phosphorylation
domain between transmembrane domains
conserved amongst all
one or
thought
across
The divalent metal cations
large cytoplasmic
are
is
the Cu/Zn
conserved
a
motif which is
cysteine/histidine/serine (CPx)
[119]. Other conserved motifs
to
substrates
charged
PAAl
assumed
are
Cu2+, Zn2+, Cd2+, Pb2+). They
pump
and HMA5.
RANI
and
organisms
at
P-type
ATPases. The metal
the N-terminal part of the
binding
protein (Fig.
5.1).
tBLASTn with A. thaliana
at
least two
potential homologues
scaffold 1184,
which
causes a
contigs 2,
CPx-type
exist in C reinhardtii
3 and 4. It is shorter than
premature stop. One EST
CPx89 is located in scaffold 89
contigs
the ESTs BE761354 and BG844651
[78].
We
identified
BE761354. Both
several
putative
ATPase PAAl
CDF
ATP-binding, phosphorylation,
ESTs
was
no.
(Fig. 5.5).
Q9SZC9)
CPxll84 is located in
expected, possibly
identified for this
showed that
due to false
putative
gene
splicing
(AV636551).
3 and 4. Part of CPx89 DNA sequence is identical to
previously
for
identified
CPx89:
homologues
metal
(Ace.
as
a
copper
transporting
AV387187, BE129393,
ATPase
BE761353
possess motifs conserved in CPx-ATPases
binding).
83
and
(CPx,
Cf>tl!H
CPx] 184
MAI
PAA]
»SMI
RAMI
CPx«»
CPxS9
CFX1184
CP-4] 1,84
PAAl
RAM
PAAl
RAMI
,
crx8§
CPx89
CPx 1.1 84
CPx]184
PAAl
PÄA1
ran:
RAMI
CPx8 9
Cl x89
cpxiia«
rpal184
£>AAJ
PAAl
RANI
RAI11
CÏX89
CPX8 9
CPXU84
CPxl1«4
PAAl
eBffH'ÄetktkdkqarBke30beBi\vswaBcB
RAN 1
<
#
ifclpI'TiPISfc] E§\"FPFEvf<i.-îRsiJc|
CPxP.9
CPxlId»
PAAl
88
; f,7
CPxl184
J^5
CPxàa
251
CPxl 184
5,13
RANI
2 91
RAMI
CPXS9
B9
L&i
RANI
CPX15Î
I AA1
¥Lsi¥i,FrrovirpHiPiFDP
251
PAAl
PAAl
cpx]184
CL'.'Ih] THI''fcl*MÄTiA-ROTRPPVl
gtEtjFe''FK§pp»JsBr<lI~srD*rSCA|îAFRPFI>ÏSI
RANI
CPx^^l
PAAl
RAH I
CPX89
~FxIl&4
_.__
WtlMtul
PAAl
LL,."W»Ct>PEMMGDWLKWA|vs|lOFV
PAM
OAQMjilPE
L
MIS IUFHrtMNPHKLPSPT V
lAKGKflsMMBhjgl,
PTC
j
CÏXS9
IsKfïllllppBlipPT Jllp
CPxilSä
616
PAA 1
8BP
KAN 1
95 1
CPxSS
8"0
î£PP"S§
MP
M—RiSFFRL
jg
PPAOGGP-AGC.RFQrLWFYpl
'-PX11P4
PAAl
RANI
CPxhp
critliH
PAAl
.HAK 1
CPx8 9
Figure
5.5
Alignment
of PAAl and RANI from A thaliana with C. reinhardtii
CPxl 184 and CPx89. Black lines indicate functional domains
[118] and
stars above
the sequence indicate the conserved GMxCxxC motif. The CPx motif is marked
a white bar above the sequence.
by
Transition metal transport in C. reinhardtii
5.3.4 The
Nramp-family (natural
Nramps
They
are
widely
distributed and
transporting proteins
in many
occur
macrophage proteins)
organisms
cloned and
assays
functionally
[30, 127]. Previous
Nramp homologues
characterized
AF515631, Rosakis A. and
12 transmembrane
(Fig. 5.1).
Köster W. in
preparation). Nramps
man
were
as
are
AtNrampl,
shown
by yeast
(DMTl,
to
predicted
Ace.
for
(GQNSTITGTLSGQIVMEGF
no.
consist of 10
to possess 11
conserved motif in the
between transmembrane domains VIII and IX, often referred to
"consensus transport motif
had
we
sequence,
predicted
highly
a
specificity.
[18, 72, 79, 82,
found.
was
genomic
DMTl from C reinhardtii is
A remarkable feature of Nramps is the presence of
cytoplasmic loop
broad
a
in C reinhardtii
Nramp homologue
an
domains, while
the release of the
to
with
from bacteria to
and 4 contribute to manganese, iron and cadmium transport
complementation
or
conserved metal
In the A thaliana genome several
103, 123, 127].
3
highly
are
resistance associated
as
the
The function of this
DMTl).
motif is not clear yet.
A search for
Nramp homologues
genes apart from the cloned DMTl
putative Nramp
located in scaffold 579
30332:34793). Nramp579,
complete
silico
open
splicing
in the genome of C reinhardtii revealed another four
reading
frame
(ORF).
The
and translation. Some
putative
regions
(scaffold_489, contig 3,
amino acid sequence
were
corrected
showed that
largest part
most
Nramp579 possessed the
of the sequence
variable part of the
Two ESTs
were
(Fig. 5.6).
The N-terminus is not
Nramp proteins and the
identified identical to
correct
similar to
DMTl
(Rosakis
prokaryotic
than to
A. and Köster W. in
Other sequences similar to
scaffold 672
contig
1
possible
reading
we
cannot
(AV628920)
was
determined
by
in
comparison
with
Alignment with
Nramps
determine
over
as
the
it is the
BLAST with
Nramp homologue is
already
as
shown for
preparation).
Nramps
are
located in scaffold 3557
contig
exclude the
Nramp672 and Nramp3557 belong
a
frame cannot be defined.
eukaryotic Nramps (not shown),
and scaffold 2132
and
of
to
sequence of this
1.
Gaps
to
the
possibility
same
identified.
85
ORF.
contigs
and truncation of the
sequence in the scaffolds do not allow identification of the
putative homologues
for
frame.
regions
pairs
probably
Nramp579 (BE724331, BE337524).
Nramp579 showed that the potential partial amino acid
more
reading
conserved
most
was
hand after
by
the DMTl sequence because the program had chosen the false
DMTl
codes most
contig 7,
base
that
complete
1
and 2,
genomic
sequences of these
Nramp672 and Nramp2132
Only
for
Nramp3557
one
or
EST
Transition metal transport in C. reinhardtii
of the 5
Alignment
conserved in most
C
reinhardtii
Nramps
Nramp homologues shows that the
also present: DPGN in
are
Nramp579, CTM-motif inNramp579 and
5.3.5 Further genes involved in metal
The green
in which
kingdom
microalga
a
kinetic characterization and
genomic
or
D. satina
(Ace
cress
no.
T10729). Further,
was
the first
identified
organism
outside the animal
[40]. Transferrins
BLAST)
[41].
similarity
we
to
In C reinhardtii
plasma
we
looked into the COPT
[70, 116]. Five homologues
in the EST
as
well
as
one
in the
results, indicating that specific
of them
we
are
family
algae
iron
are
did not
binding
given by
identify
any
of copper transporters
present in the genome of the
performed similarity
genomic library
are
membrane bound transferrin from
(COPT1, AF466373; COPT2, AF466370; COPT3, AF466371;
COPT5, AF466374). With each
another
was
detection
EST sequence which bears
identified in A thaliana
recently
no
transport
homologue
antibody
MPH in
3557;
(Fig. 5.6).
Dunaliella satina
transferrin
2132 and
involved in iron transport. Hints that transferrins exist also in other
proteins
thale
672
Nramp579,
sequence motifs
COPT4
search
AF466372;
(tBLASTn
of C reinhardtii. The search
and
produced
copper transport in C reinhardtii may be mediated
by
protein family.
Figure 5.6 Alignment of C. reinhardtii DMTl and putative Nramp homologues. The potential sequence for
Nramp579 was derived from the genomic DNA by in silico splicing and translation and some correction by hand.
Other sequences were derived by in silico splicing and translation. The alignment was performed with
ClustalW1.8 and corrected by hand. Identical residues are highlighted black and the CTM motif is boxed. In the
consensus
sequence stars mark identical residues.
86
Transition metal transport in C. reinhardtii
579
2132
3551
DMTl
-MVLGRLFARSESAAPALADÏNNEGNGSTIDTSVVAVE
672
consensus
579
2132
3557
1
DMTl
3 8
672
!
consensus
1
VEÄSGSPSSÄTVRGDSTLNEPLLEGFKDRGELPSLPESNÄSL
ffi:
B
IBS
579
2132
3557
DMTl
672
consensus
579
57
2132
48
3557
65
DMTl
155
672
{WABBAHHBflaNB^BalaBLABGaTWftE-
IB8AiBAîiHBHBNKHBvBIflAAlABTBAE¥VB^ cBAî
181
***
579
115
L
80
—
3557
65
—
DMTl
214
672
**
*
*
* *
*****
* *
-llUvwWIviaiLVSRlPLRSlAGlLiBR
T¥G-
ERRES§
SFKFLEÏLVGSfllgJLI sIImyBjVKAoIdMGkImHHyIBsKE I IINKDMll
11
1
consensus
241
r
579
163
(t(vsi
2132
PiMwagvjiBaaBv
1
consensus
2132
:«pci||i|Jgl||a
-
*
* *
M,VAGQARGAVA|aAAA|aAAAAL(iG.
AV
1s¥iqtrayprtt1grti1iklglv§stl
SI
80
3557
97
DMTl
274
t£
672
consensus
301
579
2 23
2132
80
3557
101
*
ik * *
*
*****
* -k -k -k
*
*
*
* * *
llJsWTpisBGVlGWVHTLOlHilEOiilsSSllP.
DMTl
872
consensus
MyUIa'itMgakaBs I
x
361
r§tykeraeregas|g-
579
2132
3557
DMTl
672
consensus
421
579
337
2132
-
*
*****
-wg|qg||gmgs~
3557
101
454
:s
86
|PG
consensus
* * *
80
DMTl
672
* *
481
NFANRWYASV1AWLLFLF1SALNVNLWQSA1SGSFGGLGHRRAAV
YANGC
******
87
Transition metal transport in C. reinhardtii
5.4 Discussion
In order to obtain
metals in
algae,
molecular information
more
performed
we
microalga Chlamydomonas
a
comparative
reinhardtii.
four transporter families: four ZIPs,
on
sequence
the transport processes for transition
analysis
BLAST search
By
CDF,
one
we
in the genome of the green
identified
putative
members in
CPx-ATPases, and five Nramps.
two
We
present alignments of the newly identified sequences with already characterized homologues
from A. thaliana and S. cerevisiae, which show that the sequence motives
protein
families
also
are
in the
represented
putative
probability
that the C reinhardtii sequences encode
increased.
Since the
confirmed at this
silico
point, alignments
spliced genomic
and identified ESTs
thaliana, since
expressed
to
be
quite
potential
sequences
we
corresponding
surprising
that the
in multicellular
five
metal transporter
reason
of these
the
functional in metal transport is
reading
frames cannot be
caution, serving mainly
homologues. Still,
we
for this
we now
in the
the membrane of intracellular vesicles and
study
advance
our
as
homologues
than the
homologues
of the
may be found in
is the
case
current
have
raw or
an
in
a
same
generally
A.
family
considered
different localization
while
Smf2p
in the vacuolar membrane
knowledge
plant
or
in S. cerevisiae. In the yeast the
plasma membrane,
Smf3p
good
Nramp family might be
of these transporters is
redundancy
potential proteins,
as a
(Tab. 5.2).
that the
speculate
to
specificity
Nramp homologue Smflp is localized
The results of this
often find many
interesting
since
metal transporters
may possess less
alga
the
search in the EST database of C reinhardtii
a
putative
organisms
homologues,
broad. The
expression pattern
with
interpreted
performed
to
in different tissues. It is
represented by
be
must
homologues. By this,
of the main metal transport systems in C reinhardtii. With the
on some
It is not
proteins
sequence of the identified open
precise
indication for the existence of
overview
C reinhardtii
for these
typical
is
expressed
in
[84, 86, 103].
of the metal transporter network
present in C reinhardtii and facilitate cloning of selected metal transporter genes for further
investigation
and characterization. Where EST clones
would be based
on
(rapid amplification
hybridization
of
a
RT-PCR
identified, the cloning procedure
(reverse transcription-polymerase
of cDNA
cDNA
were
chain
reaction)
and RACE
ends). Otherwise, amplification using genomic
library might be
DNA
or
the method of choice.
Acknowledgement
The
genomic sequence data were produced by the US Department of Energy Joint
Institute, http://www.jgi.doe.gov/ and are provided for use in this publication only.
88
Genome
Conclusions and perspectives
6
Chapter
perspectives
Conclusions and
Algae
far the most
by
are
interest to find out how these
important
producers
oxygen
handle
photosynthetic organisms
in their environment. The lack of molecular data
regarding
gave the
reinhardtii is
for this work.
impulse
laboratory
work and served
as a
molecular level. It should be
the
laboratory work,
hampers
research
function of the
well
accepted
protein
in
method to
as a
regulation
in the cell.
pointed
a
organism
out
drawback
major
alga itself, specificity
of the
model
considerably. Thus,
DMTl acted
study
Chlamydomonas
here
-
in the
a
reinhardtii is
after identification and
function of plant transport
metal transporter with broad
may be narrower,
cloning
provided by regulation
assays, which is
of gene
organism,
hybridization system,
was
a
-
the
expression. Thus,
may prove useful in order to further characterize the role of the
aiming
showed that the DMTl
-
-
of DMTl I studied the
In the native
specificity.
abundant and not detectable in the
in contrast to the
to
protein
elucidate
transcript
is not
transcript
of the
detectable. Treatment of the cultures with the metal chelator EGTA
several time
at
versatile in
proteins [22].
of the gene
sampling
quite
reliable gene knock-out system
a
heterologous system by yeast complementation
study
algae
suitable for
microalga
green
My preliminary results from northern blot experiments
control gene which
metal concentrations
of metal transport systems at the
that, although C
potential transcriptional regulation
and
varying
transition metal transport in
study
the lack of
earth and it is of great
on
points
after the treatment did also not allow detection of the
DMTl mRNA.
It
equal
was
for
0
to
growth
uM) [78].
was
shown that
since
[78].
I
growth
Since the
high affinity
a
iron
backup
that
uptake system
iron
uptake
is
(and
compromised
at
an
unspecific
metal
all
when
algal
cells
uptake system
89
is
medium
deficiency (-Fe/-Cu,
(-Fe)
was
where Cu
requires
copper
mechanism must exist when copper is
to
not
depleted
FOXl/FLP-FTRl
expect the backup for the FOX1 system
Measurements of the metal content of
suggested
of C reinhardtii in iron
in medium with combined iron and copper
function, it is plausible that
limited
growth
Cu
be rather
subjected
thus
to
functioning
a
high affinity system,
FOX1)
is
absent
[78].
different metal conditions
in iron
uptake
when copper
Conclusions and perspectives
(and
was
thus
F0X1)
shown
is needed
as
iron
by
shown
was
uptake measurements,
the
the
of the
affinity
uptake activity
by
in the
low
or
specificity,
might
be
a
iron
ions, needs
backup uptake system
cellular concentration of other metals
and
subsequent growth
on
low
and low
The
be
cannot
the broad
specificity
eventuality
of five
explained by
tissue
so
Nramps
in the cell
might
some
RNAi, is
a
studying
the
generation
means
a
be
quite
light
on
protein
question.
followed
proteins. Thus,
identification of
the
without
homologues
the
could be
firstly by complementation
drawbacks
compared
assays
their role
the identified members of other transporter
a
with
organism (which
homologues
two
have been
would expect all C
one
with the
putative homologues
of each
homologue,
assays, with the
the method is
sites
specific
of
can
on
and
multiple
secondly by
affinity
e.g.
by
advantage
of
inapplicable
the
for
Nramp protein
in
a
"cleaner"
be defined and the gene of
in their function
together
on
much
uptake system
function, specificity and regulation might be better performed
lot of information
follow
definitely give
unusual, especially considering
silencing
organism. Yet,
differences in the
a
RNA interference
unicellular
a
While
complementation
competition experiments. Thereby,
giving
in
experiments
Directed gene
alternative to yeast
precisely targeted
Alternatively,
would
Nramps. Only
system, like S. cerevisiae where the genetic background
interest
by
the
increasing
function, i.e. metal transport, the role of each homologue
function in the native
of mutated
efficient without
of DMTl
is at least
cerevisiae.
different. Localization
this
more
it
That way iron assimilation
specific.
Nramp homologues present
same
uptake system [61]. However,
possible.
specific expression)
have the
to
promising
essential for
all
far in humans and three in S.
reinhardtii
may shed
by
iron
high affinity
Whether it
transition metal ion
as a
iron,
be demonstrated. DMTl also has
Silencing
which is described for most
specificity
identified
is
able to transport
a
uptake experiments
its actual role in the cell. A role
information
affinity
and
well.
as
clarification
displays
to
was
as
uptake system, by measuring
uptake system.
under combined iron and copper limitation would be
(RNAi)
iron
feature described for the alternative iron
sensible if the
more
backup
affinity system,
high affinity system. Thus,
of the yeast strain deficient for
this
as
a
low
a
experiment. DMTl, being
same
towards divalent metal
affinity
broad
and not to
suggested backup
complementation
transport (fet3lfet4), might function
high
the authors referred to
limiting [61]. However,
concerning
and iron
growth
is
insertions
or
deletions.
by overexpression
metal
of each
with the localization
uptake
protein
in yeast,
assays
and
may be detected
experiments. Regarding
families, the characterization procedure could
similar route.
90
References
References
1.
2.
3.
4.
5.
Biology of the prokaryotes, ed. J.W. Lengeler, G. Drews, and H.G. Schlegel. 1999,
Stuttgart: Georg Thieme Verlag.
Altet, L., O. Francino, L. Solano-Gallego, C. Renier, and A. Sanchez, Mapping and
sequencing of the canine NRAMP 1 gene and identification of mutations in
leishmaniasis-susceptible dogs, Infect Immun 70 (2002) 2763-71.
Archibald, F.S. and I. Fridovich, The scavenging of superoxide radical by manganous
complexes: in vitro, Arch Biochem Biophys 214 (1982) 452-63.
Asamizu, E., K. Miura, K. Kucho, Y. Inoue, H. Fukuzawa, K. Ohyama, Y. Nakamura,
and S. Tabata, Generation of expressed sequence tags from low-C02 and high-C02
adapted cells of Chlamydomonas reinhardtii, DNA Res 7 (2000) 305-7.
Asamizu, E., Y. Nakamura, S. Sato, H. Fukuzawa, and S. Tabata, A large scale
structural analysis of cDNAs in a unicellular green alga, Chlamydomonas reinhardtii. I.
Generation of 3433 non-redundant expressed sequence tags, DNA Res 6 (1999) 36973.
6.
Atkins,
P. and L.
Jones, Chemistry
-
Molecules,
Matter and
Change.
3rd ed. 1997,
New York: W. H. Freeman.
7.
8.
9.
Avery, S.V.,
Metal
toxicity
Microbiol 49
(2001)
111-42.
12.
13.
of nramp and irt metal transporter genes in wild type and iron
of tomato, J Biol Chem 278
uptake
(2003)
Beyreuther, K., B. Bieseler, R. Ehring, H.W. Griesser, M. Mieschendahl, B. MüllerHill, and I. Triesch, Investigation of structure and function of lactose permease of
Escherichia coli, Biochem Soc Trans 8 (1980) 675-6.
Bloss, T., S. Clemens, and D.H. Nies, Characterization of the ZATlp zinc transporter
from Arabidopsis thaliana in microbial model
organisms and reconstituted
proteoliposomes, Planta 214 (2002) 783-91.
Böhm, B., H. Boschert, and W. Köster, Conserved amino acids in the N- and Cterminal domains of integral membrane transporter FhuB define sites important for
intra- and intermolecular interactions, Mol Microbiol 20 (1996) 223-32.
Bowen, H., T.E. Biggs, S.T. Baker, E. Phillips, V.H. Perry, D.A. Mann, and C.H.
Barton, c-Myc represses the murine Nrampl promoter, Biochem Soc Trans 30 (2002)
mutants
11.
Appl
Bannerman, R.M., Genetic defects of iron transport, Fed Proc 35 (1976) 2281-5.
Bereczky, Z., H.Y. Wang, V. Schubert, M. Ganal, and P. Bauer, Differential
regulation
10.
in yeasts and the role of oxidative stress, Adv
24697-704.
774-7.
14.
Bowen, H., T.E. Biggs, E. Phillips, S.T. Baker, V.H. Perry, D.A. Mann, and C.H.
Barton, c-Myc represses and Miz-1 activates the murine natural resistance-associated
protein
15.
1
promoter, J Biol Chem 277 (2002) 34997-5006.
Bowen, H., A.S. Lapham, E.S. Phillips, I.Y. Yenug, M. Alter-Koltunoff, B.Z. Levi,
V.H. Perry, D.A. Mann, and C.H. Barton, Characterisation of the murine Nrampl
promoter: Requirements for trans-activation by Miz-1, J Biol Chem (2003).
16.
Cellier,
M.,
A.
Belouchi,
and
P.
Gros,
Resistance
of Nramp, Trends Genet 12
17.
to
intracellular
infections:
comparative genomic analysis
(1996)
Cellier, M., G. Govoni, S. Vidal, T. Kwan, N. Groulx, J. Liu, F. Sanchez, E. Skamene,
E. Schurr, and P. Gros, Human natural resistance-associated macrophage protein:
cDNA cloning, chromosomal mapping, genomic organization, and tissue-specific
expression, J Exp Med 180 (1994) 1741-52.
91
201-4.
References
18.
Cellier, M.,
G.
Nramp defines
Prive,
a
A.
family
Belouchi,
Kwan, V. Rodrigues, W. Chia, and P. Gros,
T.
of membrane
proteins,
Proc Natl Acad Sei U S A 92
(1995)
10089-93.
19.
20.
Cellier, M.F., I. Bergevin, E. Boyer, and E. Richer, Polyphyletic origins of bacterial
Nramp transporters, Trends Genet 17 (2001) 365-70.
Chen, H., GC. Waldbieser, CD. Rice, B. Elibol, W.R. Wolters, and L.A. Hanson,
Isolation
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
and
characterization
of
channel
catfish
natural
resistance
associated
macrophage protein gene, Dev Comp Immunol 26 (2002) 517-31.
Chen, X.Z., J.B. Peng, A. Cohen, H. Nelson, N. Nelson, and M.A. Hediger, Yeast
SMFl mediates H(+)-coupled iron uptake with concomitant uncoupled cation currents,
J Biol Chem 274 (1999) 35089-94.
Cobbett, C, Heavy metals and plants model systems and hyperaccumulators, New
Phytologist 159 (2003) 289-293.
Cohen, A., H. Nelson, and N. Nelson, The family of SMF metal ion transporters in
yeast cells, J Biol Chem 275 (2000) 33388-94.
Conklin, D.S., M.R. Culbertson, and C. Kung, Interactions between gene products
involved in divalent cation transport in Saccharomyces cerevisiae, Mol Gen Genet 244
(1994)303-11.
Conklin, D.S., J.A. McMaster, M.R. Culbertson, and C. Kung, COT1, a gene involved
in cobalt accumulation in Saccharomyces cerevisiae, Mol Cell Biol 12 (1992) 3678-88.
Connolly, EX., N.H. Campbell, N. Grotz, C.L. Prichard, and M.L. Guerinot,
Overexpression of the FR02 Ferric Chelate Reductase Confers Tolerance to Growth
on Low Iron and Uncovers Posttranscriptional Control, Plant Physiol (2003).
Connolly, E.L., J.P. Fett, and M.L. Guerinot, Expression of the IRTl metal transporter
is controlled by metals at the levels of transcript and protein accumulation, Plant Cell
14 (2002) 1347-57.
Courville, P. and M.F.M. Cellier. Study of E. coli MntH transmembrane topology, in
Biometals 2002, 3rd International Biometals Symposium. 2002. London, UK.
Courville, P., R. Chaloupka, F. Veyrier, and M.F. Cellier, Determination of the
transmembrane topology of Escherichia coli Nramp ortholog [Epub ahead of print], J
Biol Chem Ace. Nr. 14607838 (2003).
Curie, C, J.M. Alonso, M. Le Jean, J.R. Ecker, and J.F. Briat, Involvement of
NRAMP 1 from Arabidopsis thaliana in iron transport, Biochem J 347 Pt 3 (2000) 749-
55.
31.
Dent, R.M., M. Han, and K.K. Niyogi, Functional genomics of plant photosynthesis in
the fast lane
32.
using Chlamydomonas reinhardtii, Trends Plant Sei 6 (2001) 364-71.
Dix, D., J. Bridgham, M. Broderius, and D. Eide, Characterization of the FET4 protein
of yeast. Evidence for a direct role in the transport of iron, J Biol Chem 272 (1997)
33.
Dix, D.R.,
11770-7.
J.T.
Bridgham,
M.A.
Broderius,
34.
35.
36.
Byersdorfer, and D.J. Eide, The FET4
protein of Saccharomyces cerevisiae, J
C.A.
gene encodes the low affinity Fe(II) transport
Biol Chem 269 (1994) 26092-9.
Donovan, A., A. Brownlie, M.O. Dorschner, Y. Zhou, S.J. Pratt, B.H. Paw, R.B.
Phillips, C. Thisse, B. Thisse, and L.I. Zon, The zebrafish mutant gene chardonnay
(cdy) encodes divalent metal transporter 1 (DMTl), Blood 100 (2002) 4655-9.
Duffus, J.H., "Heavy metals" A meaningless term? (IUPAC technical report), Pure
and Applied Chemistry 74 (2002) 793-807.
Eckhardt, U. and T.J. Buckhout, Iron assimilation in Chlamydomonas reinhardtii
involves ferric reduction and is similar to Strategy I higher plants, Journal of
Experimental Botany 49 (1998) 1219-1226.
-
92
References
37.
Eide, D.,
M.
Broderius,
J.
Fett, and M.L. Guerinot, A novel iron-regulated metal
transporter from plants identified by functional expression in yeast, Proc Natl Acad
SciUS A93
38.
39.
40.
41.
42.
43.
44.
(1996)5624-8.
Eng, B.H., M.L. Guerinot, D. Eide, and M.H. Saier, Jr., Sequence analyses and
phylogenetic characterization of the ZIP family of metal ion transport proteins, J
MembrBiol 166(1998) 1-7.
Feng, J., Y. Li, M. Hashad, E. Schurr, P. Gros, L.G. Adams, and J.W. Templeton,
Bovine natural resistance associated macrophage protein 1 (Nrampl) gene, Genome
Res 6 (1996) 956-64.
Fisher, M., I. Gokhman, U. Pick, and A. Zamir, A structurally novel transferrin-like
protein accumulates in the plasma membrane of the unicellular green alga Dunaliella
salina grown in high salinities, J Biol Chem 272 (1997) 1565-70.
Fisher, M., A. Zamir, and U. Pick, Iron uptake by the halotolerant alga Dunaliella is
mediated by a plasma membrane transferrin, J Biol Chem 273 (1998) 17553-8.
Fleming, M.D., C.C. Trenor, 3rd, M.A. Su, D. Foernzler, D.R. Beier, W.F. Dietrich,
and N.C. Andrews, Microcytic anaemia mice have a mutation in Nramp2, a candidate
iron transporter gene, Nat Genet 16 (1997) 383-6.
Forbes, J.R. and P. Gros, Iron, manganese, and cobalt transport by Nrampl (Slcllal)
and Nramp2 (Slcl la2) expressed at the plasma membrane, Blood 102 (2003) 1884-92.
Fraüsto da Silva, J. J.R. and R.J.P. Williams, The biological chemistry of the elements.
1991, Oxford: Clarendon Press.
45.
Eide, Functional expression of the human hZIP2 zinc
(2000) 5560-4.
Gaither, L.A. and D.J. Eide, Eukaryotic zinc transporters and their regulation,
Biometals 14(2001)251-70.
Garnham, G.W., G.A. Codd, and G.M. Gadd, Kinetics of Uptake and Intracellular
Location of Cobalt, Manganese and Zinc in the Estuarine Green-Alga Chlorella-Salina,
Applied Microbiology and Biotechnology 37 (1992) 270-276.
Gattiker, A., E. Gasteiger, and A. Bairoch, ScanProsite: a reference implementation of
a PROSITE scanning tool, Applied Bioinformatics 1 (2002) 107-108.
Gitan, R.S. and D.J. Eide, Zinc-regulated ubiquitin conjugation signals endocytosis of
the yeast ZRT1 zinc transporter, Biochem J 346 Pt 2 (2000) 329-36.
Goswami, T., A. Bhattacharjee, P. Babal, S. Searle, E. Moore, M. Li, and J.M.
Blackwell, Natural-resistance-associated macrophage protein 1 is an H+/bivalent
cation antiporter, Biochem J 354 (2001) 511-9.
Grass, G., M.D. Wong, B.P. Rosen, R.L. Smith, and C. Rensing, ZupT is a Zn(II)
uptake system in Escherichia coli, Journal of Bacteriology 184 (2002) 864-866.
Grossman, A.R., E.E. Harris, C. Häuser, P.A. Lefebvre, D. Martinez, D. Rokhsar, J.
Shrager, CD. Silflow, D. Stern, O. Vallon, and Z. Zhang, Chlamydomonas reinhardtii
at the Crossroads of Genomics, Eukaryot Cell 2 (2003) 1137-1150.
Grotz, N., T. Fox, E. Connolly, W. Park, M.L. Guerinot, and D. Eide, Identification of
a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency,
Proc Natl Acad Sei U S A 95 (1998) 7220-4.
Gruenheid, S., F. Canonne-Hergaux, S. Gauthier, D.J. Hackam, S. Grinstein, and P.
Gros, The iron transport protein NRAMP2 is an integral membrane glycoprotein that
colocalizes with transferrin in recycling endosomes, J Exp Med 189 (1999) 831-41.
Gruenheid, S., E. Pinner, M. Desjardins, and P. Gros, Natural resistance to infection
with intracellular pathogens: the Nrampl protein is recruited to the membrane of the
phagosome, J Exp Med 185 (1997) 717-30.
Gaither,
L.A.
and
D.J.
transporter, J Biol Chem 275
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
93
References
56.
57.
58.
Guerinot, M.L., The ZIP family of metal transporters, Biochim Biophys Acta 1465
(2000) 190-8.
Gunshin, H., B. Mackenzie, U.V. Berger, Y. Gunshin, M.F. Romero, W.F. Boron, S.
Nussberger, J.L. Gollan, and M.A. Hediger, Cloning and characterization of a
mammalian proton-coupled metal-ion transporter, Nature 388 (1997) 482-8.
Haring, M.A. and CF. Beck, A promoter trap for Chlamydomonas reinhardtii:
development of a gene cloning method using 5' RACE-based probes, Plant J 11 (1997)
1341-8.
59.
Harris, E.H.,
The
Chlamydomonas
Sourcebook.
1989,
San
Diego,
California:
Academic Press, Inc.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
Heginbotham, L., Z. Lu, T. Abramson, and R. MacKinnon, Mutations in the K+
channel signature sequence, Biophys J 66 (1994) 1061-7.
Herbik, A., C. Boiling, and T.J. Buckhout, The Involvement of a Multicopper Oxidase
in Iron Uptake by the Green Algae Chlamydomonas reinhardtii, Plant Physiol 130
(2002) 2039-48.
Herbik, A., S. Haebel, and T.J. Buckhout, Is a ferroxidase involved in the high-affinity
iron uptake in Chlamydomonas reinhardtii?, Plant and Soil 241 (2002) 1-9.
Hill, K.L., R. Hassett, D. Kosman, and S. Merchant, Regulated copper uptake in
Chlamydomonas reinhardtii in response to copper availability, Plant Physiol 112
(1996) 697-704.
Hirayama, T. and J.M. Alonso, Ethylene captures a metal! Metal ions are involved in
ethylene perception and signal transduction, Plant Cell Physiol 41 (2000) 548-55.
Hirayama, T., J.J. Kieber, N. Hirayama, M. Kogan, P. Guzman, S. Nourizadeh, J.M.
Alonso, W.P. Dailey, A. Dancis, and J.R. Ecker, RESPONSIVE-TO-ANTAGONIST1,
a Menkes/Wilson disease-related copper transporter, is required for ethylene signaling
in Arabidopsis, Cell 97 (1999) 383-93.
Holden, M.J., T.J. Crimmins, Jr., and R.L. Chaney, Cu2+ Reduction by Tomato Root
Plasma Membrane Vesicles, Plant Physiol 108 (1995) 1093-1098.
Hu, S.X., K.W.K. Lau, and M. Wu, Cadmium sequestration in Chlamydomonas
reinhardtii, Plant Science 161 (2001) 987-996.
Jabado, N., A. Jankowski, S. Dougaparsad, V. Picard, S. Grinstein, and P. Gros,
Natural resistance to intracellular infections: natural resistance-associated macrophage
protein 1 (Nrampl) functions as a pH-dependent manganese transporter at the
phagosomal membrane, J Exp Med 192 (2000) 1237-48.
Kamizono, A., M. Nishizawa, Y. Teranishi, K. Murata, and A. Kimura, Identification
of a gene conferring resistance to zinc and cadmium ions in the yeast Saccharomyces
cerevisiae, Mol Gen Genet 219 (1989) 161-7.
Kampfenkel, K., S. Kushnir, E. Babiychuk, D. Inze, and M. Van Montagu, Molecular
characterization of a putative Arabidopsis thaliana copper transporter and its yeast
homologue, J Biol Chem 270 (1995) 28479-86.
Kehres, D.G., A. Janakiraman, J.M. Slauch, and M.E. Maguire, SitABCD is the
alkaline Mn(2+) transporter of Salmonella enterica serovar Typhimurium, J Bacteriol
184(2002)3159-66.
Kehres, D.G., M.L. Zaharik, B.B. Finlay, and M.E. Maguire, The NRAMP proteins of
Salmonella typhimurium and Escherichia coli are selective manganese transporters
involved in the response to reactive oxygen, Mol Microbiol 36 (2000) 1085-100.
Kobayashi, H., S. Odani, and Y. Shiraiwa, A high-C02-inducible, periplasmic
polypeptide in a unicellular green alga Chlamydomonas reinhardtii., Plant Physiology
114(1997)493-493.
94
References
74.
Korshunova, Y.O.,
D.
Eide,
W.G.
Clark,
M.L.
Guerinot, and
H.B.
Pakrasi, The
IRTl
Arabidopsis thaliana is a metal transporter with a broad substrate range,
(1999) 37-44.
Köster, W. and V. Braun, Iron hydroxamate transport of Escherichia coli: nucleotide
sequence of the fhuB gene and identification of the protein, Mol Gen Genet 204 (1986)
protein
from
Plant Mol Biol 40
75.
435-42.
76.
Krogh,
B.
Larsson,
G.
von
Heijne,
and
Sonnhammer,
Predicting
protein topology with a hidden Markov model: application to complete
genomes, J Mol Biol 305 (2001) 567-80.
La Fontaine, S., J.M. Quinn, and S. Merchant, Comparative analysis of copper and
iron metabolism in photosynthetic eukaryotes vs yeast and mammals, in Handbook of
copper pharmakology and toxicology, E.J. Masaro, Editor. 2002, Humana Press Inc.:
A.,
E.L.
transmembrane
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
Totowa,NJ. p. 481-502.
Fontaine, S., J.M. Quinn, S.S. Nakamoto, M.D. Page, V. Gohre, J.L. Moseley, J.
La
Kropat, and S. Merchant, Copper-dependent iron assimilation pathway in the model
photosynthetic eukaryote Chlamydomonas reinhardtii, Eukaryot Cell 1 (2002) 736-57.
Lam-Yuk-Tseung, S., G. Govoni, J. Forbes, and P. Gros, Iron transport by
Nramp2/DMT1 : pH regulation of transport by 2 histidines in transmembrane domain 6,
Blood 101 (2003) 3699-707.
Li, L. and J. Kaplan, Defects in the yeast high affinity iron transport system result in
increased metal sensitivity because of the increased expression of transporters with a
broad transition metal specificity, J Biol Chem 273 (1998) 22181-7.
Liu, X.F. and V.C. Culotta, The requirement for yeast superoxide dismutase is
bypassed through mutations in BSD2, a novel metal homeostasis gene, Mol Cell Biol
14 (1994) 7037-45.
Liu, X.F. and V.C. Culotta, Mutational analysis of Saccharomyces cerevisiae Smflp, a
member of the Nramp family of metal transporters, J Mol Biol 289 (1999) 885-91.
Liu, X.F. and V.C. Culotta, Post-translation control of Nramp metal transport in yeast.
Role of metal ions and the BSD2 gene, J Biol Chem 274 (1999) 4863-8.
Liu, X.F., F. Supek, N. Nelson, and V.C. Culotta, Negative control of heavy metal
uptake by the Saccharomyces cerevisiae BSD2 gene, J Biol Chem 272 (1997) 11763-9.
Locher, K.P., A.T. Lee, and D.C Rees, The E. coli BtuCD structure: a framework for
ABC transporter architecture and mechanism, Science 296 (2002) 1091-8.
Luk, E.E. and V.C. Culotta, Manganese superoxide dismutase in Saccharomyces
cerevisiae acquires its metal co-factor through a pathway involving the Nramp metal
transporter, Smf2p, J Biol Chem 276 (2001) 47556-62.
MacDiarmid, C.W., L.A. Gaither, and D. Eide, Zinc transporters that regulate vacuolar
zinc storage in Saccharomyces cerevisiae, Embo J 19 (2000) 2845-55.
Mackenzie, B. and M.A. Hediger, SLC11 family of H(+)-coupled metal-ion
transporters NRAMP 1 and DMTl, Pflugers Arch (2003).
Manning, D.L., R.A. McClelland, J.M. Knowlden, S. Bryant, J.M. Gee, CD. Green,
J.F. Robertson, R.W. Blarney, R.L. Sutherland, C.J. Ormandy, and et al., Differential
of oestrogen regulated genes in breast cancer, Acta Oncol 34 (1995) 641-6.
McGowan, S.J., H.C. Gorham, and D.A. Hodgson, Light-induced carotenogenesis in
expression
90.
91.
Myxococcus xanthus: DNA sequence analysis of the carR region, Mol Microbiol 10
(1993)713-35.
Moens, L., J. Vanfleteren, Y. Van de Peer, K. Peeters, O. Kapp, J. Czeluzniak, M.
Goodman, M. Blaxter, and S. Vinogradov, Globins in nonvertebrate species: dispersal
by
horizontal gene transfer and evolution of the structure-function
BiolEvol
13(1996)324-33.
95
relationships,
Mol
References
92.
Morris, CA.,
B.
Nicolaus,
characterization of
vesiculosus,
93.
a
V.
Sampson,
J.L.
Harwood, and
recombinant metallothionein
Biochem J 338
(
Pt
2) (1999)
protein
P.
Kille, Identification and
from
a
marine
alga,
Fucus
553-60.
Mourez, M., M. Hofnung, and E. Dassa, Subunit interactions in ABC transporters:
a
conserved sequence in hydrophobic membrane proteins of periplasmic permeases
defines an important site of interaction with the ATPase subunits, Embo J 16 (1997)
3066-77.
94.
95.
96.
97.
98.
99.
100.
101.
102.
Nelson, J.A. and P.A. Lefebvre, Targeted disruption of the NIT8 gene in
Chlamydomonas reinhardtii, Mol Cell Biol 15 (1995) 5762-9.
Nucifora, G., L. Chu, T.K. Misra, and S. Silver, Cadmium resistance from
Staphylococcus aureus plasmid pI258 cadA gene results from a cadmium-efflux
ATPase, Proc Natl Acad Sei U S A 86 (1989) 3544-8.
Ooi, CE., E. Rabinovich, A. Dancis, J.S. Bonifacino, and R.D. Klausner, Copper-
dependent degradation of the Saccharomyces cerevisiae plasma membrane copper
transporter Ctrlp in the apparent absence of endocytosis, Embo J 15 (1996) 3515-23.
Orgad, S., H. Nelson, D. Segal, and N. Nelson, Metal ions suppress the abnormal taste
behavior of the Drosophila mutant malvolio, J Exp Biol 201 ( Pt 1) (1998) 115-20.
Palmiter, R.D., T.B. Cole, and S.D. Findley, ZnT-2, a mammalian protein that confers
resistance to zinc by facilitating vesicular sequestration, Embo J 15 (1996) 1784-91.
Palmiter, R.D. and S.D. Findley, Cloning and functional characterization of a
mammalian zinc transporter that confers resistance to zinc, Embo J 14 (1995) 639-49.
Patzer, S.I. and K. Hantke, Dual repression by Fe(2+)-Fur and Mn(2+)-MntR of the
mntH gene, encoding an NRAMP-like Mn(2+) transporter in Escherichia coli, J
Bacteriol 183 (2001)4806-13.
Pinner, E., S. Gruenheid, M. Raymond, and P. Gros, Functional complementation of
the yeast divalent cation transporter family SMF by NRAMP2, a member of the
mammalian natural resistance-associated macrophage protein family, J Biol Chem 272
(1997)28933-8.
Portnoy, M.E., L.T. Jensen, and V.C. Culotta, The distinct methods by which
manganese and iron regulate the Nramp transporters in yeast, Biochem J 362 (2002)
119-24.
103.
104.
Portnoy, M.E., X.F. Liu, and V.C. Culotta, Saccharomyces cerevisiae expresses three
functionally distinct homologues of the nramp family of metal transporters, Mol Cell
Biol 20 (2000) 7893-902.
Que, Q. and J.D. Helmann, Manganese homeostasis in Bacillus subtilis is regulated by
regulator related to the diphtheria toxin repressor family of
proteins, Mol Microbiol 35 (2000) 1454-68.
Reeve, I., D. Hummel, N. Nelson, J. Voss, and D. Hummell, Overexpression,
purification, and site-directed spin labeling of the Nramp metal transporter from
Mycobacterium leprae, Proc Natl Acad Sei U S A 99 (2002) 8608-13.
Rensing, C, B. Mitra, and B.P. Rosen, The zntA gene of Escherichia coli encodes a
Zn(II)-translocating P-type ATPase, Proc Natl Acad Sei U S A 94 (1997) 14326-31.
Robinson, N.J., CM. Procter, E.L. Connolly, and M.L. Guerinot, A ferric-chelate
reductase for iron uptake from soils, Nature 397 (1999) 694-7.
Rochaix, J.D., M. Goldschmidt-Clermont, and S. Merchant, The molecular biology of
chloroplasts and mitochondria in Chlamydomonas. Advances in photosynthesis, ed.
Govindjee. Vol. 7. 1998: Kluwer Academic Publishers.
Rodrigues, V., P.Y. Cheah, K. Ray, and W. Chia, malvolio, the Drosophila homologue
of mouse NRAMP-1 (Beg), is expressed in macrophages and in the nervous system
and is required for normal taste behaviour, Embo J 14 (1995) 3007-20.
MntR,
105.
106.
107.
108.
109.
a
bifunctional
96
References
D.J.
Eide, and
Guerinot, Altered selectivity
Natl Acad Sei U S A 97 (2000) 12356-60.
110.
Rogers, E.E.,
111.
metal transporter, Proc
Rosakis, A. and W. Köster,
M.L.
Transition
metal
in
an
Arabidopsis
transport in the green microalga
Chlamydomonas reinhardtii Genomic sequence analysis, Research in Microbiology
(in press).
Rubinelli, P., S. Siripornadulsil, F. Gao-Rubinelli, and R.T. Sayre, Cadmium- and
iron-stress-inducible gene expression in the green alga Chlamydomonas reinhardtii:
evidence for H43 protein function in iron assimilation, Planta 215 (2002) 1-13.
Sacher, A., A. Cohen, and N. Nelson, Properties of the mammalian and yeast metalion transporters DCT1 and Smflp expressed in Xenopus laevis oocytes, J Exp Biol
204(2001) 1053-61.
Saier, M.H., Jr., A functional-phylogenetic classification system for transmembrane
solute transporters, Microbiol Mol Biol Rev 64 (2000) 354-411.
Sambrook, J., E.F. Fritsch, and T. Maniatis, Molecular Cloning : A Laboratory Manual.
1989: Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory.
Sancenon, V., S. Puig, H. Mira, D.J. Thiele, and L. Penarrubia, Identification of a
copper transporter family in Arabidopsis thaliana, Plant Mol Biol 51 (2003) 577-87.
Sasaki, T., N. Kurano, and S. Miyachi, Cloning and characterization of high-C02specific cDNAs from a marine microalga, Chlorococcum littorale, and effect of C02
concentration and iron deficiency on the gene expression, Plant Cell Physiol 39 (1998)
-
112.
113.
114.
115.
116.
117.
131-8.
118.
Muller-Moule, Y. Munekage, K.K. Niyogi, and M. Pilon, PAAl, a PArabidopsis, functions in copper transport in chloroplasts, Plant Cell
15(2003)1333-46.
Solioz, M. and C. Vulpe, CPx-type ATPases: a class of P-type ATPases that pump
heavy metals, Trends Biochem Sei 21 (1996) 237-41.
Sunda, W.G. and S.A. Huntsman, Interactions among Cu2+, Zn2+, and Mn2+ in
Shikanai, T.,
P.
type ATPase of
119.
120.
121.
122.
123.
124.
controlling cellular Mn, Zn, and growth rate in the coastal alga Chlamydomonas,
Limnology and Oceanography 43 (1998) 1055-1064.
Supek, F., L. Supekova, H. Nelson, and N. Nelson, A yeast manganese transporter
related to the macrophage protein involved in conferring resistance to mycobacteria,
Proc Natl Acad Sei U S A 93 (1996) 5105-10.
Tabata, K., S. Kashiwagi, H. Mori, C. Ueguchi, and T. Mizuno, Cloning of a cDNA
encoding a putative metal-transporting P-type ATPase from Arabidopsis thaliana,
Biochim Biophys Acta 1326 (1997) 1-6.
Tabuchi, M., T. Yoshida, K. Takegawa, and F. Kishi, Functional analysis of the
human NRAMP family expressed in fission yeast, Biochem J 344 Pt 1 (1999) 211-9.
Tabuchi, M., T. Yoshimori, K. Yamaguchi, T. Yoshida, and F. Kishi, Human
NRAMP2/DMT1, which mediates iron transport across endosomal membranes, is
localized to late endosomes and lysosomes in HEp-2 cells, J Biol Chem 275 (2000)
22220-8.
125.
126.
127.
Takahashi, M., Y. Terada, I. Nakai, H. Nakanishi, E. Yoshimura, S. Mori, and N.K.
Nishizawa, Role of nicotianamine in the intracellular delivery of metals and plant
reproductive development, Plant Cell 15 (2003) 1263-80.
Thomine, S., F. Lelievre, E. Debarbieux, J.L Schroeder, and H. Barbier-Brygoo,
AtNRAMP3, a multispecific vacuolar metal transporter involved in plant responses to
iron deficiency, Plant J 34 (2003) 685-95.
Thomine, S., R. Wang, J.M. Ward, N.M. Crawford, and J.I. Schroeder, Cadmium and
by members of a plant metal transporter family in Arabidopsis
Nramp genes, Proc Natl Acad Sei U S A 97 (2000) 4991-6.
iron transport
homology
to
97
with
References
128.
Tottey, S., P.R. Rich, S.A. Rondet, and N.J. Robinson, Two Menkes-type atpases
supply copper for photosynthesis in Synechocystis PCC 6803, J Biol Chem 276 (2001)
19999-20004.
129.
130.
131.
der
Zaal, B.J., L.W. Neuteboom, J.E. Pinas, A.N. Chardonnens, H. Schat, J.A.
Verkleij, and P.J. Hooykaas, Overexpression of a novel Arabidopsis gene related to
putative zinc-transporter genes from animals can lead to enhanced zinc resistance and
accumulation, Plant Physiol 119 (1999) 1047-55.
Van Ho, A., D.M. Ward, and J. Kaplan, Transition metal transport in yeast, Annual
Review of Microbiology 56 (2002) 237-261.
Vidal, S.M., D. Malo, K. Vogan, E. Skamene, and P. Gros, Natural resistance to
infection with intracellular parasites: isolation of a candidate for Beg, Cell 73 (1993)
van
469-85.
132.
Lepage, S. Gauthier, and P. Gros, Natural resistance to
Nrampl encodes a membrane phosphoglycoprotein absent in
macrophages from susceptible (Nrampl D169) mouse strains, J Immunol 157 (1996)
Vidal, S.M.,
E.
Pinner,
P.
intracellular infections:
3559-68.
133.
Welch, R.M., W.A. Norvell, S.C Schaefer, J.E. Shaff, and L.V. Kochian, Induction of
Iron(Iii) and Copper(Ii) Reduction in Pea (Pi sum-Sativum L) Roots by Fe and Cu
Status
Does the Root-Cell Plasmalemma Fe(Iii)-Chelate Reductase Perform a
General Role in Regulating Cation Uptake, Planta 190 (1993) 555-561.
Wilde, E.W. and J.R. Benemann, Bioremoval of heavy metals by the use of
microalgae, Biotech Adv 11 (1993) 781-812.
Williams, L.E., J.K. Pittman, and J.L. Hall, Emerging mechanisms for heavy metal
transport in plants, Biochim Biophys Acta 1465 (2000) 104-26.
Wood, M.W., H.M. VanDongen, and A.M. VanDongen, Structural conservation of ion
conduction pathways in K channels and glutamate receptors, Proc Natl Acad Sei U S
A 92 (1995) 4882-6.
Yuan, D.S., A. Dancis, and R.D. Klausner, Restriction of copper export in
Saccharomyces cerevisiae to a late Golgi or post-Golgi compartment in the secretory
pathway, J Biol Chem 272 (1997) 25787-93.
Zhao, H. and D. Eide, The yeast ZRTl gene encodes the zinc transporter protein of a
high-affinity uptake system induced by zinc limitation, Proc Natl Acad Sei U S A 93
(1996)2454-8.
Zhao, H. and D. Eide, The ZRT2 gene encodes the low affinity zinc transporter in
Saccharomyces cerevisiae, J Biol Chem 271 (1996) 23203-10.
Zwilling, B.S., D.E. Kuhn, L. Wikoff, D. Brown, and W. Lafuse, Role of iron in
Nrampl-mediated inhibition of mycobacterial growth, Infect Immun 67 (1999) 1386-
134.
135.
136.
137.
138.
139.
140.
92.
98
Danksagung
Mein
besonderer
Dank
gilt Wolfgang Köster,
für
durchgeführt wurde,
sein
fortwährendes
Hilfsbereitschaft sowie den dabei
Ein
weiteres
Möglichkeit
Interesse
an
grosses
hier
am
gewährten
Dankeschön
Institut
zu
an
unter
Interesse
dessen
an
meiner
Arbeit,
seine
Arbeit
stete
Freiraum.
Prof.
promovieren,
Dr.
Alexander
Zehnder
für
die
gegebene
sowie für die Diskussionsbereitschaft und das
meiner Arbeit.
Prof. Dr. Thomas Buckhout danke ich für seine Bereitschaft die
zu
diese
Betreuung
Begutachtung
meiner Arbeit
übernehmen.
Besonders möchte ich Annette danken für die wertvolle
Atmosphäre
Kollegialität,
die sehr
angenehme
im Labor und die immerwährende Hilfsbereitschaft.
Ganz herzlichen Dank
an
alle, die diese Doktorarbeit ermöglicht und begleitet haben.
99
CURRICULUM VITAE
Date of birth:
23.8.1976
Place of birth:
Athens, Greece
Nationality:
German
1982-1988
Greek-German
1988-1994
German
1994-1999
Studies in
2000-2004
Doctoral studies at the
primary school, Athens,
Gymnasium, Athens,
Biology
Science and
at the
Greece
Greece
University
of Konstanz,
Diploma
Biology
Swiss Federal Institute for Environmental
Technology (EAWAG/ETH), Dübendorf,
100
in
Switzerland