, '4
Functional Organization of the
Chloroplast in the Diatom Phaeodactylum tricornutum.
Andrew M. Pyszniak
Department of Biology
l'icGill Unlversity, Montreal
Novernber 1990
A thesis submitted to the Faculty of Graduate Studies and
Research in partial fulfillment of the requirements for
the degree of Master of Science.
(c)
Andrew M. Pyszniak,
1990
DEDICATED
TO
MOM
AND
i
DAD
------------------------------------------------------------------------------~.~----.
1
Abstract
The technique of protein A-gold imrlllnoc lect '-(,Il !11 i (' l"Cl:"
'llpy
was used ta determine the distributions of t\Vo photn:;yntlwt il'
complexes
in
the
"Chylakoid
Phaeodactylum tricornutum.
light-harvesting
photo system II,
complex,
membranes
of
more
suggest that
believed
was found to
concentrated
in
diatoms,
to
be
ùssociùtcd
be equally distributed
in
the
the
two
latter.
Thef;(\
photosystcnuj
algae.
Furtherrnore,
it
was
expectcd
postulated
to
of
the
be
CqUd
0
f
periplastidal
involved
in
the [ueu>: ilt1th i 11-
reticulum,
protein
prc!;ollt
wh i ch
transport
these proteins were absent from the pedplastidi11
ii
l',
1 Il
chloroplasts. Immunolabell ing resul ts, however, i nù jc"t.ed
J
1 1Y
cont'-i1~;t
thilt
chlorophyll g/Q light-harvesting complex would be
vesicles
th!" i ,-
observed in highcr pli1nh; i111d
trico:;:-nutum, the nuclear-coded proteins
the
,111l0nq
n":,11 1 t:;
ilnd
distributed on the two types of membranes, in morkcc1
to the lateral heterogeneity
\Vith
rhotosy~;t0111 1 V:d:;
associated li9ht-harvesting complexes are essenti a 11y
green
,_'-/1'
The fucoxanthin-chlorophy 11
appressed and unappressed membranes, whereas
slightly
di,1tn!11
U1C'
i Il
vJO t'p
i nto
t.l1d
v('~~i('I(";.
t
Résumé
La technique de microscopie electronique utilisant la
protéine A couplée a l ' or colloldal a ete util isee dans le but
de
deterP1iner
localisation
la
photo synthetiques dans
diatornee
de
complexes
deu>:
les membranes des thylakoldes
Phaeodactylum tricornutum.
de la
Le complexe collecteur
d'énergie composé de fucoxanthine et de chlorophylle
~
et ç
semble associé au photosysterne I I et est distribue de façon
egaIe
parmi les membranes des thylakoldes,
j uxtaposees
ou
non.
Le
photosystème
l,
qu'elles
quant
a
soient
lui 1
se
retrouve pluo fréquemment dans les membranes non juxtaposees.
Ces
résultats
suggèrent <::t.e
dans
les diatomees,
les
cleux
photosystèmes ainsi que leurs complexes collecteurs de lumiere
associes sont essentiellement distribues de la même façon sur
les deux
types de membrane,
ce qui
est tres di fferent
de
l' hetérogénei te laterale observée chez les plantes super ieures
et les algues vertes. De plus, il a éte postule que dans
E.
tricornutum, les protéines nucleaires du complexe collecteur
de lumiere compose de fucoxanthine et de
seraient
presentes
dans
les
chlorophyl~e
vésicules
du
g et ç
reticulum
periplastidal, lesquels semblent impliqués dans le transport
de protéines dans les chloroplastes. Toutefois, des resultats
de
marquages
iwmunologiques
indiquent
que
ces
étaient absentes des vésicules périplastidales.
iii
proteines
Preface
This thesis is divided into three
chapt('r~;.
Ch,lptC'l- 1
a general review of the literature descrlbing the lm',,1 i
of photosynthetic
complexes
in the
thylako i cl
,',ü
1110111bt',1 tH":;
i:;
ion
()
t
higher plants and various groups of algae.
Chapter
II
contains
"Immunocytochemical
a
manuscript
Lot::alization of
ont. 1 t loli
Photosystem
th ..
,1Ild
Fucoxanthin-Chlorophyll .ê./Q Light-harvestinrJ Comp 1 ox
1n
t Ill'
oiatom Pha80dactylum tricornutum ", which ha;, hoc'n :;llhm i tt'('d
for publication to the journal Protopl :'lsmêl.
this
manuscript
was
a
collaborative
Pn'p,ll,,,t 1 (l11
effort
lJC't\JN'n
() t
tll('
candidate and Dr. SarGh P. Gibbs.
Chapter
III
describes
the
resul ts
of
é1
pn' 1 i m i 11<1 t"y
project on protein transport into chloroplasts.
English
and
French
abstracts,
as
well
il;'
en ne ltlr! i nq
remarks, are also included in the thesis.
The references ci ted
in
the three chélpte r~;
h<lVI'
)J(>('11
cOTl'bined into one bibl iography, and placed at the ('nd () f the'
thesis.
iv
contributions to original Knowledge
1. This study is the first to determine whether photosystem l
and light-harvesting proteins associated with photosystem II
are localized on the appressed or the non-appressed thylakoid
membran~s
in a diatom, or, in fact,
in any chrornophyte alga
lacking phycobiliproteins.
2. Electron microscopie immunocytochemistry was used to show
that the fucoxanthin-chlorophyll Q/ç light-harvesting complex
is I::!qually distributed on the
non-appressed and appressed
thylakoid membranes of the diatorn Phaeodactylum tricornutum.
3. Electron microscopie immunocytochemistry was used to show
that photosystem l
membranes,
is
located
on both types
of thylakoid
but is slightly more concentrated on the outer,
non-appressed membrane.
4. The DAB reaction was used to show cytochemically that the
two outer thylakoids of each band contain more Photosystem l
activity than does the central thylakoid of the band.
v
Ac~nowledgements
l
wish to express my thanks
helpful
and
patient
investigation,
to Dr.
guidance
in
for financial support,
S.P.
C;lbh:;
the
ideal
env ironment
in
th
l:;
<lncl tor pr()\, id i I1q t 111'
opportuni t ies to attend scienti f ic con feronce~; creating
l~; 1 nq
superv
hl'I
IDI'
wh i ch
1n
ta
';11l11 t,
1 () 1
1(11)
111 Y
<1(,\/1'
scientific skills.
Many thanks go to Mike McKay for numerolls i 1 l!11ll i n .. t i
discussions, and for his patience and effort in
nq ll1"
tC'I'ldll
Il( 1
t
(l
"get my hands wet" in the labo
l
would like to thank my parents élncl
~;i!;to!-,
Ok'.dn .. , 1 (l!
their constant encouragement and support clurinq tlH' \"!-It' Inq
(li
this thesis.
I11Y
My Most heart-felt thanks :1re rcsc'r-\'()d
lo!'
fiancée, Cindy, whose unfailing love, patience, lInd0r!;tdndll)rJ,
moral support, and encouragement, throuqhout th .. !;p Y"d l".,
d Il' 1
dur ing the preparation of this thes is, has m,HIC' my t d·.1
1 () t
,j
easier.
Finally,
l
would like to express my qrflt i
Lussier for the French translation of the
Robert
Lamarche
and
Guy
L'Heureux
assistance.
vi
for
t\llir'
Ab~;trdl't
the i r
trl
1
r~d n'
rlnrl
tfJ
tpr'hn i ('" 1
Table of Contents
ii
Abstract
iii
Resumé
iv
Preface
contributions to original Knowledge
v
vi
Acknowledgements
CHAPTER 1:
1. Introduction
2.
1
Higher Plants and Green Algae
2.1 Photosystem II
3
2.2 Photosystem l
8
2.3 Light-Harvesting Complexes
11
2.4 CFa-CF, ATP Synthase
16
2.5 Cytochrome b 6/f Complex
18
3 • Chlorophyll ~-containing Algae -
Chromophytes
21
CHAPTER II:
1. Introduction
25
2. Materials and Methods
2.1 Cell Culture
28
2.2 Antisera
28
2.3 protein A-gold Preparation
28
2.4 Electron Microscopy
29
2.5 Quantitation of Gold Label
31
2.6 Cytochemical staini.ng For PS l
33
vii
3. Resul ts
4. Discussion
1.'
CHAPTER IIi:
1. Introduction
2.
~aterials
1H
and Methods
2.1 Cell Culture
'1 \
2.2 Fixation and Embedding
"
\
2.3 Immunolabelling
3. Results and Discussion
conclusions
Literature Cited
•
viii
t.',
,
l
CHAPTER
I
1. Introduction
In
photosynthetic
eukaryotes
1
the
componcnh-;
() t
apparatus responsible for catalyzing the l iCJht t'cIlet
t hl'
ll)n~;
photosynthesis (i. e. the conversion of sGlar enerqy i nto
(1
t
NI'\,
and NADPH) are located in thylakoid membranes of ch 1Ol'Op 1d:;t :',.
These
components
are
comprised
of
vad ous
complexes including photosystems land IJ
photochemical reaction centres),
SUplïl1110
(PS 1
l\~;
1
1l't'Ill d
l'
1 l , t 111'
chlorophy ll-pt'ote i Il 1 i qM-
harvesting complexes (LHC-II and LHC-I, associatcd w i th
l'r;
11
and PS l respectively), a cytochrome b 6 /f complex involv0d
ln
electron transport,
for
convert.ing
and an ATP synthase complcx
the
prot.t,n
gradient
photosynthetic electron transr ort into ATP
i Il: 0
rc:;pon~.
farmcd
~;ynthc~;
dlll'
i nq
i ~;.
In higher plants and qrenn algae, the thy la}:o i d
!1Wllllll" Ilf':.
containing these components are made up of two type:;, CJ rd n. 1
and stroma thylakoids, both
0
t which contr ibute ta
ê1
camp 1C'>' ,
physically continuous membrane system. stroma thyléü:oid:;
by def ini tion,
exposed
to
the
unstacked
and
chloroplast
their
externt:1l
stroma.
Grana
thylakoid membranes adhering at their outer
adhesion
is
probably
mediated
through
,'l'C',
:;11 r f ,)('('!.
il
re
~;ll
LHC-I
oIt-(·
t c) !l'lI ri
r f d<
r
(>!..
'l'h
hy
1 !.
comp 1('/('~:
(Bennett 1983) and resul ts in stacking of membrane;-;.
'i'I\('!~('
regions are interconnected within the chlorapld"t by !:tromd
thylakoids.
A
considerable
body
of
1
ev idence
has
fJeC'n
d 11"~.' J" j
indicat~ng
that
a
selective
distribution
(or
lateral
heterogeneity) of the various thylakoid membrane complexes
exisLs between appressed and unappressed membranes of higher
plants and various groups of algae. This evidence has been
extensively documented in several recent reviews
(Park and
Sane 1971; Anderson and Andersson 1982; Staehelin and Arntzen
1983; Staehe1in 1986; simpson and von Wettstein 1989).
The idea of a non-random distribution of photosynthetic
components within thylakoid membranes originated from early
work designed to characterize the thylakoids of higher plants.
Numerous investigators using a variety of techniques including
fractionation, freeze-etch electron microscopy, heavy-metal
shadowing, negative staining, x-ray diffraction and antibody
studies, have shown that structural differences, in terp.s of
membrane constituents, exist along the length of thylakoid
membranes (see Park and Sane 1971 for review). This structural
non-uniformity
of
thylakoids
was
extended
differences as well, as shown by Sane et al.
workers
were
able
to
mechanically
to
functional
(1970). These
separate
spinach
chloroplasts into stroma and grana fractions and test for PS
land PS II activities in these fractions. Thus, PS l activity
alone was assigned to stroma lamellae and both PS land PS II
activities to grana lamellae (with PS II being slightly more
concentrated than PS l in grana). These results led to the
paradigm that the two photosystems were located in close
physical association in grana, thereby allowing for linear
2
electron transport between PS II and PS 1 to proceed. The
results were also in accordance with the available data from
various other types of experimental work such as cytochemical
evidence, photochemical experiments in greening chloroplasts
and studies involving mutants (see Park and Sane 1971). In the
ensuing years,
a
plethora
of
evidence
has
emerged
which
suggests that this view is no longer tenable, and indicates
beyond
doubt
that
a
true
functional
and
structural
heterogeneity exists along thylakoid membranes. Furthermare,
this heterogeneity involves not only PS 1 and PS II, but aIl
the other components of photosynthesis as weIl. The fallawing
is a synopsis of the various experimental approaches used that
have
led
to
the
current
understanding
of
supramolecular
thylakoid organjzation.
2. Higher Plants and Green Algae
2.1 Photosystem II
Of aIl the photosynthetic thylakoid membrane complexes,
localization of photosystem II probably presented the least
ambiguity. The majority of the evidence indicates that PS II
is located predominantly in the appressed membranes af grana
stacks, with approximately 15% present in stroma thylakoid
membranes. In 1976, Akerlund et al. used the novel technique
of aqueous polymer two-phase partition ta separate membrane
vesicles
(derived
destabilized
from
grana)
Y'eda press treatment of
into
three
3
fractions
low saI t-
according
ta
differences in the surface properties of these vesicles. Their
results, along with those of Svensson and Albertsson (1989),
indicated
that
a
distinct
sub-popu1ation
of
vesic1es,
originating from the appressed membrane (or partition) regions
of grana, were highly enriched in PS II and depleted in PS 1.
This suggested that native grana have membrane regions of
mainly PS I I character (Akerlund et al. 1976). Thus, this was
one of the first indications that the notion of PS I I and PS
l
being
intimate1y associated in grana may not have been
accurate.
Andersson and Anderson (1980), using the same technique,
confirrned
these
results
Ly
analyzing
fractions
for
the
composition of chlorophyl1-protein complexes. They conc1uded
that. PS I I was highly concentrated in partition regions (80%)
with a little in stroma membranes (20%), and that the observed
PS l
activity in grana
(Sane et al. 1970) was due to PS l
located in the end-membranes and margins of grana.
These biochemical results were strengthenod by various
cytochemical
studies.
demonstrated
by
the
Photosystem
activity
II
photoreduction
of
one
of
can
be
several
tetrazolium salts to insoluble formazan deposits (see Vaughn
1987) which are readi1y visible under the e1ectron microscope.
Using photoreduction of thiocarbamyl nitroblue tetrazolium
(TCNBT),
Wrischer
(1988)
showed
that
PS
II
activity
was
present main1y in appressed membranes (partition regions) of
the grana in greening bean chloroplasts. The deposi tion of
(
4
formazan correlated wi th the formation of granal ini tials \\Ihon
etiolated
chloroplasts
were
placed
into
the
light,
and
increased with increasing illumination time during greening.
Scattered
reaction product
was also observed
over
stroma
thylakoids. These results directly confirm other cytochcmical
studies in Coleus blumei (Marty 1977), barley (Vallghn ct ïl1 .
1983),
and
maize
(Wrischer
ferricyanide reduction
yielded
discordant
distribution
membranes
of
PS
a~
(Hall et al.
other
studies,
lIsing
a measure of PS II activity, have
results
II
1989).
by
exists
1971;
indicating
between
that
grana
an
and
o(juill
strom,)
Nil' and Pease 1973). lIowcvor,
such results are dubious since ferricyanide can be reduced by
PS l
as weIl as PS II (Izawa 1980).
Ad~itional
of
thylak~")id
data for PS II localization come from studics
membrane complexes as revealed by freeze- fracture
and freeze-et.ch el p:.;tron
microsco~,y
(Staehel in 1986).
results stem from the fact that distinct classes of
rl'h~sc
pé1rticlc~
exist on the various surface and fracture faces of thylakoid
membranes. The non-uniform distribution of these particles ca n
then form the basis on which ta distinguish appresscd (rom
unappressed membranes. In higher plants and green algae,
represents the only complex that frac"':ures with the
or
...
E-face 1
(EF
particles)
of
freeze-fractured
P~i
l T
cxop1;'1~;mi
c
thyla~:oitb~
1In freeze-fracture nomenclature, E-face correspondf; to
the face of the unit membrane leaflet closest ta the lumen of
the thylakoid after the intact unit membré!.ne is fractured. 'l'he
outer membrane leaflet exposed by the fracture is referred ta
as the protoplasrnic or P-fàce •
5
(
(staehelin 1986). That the EF particles are equivalent ta PS
II complexes was shown by a number of correlative studies
(staehelin 1986). For example, mutants specifically deficient
in PS II activity characteristically have reduced levels of EF
particles in stacked membrane regions (EF s particles) (simpson
et al. 1988, 1989). Experimentally-induced
thylakoids
particles
results
wi th
in
the
(Staehelin 1976).
the
other
random
classes
intermixing
of
This topography is
intermediate light-grown plants
unstac)~ing
of
membrane
of grana
the
EFs
particles
identical ta that of
(Armond et al.
1977). Upon
restacking, induced by incubation in solution containing monoor divalent ions, or during greening in continuous light, the
EFs particles resegregate into their native patterns which are
indistinguishable from untreated specimens
Armond et al.
PS
II
1977). Furthermore, reconstitution of isolated
complexes
particles
(Staehelin 1976;
into
corresponding
(Sprague et al.
liposomes
in size
to
yields
those
freeze-fracture
of PS
II
cores
1985). Therefore, the finding by Staehelin
(1976) that the distribution of EF particles in spinach (8085%
in
grana
membranes
and
15-20%
in stroma
thylakoids)
corresponds to the biochemical results on PS II activities
described above lent strong support for the notion that PS II
is predominantly localized in grana.
Immunocytochemical work has also proved to be of value in
localizing PS II and other thylakoid membrane components. This
method is probably one of the most direct in local izing a
c·
6
prote in in the membrane. It is based on the use of a co 1] 0 id,ll
gold label that has been conjugated (usually indirectly) to
lln
antibody directed against the protein in question (sec koth
1982). Antibodies raised against polypeptides 01
(herbicide
binding rE'ceptor) and 02, both of which are assor.iated wi th
PS
membrHnc~
in
II, labelled mostly (approximately 90%) stacked
spinach and the green alga Chlamydomonr.ts (Vallon et al. 198(,).
Immunoblot analysis of Spirodela 01 igorrhiza y ielded Sil1111él r
results (86-88% in grana membranes, Callahan et al. 1989), nnd
also showed that the high potential form of cytochromc
associated
largely
1989).
with
active
associated
This
PS
with
result was
II
reaction
grana
centres,
membranes
Wil~,
a IF;{)
(Callahan
support ive of previous
bs~'
ct
il
1.
biochcl11ic,ll
studies (Cox and Andersson 1981; Anderson 1982). In addition,
polypeptides of the oxygen evol v ing system of photosynthcs if;,
which has been correlated with the presence of EF.,
(and,
by extension, with PS II)
(Seibert et al.
partjclC'~;
1987)
h<1V0
been irnmunolocalized predominantly in grana rerJ ions (Goodeh i 1d
et al. 1985b; Vallon et al. 1985), along wi th the 43 and
PS II proteins of Spirodela
together,
these
results
(Callahan et. al.
again
support
~)
1 ~:()
1989). Ti1kcn
the
prcdominllnt
localization ot PS II in the grana.
Evidence has
accumulated
to support
the existence of
heterogenei ty wi thin the PS II population i tsel f. l t hcH; bren
shawn (Armand and Arntzen 1977) that the EF partiel es of qrdni1
regians
are,
an
average,
larger
7
than
those
of
stroma
membranes.
Moreover,
PS II activity in grana was saturated
with moderate light intensities, while that of stroma lamellae
did not saturate. These results had been attributed to PS II
centres containing variable amounts of bound light-harvesting
proteins (Armond and Arntzen 1977) . However, other differences
between
granal
documented.
and
stromal
Photo system
II
PS
II
from
have
been
subsequently
non-appressed
regions
was
shown to be less sensitive to photoinhibition than that from
appressed membranes (Maenpaa et al. 1987). Kinetic analysis of
PS
II
photoactivity
revealed
a
fast
anà
nonexponential
component (termed PS lIa) and a slower exponential component
(PS II(\)
(Melis and Hamann 1978). The PS II of non-appressed
membranes has been shown to have fluorescence properties of PS
lIB (Anderson and Melis 1983), as did PS II of mature maize
bundle sheath chloroplasts, which lack grana stacks (Ghirardi
and
Melis
1983).
Thus,
it
would
appear
that
the
large
proportion of grana-resident PS II is PS lIa' while that in
the stroma lamellae is PS lIB (Anderson and Melis 1983).
2.2 Photosystem l
The localization of PS l
has been the subject of sorne
controversy. To date, it is still not entirely clear where in
the
thylakoid
membrane
this
complex
is
located,
but
the
available evidence suggests that it is present predominantly,
if not exclusively, in the unappressed membranes.
Early
fractionation
studies
8
suggested
that
PS
l
was
r
present in both grana and stroma thylakoids (Sd ne ct
..
,~I.
Il) / () ;
Park and Sane 1971). As mentioned previously, the pn'v,1 il i nq
paradigm at the time was that PS 1 and PS Il
h~ct
ta bc
in
~llaw
close physical association with each other in arder ta
for linear electron transport between the two photosystems to
proceed.
However,
two-phase
with the introduction of aqueaus polymor
parti tioning
to
separate
di f ferent
thy l ,1 ka i li
membrane fractions came the realization that PS 1 was scvcrely
depleted in grana partition regions as compared to jsolîlted
grana stacks (Akerlund et al. 1976). In a complementary study,
Andersson and Anderson (1980) compared the relative content 01
chlorophyll-protein complexes
in these fractj ons,
Il
nd
cl
1:;0
found that most PS 1 was present in stroma-expos0(\ m0mhrdIW:; 1
rather than in appressed regions. Furthermore,
me~Hur0ment
P700
by
(found
only
spectrophotometry
in
PS
yielded
1)
concentration
similar
resul ts
djfforonC0
(Anderson
19B2;
Anderson and Melis 1983). Consistent in aIl these stud j es
the presence of a residual am ou nt (20%)
of PS 1
with vesicles derived from qrana partitions.
attributed
to
contamination
of
this
W<l:;
as~,ociîlt('d
'l'hjs P:-;
fract ion
01
by
1
\thl:;
:;tromd
thylakoids since the coupling factor of ATP synthase, whjch
likely restricted to unappressed membranes (see below)
1
j~
WFl:;
also found in this fraction in similar amounts (Anden,on
.o~r;d
Melis 1983). On the basis of these results, the proponi1\
WFl!,
made that PS 1 was entirely excluded from appresscd mcmbr'l/lf'
reg ions in vivo (Andersson and Anderson 1980; Amle rr;on ] 'JH ] ) •
9
-~
1
l
Other investigations have supported the total exclusion
of
PS
l
from
micrographs,
appressed
PS
l
membranes.
complexes
have
ln
been
freeze-fracture
correlated
with
protoplasmic face (PF) particles mainly through the Rnalysis
organis~s
of PS l mutants deficient in PS l activity. These
characteristically have reduced levels of a class of 10-13 nm
particles in the unstacked membrane regions
(PFu particles)
when compared to wild type plants, while no differences are
seen
between
granal
membranes
of
mutant
thylakoids (Miller 1980; Olive et al.
1988).
l
~urther
complexes
isolated
PS
wild
type
1983; simpson et al.
evidence that these particles correspond to PS
is
l
and
given by
reconstitution
experiments
where
complexes were inserted into 1 iposomes.
The
resulting particles are of a size consistent with that of PFu
particles in intact thylakoids (Mullet et al.
the se
results
suggest
that
PS
l
resides
1980). Thus,
in
unappressed
membranes.
More
direct
evidence
for
this
proposaI
cornes
from
immunolabelling experiments on isolated thylakoids. Antibody
directed against the CP l
apoprotein of the PS l
reaction
center of spinach and Chlamydomonas labelled unstacked regions
much more heavily than stacked areas (Vallon et al. 1986). In
addition, the low amount of label found over stacked areas was
considered insignificant since a mutant lacking CP l exhibited
similar levels of labelling over these regions. These results
were
interpreted as meaning total
10
exclusion of
PS
l
from
appressed regions (Vallon et al.
1986).
l'~~
Despite this evidence favoring exclusion of
partition
regions,
other
investigations
t
1
1"01'1
havc
conf1icting data. Cytochemica1 studies designed to dctonn i
PS
location
l
through
diaminobenzidine
(DAB)
the
or
photooxidation
(Vaughn
1987)
clenrly
11C'
J, J ,-
rs
show
activity to be present in both stroma and grana mCmbrilI10!-;
spinach
greening
(Nir and Pease 1973),
bean
leaf
Co1eus blumei
chloroplasts
(M'"\I-ty
(\'Jrischer
1n
1'1//),
h.1 r 1 l'y
1(78),
(Vaughn et al. 1983), and maize (Wrischer 1989). flowcvol", t Ill'
DAB reaction product appears more dense in the stromél-c>:po!;od
membranes of grana than in appressed membranes (Wrischcr l ')
1989; Cal1ahan et al. 1989), suggesting an enr ichmcnt
in
the
former.
(Vaughn et al.
Furthermore,
1983)
immunocytochem i ca 1
0
('V
n~,
fI':;
1
i dene'('
a1so argues in favor of PS T b0 i nq
1n
both types of membrane, and Staehelin (1986) has obf;crvod th<lt
freeze-fracture particles representing PS l
cores mily onter
grana partitions.
In any event, whether or not future stud ies
l
to be definitively absent from appressed
existing
data
clearly
localization of PS l
support
at
VI
i 1 1 r;how l'!;
thy]()J.:oid~;,
least
a
thr'
pre'dom in" nt
in unappressed membranes.
2.3 Light-Harvesting Complexes
As may be deduced from their name, the primary function
of
light-harvesting
complexes
Il
(LHC)
is to
absorb
j
ne 1 d,'nt
light,
and transfer the resulting excitation energy to the
photosystems where i t
photosynthesis.
various
The
pigment
carotenoids,
can be used to dr ive the process of
absorption of
molecules
etc.)
(e.g.
that are
light
is
facilitateà
~
chlorophylls
and
by
Q,
non-covalently complexed with
proteins to form chlorophyll-pretein (CP) complexes. There are
two maj or
populations of
chlorophyll .9./Q l ight-harvesting
complex in higher plants, LHC-I and LHC-II, which function in
concert with PS land PS II respectively (Thernber 1986). In
light of the previous discussion on the location of the two
photosystems,
it
follows
that
LHC-I
should
be
located
predominantly in unappressed membranes, and LHC-II and ether
PS II associated CP's in the appressed membranes.
The majority of the evidence indicating that this is, in
fact,
the case cornes from immunological studies.
analysis using monoclonal
Immunoblot
antibodies against two different
denatured LHC-II apoproteins showed that they were
prese~t
in
both appressed and unappressed membranes, although they were
more
concentrated
in
the
former
(77-81%
versus
19-23%)
(Callahan et al. 1989). In a similar study, Di Paolo et al.
(1990) used a battery of antibodies raised against a variety
of CP's associated with PS l or PS II to probe western blots
of stroma- and grana-derived membrane complexes. Their resul ts
conclusively indicate that the CP's associated with PS II (CP
2'), CP 26, CP 24, representing minor PS II LHC' s) are confined
to grana regions. Chlorophyll-proteins of PS l
12
(LHC 1-730 and
LHC 1-680)
were detected only in stroma membrane
l",let
t
1011:;.
Thus, both of these studies y ielded resul ts that pi.1 1',111 C' 1 thf'
proposed
spatial
thylakoids,
and
heterogeneity
substantiate
of
PS
earl ier
,lnd
1
stud iC's
11
1 Il
l 'lB:~
(c 1 i nco
;
simpson et al. 1987, 1988).
Further support for these conclusions
WélS
Andersson and Anderson (1980) who showed that on the
LHC-II-associated CP concentration,
i drd hy
pn1V
!J.l:; 1:; ()
1
grana pi1rtition-dC'l"iv!'d
fracticns have a much higher proportion of LHC êlpoprotr i n thdll
do stroma membranes. Indeed, strorna-exposcd thyLlkoid \11c1I"qin"
completely
lack
LHC-II-associated
proteins
(Webbrr
(,t
,II.
1988).
Furthermore, immunocytochemical experimcnü; hy \)c1h 1 111
(1989)
indicated that in wheat chloroplasts, wh i ch h,ld h('('11
treated with a herbicide meant to induce diffcrC'nt
grana
stacking,
the
amount
of
LHC-II
lêlbcll inq
correlated with the degree of stacking, and wa:-:;
much greater over appressed
thylakoids,
c!c'qn'f>', 01
Oll';('IVf'r!
con~;
membranes than ovcr
i :;trnt 1y
un!;t.lC'~:('<I
except at the highest herbicide concrnt_l"ilt ion!;
useG. Other immunocytochemical investigations yieldC'd
results
(Vallon
et al.
simpson
1986,
et
al.
~;lmi Idr
le>B/).
results conflicted with those of Shaw and I/cmJOod
{1 (Jil',)
used immunogold labelling to show an even distrj but J on
II amongst paired and unpaired membranes in pea. Tt
'1'1)(':;('
01
~;ho\ll
v/tHl
l,fW-
d br'
noted, however, that these authon; qualify their rcsult:; tJy
acknowledging that a subgroup of LHC-II exjstfj thilt
tightly bound to
PS
II,
but
rather has
'!
i
13
the
i:;
capdo 1 l i t '/
11()t
(d
---------------------
- -
---
migrating within the plane of the thylakoid membrane depending
on
the
l ight
conditions
(see
below).
Their
resul ts
may,
therefore, be reflective of this migration.
Perhaps, the most convincing evidence for the nature of
the stacked
membranes was provided
by Hinshaw and Miller
(1989). These investigators succeeded in exposing the outer
surface
of
previously
the
appressed
accomplished,
freeze-etch
membranes
and
(PS s ) '
analyzed
electron microscopy.
The
this
a
feat
region
not
using
resul ting micrographs
displayed a smooth topography of stacked membrane surfaces,
while the
adjacen~,
unstacked membrane surfaces were studded
with several particles. This result supported the concept of
lateral
heterogeneity
since
none
of
the
transmembrane
complexes that protrude significantly from the membrane (e.g.
ATP synthase,
PS 1) appear in this are a
(Hinshaw and Miller
1989). Moreover, a monoclonal antibody directed against the
amino
terminus of
LHC-II
was
used to
locate this complex
within the thylakoids, and showed a high preponderance of it
in the stacked regions. Only small amounts of label were seen
over the unstacked surface. These results again support the
presence of LHC-II mainly in granal regions, and strengthen
previous
freeze-fracture
data
that
revealed
a
correlation
between EF particle size and the amount of LHC bound to PS II
cores (e.g. Armond
a~d
Arntzen 1977; Armond et al. 1977).
The foregoing discussion centered largely on LHC-II. This
was rnainly due to the fa ct that it is by far the best-studied
(
14
...
'
r
1
chlorophyll-protein
1
cOiuplex,
probably
becallse
interesting properties. As mentioned above,
LHC-II,
referred
to
as
"mobile"
ot
a proportion
is
LHC·-II,
01
capi1blc
01
migrating between grana and stroma regions in responsc ta thl'
available
Arntzen
light
1983).
conditions
The
dogma
(Bennett
is
that
staehcl in
,1I1d
chloroplnsts
nrc
1983 =
when
illuminated by light of a wavelength that is prefercntially
absorbed by PS II, mobile IHC-II
d~
c;sociates from PS TT
in
grana, and migrates to the stroma where i t can transfcr l i {Jht
energy to PS 1
(this idea is not ubiqui tOllsly acccptcd;
happcn~-;
c' t •
in
1 i qht
preferentially absorbed by PS 1. Thus, this mcchani ~;m
~-;ervc:;
Anderson
and
Melis
1983).
The
reverse
to balance light absorption between PS 1 and PS Il, thcrcby
optjmizing the efficiency of photosynthesis. Phospharylation
;".md dephosphorylation of LHC-II appears ta be the
mCéll1f;
which its migration is effected (Bennett 1°83; Stachcl in
Arntzen
1983).
Most
of
the
studies
ci ted
above
é1
i J11cd
by
Ilnrl
ilt
localizing LHC-II showed that a small amount of thü; comp 10X
was
found
in
stroma
lamellae.
This
finding
io
entjrely
consistent with the notion of LHC-II migration. Severai
linc~
of evidence indicate that LHC-II is also involved in membr,lnc
stacking (see Staehelin 1986). If this is the case, th0n lt
not surprising to find that a lùrge proportion of LlIC-! 1 i:;
associated with appressed membranes.
15
2.4 CF o-CF 1 ATP Synthase
The ATP synthase comp1ex is comprised of
subunit, termed coupling factor l
a cata1ytic
(CF,), and an anchor (CF o).
This complex was the first of the thylakoid membrane complexes
to be positively identified (Staehe1in 1986). Its 10calization
in
the
thylakoid
membrane
was
also
relatively
easy
to
ascertain. This is undoubtedly due, at least in part, to the
very characteristic shape and size of the complex.
The work of Miller and Staehelin (1976) was one of the
most instrumental in localizing ATP synthase.
class of
large particles
(15
nm diameter)
Removal of a
from the outer
thylakoid surface by washing membranes in solutions of low
ionic strength resulted in the 10ss of CF 1 activity. However,
incubation of these membranes with purified
CF, particles
resulted in the reassociation of the complex in the membrane,
and reappearance
~f
the large particles and of CF 1 activity.
Further, treatment of membrane preparations with an antibodyferritin
conjugate
showed
label
clearly
associated
with
stroma-exposed membranes, but none in grana stacks. This was
not due to the inabi1ity of the
antigen
in
grana
partitions
ferritin
as
shown
label to access
by
unstacking
experiments. The authors reasoned that if CF, is excluded from
appressed regions, unstacking of the membranes would result in
a redistribution of the CF 1 particles su ch that the
density of CF 1 would be reduced. This effect was,
observed.
Therefore,
from
the
16
above
data,
the
overal~
in fact,
authors
concluded that CF, was present only in unappressed membrdnes
(Miller and Staehelin 1976).
Support ive evidence
variety of work.
for
Immunoblot
this conclusion cornes
from a
analysis of detergent-der i vcd
granal and stromal lamellae showed that the a and B subunits
of
the
CF,
were
(Callahan
et
located
al.
exclusively
in
This
1989) •
stromal
was
lamcl\ ae
confirmed
by
immunocytochemistry, which revealed the exclusive labell ing of
unstacked
membranes,
membranes,
by
including
anti-cF,
grana
antibody
margins
in
both
and
end-
spinach
ilnc1
Chlamydomonas (Allred and Staehelin 1985; Vallon et al. 198G),
as weIl as in barley (Shaw and Henwood 1985) .
Webber et al.
(1988)
used a
detergent
(Tween-20)
to
selectively solubilize thylakoid marginal regions and break
the
membranes
at
the
junction
of
stroma
and
partition
membranes (i.e. at the membrane region connecting grana and
stroma
thylakoids,
hereinafter referred
to
as
the
border
region), leaving the appressed partitions intact. After hjgh
speed centrifugation to pellet down membranous material, and
analysis
associated
of
this
with
material,
no
partition-derived
CF,
particles
vesicles.
were
seen
Rather,
they
studded the surface of membranes of the border reg iun.
Jn
addition, CF, polypeptides were predominant in the supernrltilnt
after Tween-20 solubilization,
indicating their presence in
stroma-exposed margins of the grana thylakoids. ImportantJy,
these results disprove the hypothesis of Murphy
17
(1986)
who
•
~
suggested that no proteins resided in these regions.
(
2.5 cytochrome btJf Complex
The
local i zation
of
the
cytochrome b 61 f
complex,
an
interrnediate in the transport of electrons from PS II to PS l,
proved
to
e1icit more
contention than
any
of
the
other
thylakoid membrane complexes. At present, the most prevalent
hypotheses concerning i ts intra-membrane locale are that i t is
equa11y
distributed
membranes, or that
J. t
amongst
appressed
and
unappressed
resides at the interface of stroma1 and
grana1 lame11ae (the border region).
Investigations centering on biochemical fractionation of
thy1akoids into grana and stroma fractions have yie1ded rather
ambiguous resul ts. Spinach thylakoids subj ected to Yeda press
treatment, followed by aqueous polymer two-phase parti tioning,
conta in cytochrome
i, which acts as a marker for cytochrome
b 6/ f, in both appressed and unappressed membrane fractions in
equal proportions (Cox and Andersson 1981). This result was
verified when fractions from similarly treated thylakoids were
ana1yzed
for
cytochrome
.1
as
a
function
of
chlorophy11
concentration (Anderson 1982).
These resul ts did not go uncontested, however. Comparison
of mesophyll and agranal bundle shea+:h cells of maize in terms
of membrane constituents showed that the latter were dep1eted
in complexes believed to be associated wi th grana stacks (i. e.
PS II and LHC-II) (Ghirardi and Melis 1983) . No such depletion
(
18
was observed for PS I,
.
stroma
thylakoids.
which
Moreover,
is thought to be present
since the
ratio of
PS
T
in
ta
cytochrome i remained equal in bundle sheath chloroplnsts as
compared to mesophyll chloroplasts, the authors suggested that
cytochrome
b 6/f
otherwise,
a
could
not
depletion
in
be
this
present
in
grana
complex would
stacks;
have
bC'C'n
observed as was the case for PS II and LHC-II. In order ta
reconcile these results with those of previous studies (Cox
and Andersson 1981; Anderson 1982), the duthors postu.atcd
that the
cytochrome
b 6/f
complex resides
in
the
membrùne
reg ion connecting grana and stroma thylakoids (border reg ion) .
Thus, detergent fractionation, which
wou~d
thylakoid continuity at the se regions,
tend to break the
may not efficient1y
separate grana stacks from the border region,
leading to the
detection of cytochrome b 61 f in both grana and stroma lame ll?1e
(Ghirardi and Melis 1983).
In order to alleviate this problem, aqueous polymer twaphase partition-separated vesicles were subjected to a further
treatment
current
consisting
distribution
of
sonication,
(Helis et
al.
followed
1986).
by
This
countor
procedure
purportedly separates parti tion-derived membranes from stromaexposed
membranes
with
suggested an enrichment
higher
resolution.
in cytochrome b/f
membrane corresponding to the border region,
support for the ex.i.stence of a
enriched in cytochrome b6/ f .
.,
19
The
in an
re::;ult~;
area
of
addinq furthor
specialized membrane reqi on
,
i
c
In a subsequent study, Morrissey et al. (1986) incubated
thylakoids with progressively increasing concentrations of
detergent,
and
found
that
solubilization
of
membrane
components occurred in a step-wise fashion: cytochrome bJf
was first to be solubilized, followed by PS land finally by
PS II. These results were interpreted ta mean that cytachrame
b 6 /f probably resides in an area of membrane that would be
most susceptible to
disruption
d~tergent
(i. e.
the border
region). In contrast, grana partition regions would be most
resistant to the detergent; this is the rationale given for PS
II being solubilized last (Morrissey et al. 1986).
In contra st
fractionation
to the
ambiguity
procedures,
immunocytochemical
presented by
data
experiments
unappressed
thylakoids
generated
unequi vocally
homogeneous distribution of cytochrome
(Allred and
indirect
b~ f
from
show
a
in appressed and
Staehelin 1985,
1986;
Goodchild et al. 1985a; Shaw and Henwood 1985; Vallon et al.
1986) including grana margins (Webber et al. 1988). Moreover,
a freeze-fracture study offered no support for the presence of
a
distinct
membrane
region
enriched
in
cytochrome
b 6/f
(Hinshaw and Miller 1989).
Thus, it is apparent that further studies will need to be
done in order to determine the exact location of cytochrome
b6 /f,
and reconcile the
discrepancies arnong the
data.
(
20
existing
3. Ch1orophyll Q-containinq A1qae - Chromophytes
In contrast to the situation in higher plants ëlnd
(Jl"CCn
algae, little is known about the supramolecular organ i zat jan
of the photosynthetic apparatus in the chromophyte algae. 'l'he
distinction between appressed and unappressed membranes
is
retained in these a 19ae al though their organization var i es
between
the
groups
(Gibbs
Staehelin
1970;
'l'he
1986).
classification of thylakoids into grana and stroma does not
apply in these organisms.
A large proportion of the work designed to chùracterize
the
supramolecular
organization
of
the
photosynthctic
components in chromophytes has been in the form of
fracture electron microscopy
however,
are
prel iminary
,staehelin 1986).
and
should
be
frcczc-
'1'hesc d"ti1,
interprcted
w i th
reservation.
Freeze-fractured
Gonyaulax
revealed
a
thylakoids
of
heterogeneous
the
dinofla cjC!11atc
particle
distribution
(Sweeney 1981). In this alga, EF particles were present
greater abundance
in stacked lllembrane regions
thi1n
in
in th"
unappressed areas. However, the author resisted the temptilt j on
to
draw
a
complexes
paraI leI
as has
between
been done
particle distributions have
the
EF
particles
in higher plants.
also
been observed
and
PS
TI
Non-unj form
in
sever,)]
cryptomonads (Dwarte and Vesk 1983; Rhiel et al. 1985; SpcarBernstein and Miller 1985; Lichtlé et al. 1986). Con::.; istcnt
ln
aIl
a~;
these
studies
was the higher
:>1
EFs
particle dens i ty
compared to the EFu particle density.
apparent
correspondence
complexes
between
in higher plants,
On the basis of the
particles
EF
as weIl
and
as differences
PS
II
in EF
particle densities between high- and low-light illuminated
cryptomonads (Lichtlé et al. 1986), the proposaI was made that
PS II complexes are partially segregated to appressed membrane
regions (Dwarte and Vesk 1982, 1983; Lichtlé et al. 1986). The
data concerning PS l
distribution are less clear in
studies, although Lichtlé et al.
these
(1986) suggest that PS l may
be associated with PFu particles. In any event, this evidence
appears to point to an incomplete segregation of PS II and PS
l
in
these
organisms.
similar
resul ts
were
obtained
for
members of the brown algae (Berkaloff et al. 1983) and diatoms
(Dwarte and Vesk 1982).
Two other studies depicting immunocytochemical attempts
to localize photosynthetic components to appressed or nonappressed membranes of Chromophytes have been publ ished. Rhiel
et al.
~Q
(1989) investigated the localization of a chloraphyll
light-harvesting complex in a cryptomonad. Their results,
although not strictly quantitative,
the
camplex
is
equally
appear to indicate that
distributed
over
the
thylakoids,
showing no preference for appressed or unappressed membranes.
Lichtlé
et
al.
(1991),
examining
the
localization
of an
equivalent light-harvesting complex in another cryptomonad,
also showed label to be distributed on both membrane types. In
this study,
however,
the appressed membranes were slightly
22
enriched in the complex. In addition, these investigdtors hùve
shown a sI ight enrichment of PS l
to exist in
unùpprc~-;::.~C'd
membranes. Cursory examination of go Id particle distribution
in a third study (Grevby et al. 1989) appears to suqgcst
equal
~n
distribution of the chlorophyll Ël/f! l ight-harvest i mJ
complex in the brown alga Laminaria, although this study dict
not focus on supramolecular thylakoid organization.
Thus,
it
seems
that
in
chromophytes,
11
1 i:\ te rI' \
heterogenei ty of photosynthetic complexes may be present, but,
if
50,
it is evidently not as pronounced as that in hj(JhC't"
plants and green algae. Obviously, much more work rcmdins ta
be done before an accu rate picture of the organizatian
of
intra-thylakoid components in these organisms is establishcd.
23
(
CH.A1?'I'ER.
(
24
II
1. Introduction
The ability of photosynthetic organisms to convert solar
energy
into ATP and NADPH through the
light reactions of
photosynthesis is dependent on the concerted action of scverù 1
supramolecular
photosystems
complexes.
(PS)
l
and
These
II
complexes,
and
their
which
include
associated
light-
harvesting complexes (LHC-I and LHC-II), cytochrome b 6 /f and
an
ATP
synthase,
are
known
to
reside
in
the
thylakoid
membranes of chloroplasts of photosynthetic eukaryotes.
recent years,
In
a large body of evidence has accumulated ta
indicate that the distribution of the se complexes within the
thylakoid membranes of higher plants and green algae is nonrandom
(for
reviews,
see
Anderson
and
Andersson
1982;
staehelin and Arntzen 1983; Staehelin 1986: Simpson and Von
Wettstein 1989). Thus, PS II, along with LHC-II, is localized
predominantly
whereas
PS
in the
land
appressed
LHC-I
membranes
are largely,
confined ta unappressed membranes
of
grana
stéJcks
if not
exclusively,
(Andersson
and Anderson
1980; staehelin 1986; Vallon et al. 1985, 1986; Callahan ct
al. 1989). The ATP synthase complex resides exclusively in
non-appressed membranes (Miller and staehelin 1976: Allred and
staehelin 1985: Shaw and Henwood 1985: Vallon et al.
1986:
Webber et al. 1988; Cal1ahan et al. 1989), whereas cytochrorne
b~f
is
the
sole
complex
that
appears
to
be
cqually
distrlbuted among both types of membrane (Cox and Andcrsson
25
•-,
1981; Allred and Staehelin 1985, 1986: Goodchild et al. 1985a:
(
Shaw and Henwood 1985; Vallon et al. 1986).
In contrast,
comparatively little is known about the
supramolecular organization of
chromophyte algae.
thylc....koid
membranes
in the
These algae possess chlorophyll Q instead
of chlorophyll 12 and their thylakoids are arranged in extended
bands
of
three
cryptomonads,
in
appressed
pairs
of
thylakoids,
loosely
or
in
the
case
of
associated thylakoids.
Freeze-fracture studies of representative species of brown
algae (Berkaloff et al. 1983), dinoflagellates (Sweeney 1981),
prymnesiophytes, chloromonads,
and diatoms
(Dwarte and Vesk
1982), and cryptomonads (Dwarte and Vesk 1983: Rhiel et al.
1~86)
1985; Spear-Bernstein and Miller 1985; Lichtlé et al.
have shown that these algae possess heterogeneous EF fracture
faces
resembl ing those of the
appressed and non-appressed
thylakoid membranes of higher plants and green aJgae.
On the
basis of these observations, Dwarte and Vesk (1982, 1983) and
Lichtlé et al.
(1986)
have suggested that
associated
might
be
LHC
preferentially
appressed thylakoid membranes and PS
thylakoid membranes of chromophytes.
l
PS
II
located
and
its
on
the
on the unappressed
However, the only direct
evidence for the supramolecular organization of PS land PS II
in chromophyte chloroplasts is a recent immunocytochemical
study of Cryptomonas rufescens (Lichtlé et al. 1991).
study showed that a PS II-associated
chlorop~yll
harvesting protein was slightly enriched
«
26
This
2/Q light-
in the appressed
1
membranes,
whereas PS l
appressed membranes.
was more concentratcct
in tilt"
non-
Thus, while a lateral heteroC)encity nt
photosynthetic complexes may exist
in cryptomoncllh-;,
'\
"
"
evidently not as pronounced as that in higher plants l1nd qt'cc'n
algae.
In
this
report,
immunocytochemical
present
we
experiments
the
result"
01
to
detcrm i ne
tho
designed
localization of PS l and the major fucoxanthin-chlorophy 11
light-harvesting
pigment-protein
complex
( FCPC)
thylakoids of the diatom Phaeodactylum tricorxt1JtJ.1J].
that
FCpe
unappressed
is
equally
membranes,
distributed
among
whereas
l
concentrated in the latter.
27
PS
is
in
<l/e
t
Ill'
\'.Je
:;!1mv
ê1ppre~,sed
,1I1t!
slightly
moro
2. Materials and Methods
2.1 Cell Culture
phaeodactylum tricornutum Bohlin was obtained from the
University of Texas Culture Collection of Algae (UTEX 646).
Cells
were
grown
at
20°C
in
250
ml
Erlenmeyer
flasks
containing 75 ml of f/2 medium (see McLachlan 1973) under a
bank of cool-white
fluorescent lamps
(::::::40 /-LEm- 2 s-')
on a
16
hour light: 8 hour dark cycle. Culture flasks were continuously
agitated using a rotary shaker.
2.2 Antisera
Antiserum directed against a mixture of the 19 and 19.5
kilodalton (kDa) polypeptides of the fucoxanthin-chlorophyll
Ël/ç light harvesting complex of
g. tricornutum (Fawley and
Grossman 1986)
was the generous gift of A.
Plant Biology,
Carnegie Institute of
CA).
Antiserum
against
sucrose
Grossman
Washington,
(Dept.
stanford,
gradient-purified
maize
photosystem l particles was kindly donated by A. Barkan (Dept.
Botany, Univ. of California, Berkeley, CA).
2.3 protein A - gold preparation
The protocol used in preparing protein A complexed ta
small gold particles was based on that outlined in Lud\vig and
Gibbs (1989). A solution of colloidal gold particles (3-10 nm
diameter)
was
prepared fOllowing the
procedure of Tschopp
(1984). After complexing to protein A (Roth 1982), protein A-
,.1
"
28
gold complexes were isolated by centrifugation in a Reckman 'ri
70.1 rotor at 30,000 rpm for 35 min at 4°C. The protein A-qold
was resuspended in phosphate-buffered saI ine (PBS) contù in i nq
0.02% polyethylene glycol.
This suspension was sUbsequently
separated into fractions containing different gold particl0
diameters according to the method of Slot and Geuze (J9Rl).
Briefly, a 1 ml aliquot of the protein A-gold sol ution
W.1~;
layered on a 10%-30% continuous sucrose gradient in PBS,
~nd
centrifuged at 30,000 rpm for 85 minutes (4°C)
in a Rcckman
SW41 rotor. One ml fractions were collected from the qracl i ent ,
dialyzed
against
fraction,
PBS
at
for
4°C
polyethylene glycol
overnight 1
(MW 20,000)
and,
to
ci1cll
and sodium i1zido
were added to give a final concentration of 0.02% each. Two
fractions containing gold particles of
~
4-5 nm and 5-6 nm in
diameter respectively (as determined by measurements of qold
particles
on
micrographs)
were
used
in
sub~-;cqll('nt
immunolabelling experiments.
2.4 Electron Microscopy
For routine ultrastructural observation, late lorJélrithmic
phase cells of
light
period
~.
of
tricornutum were harvested early
the
growth
cycle,
and
fixed
glutaraldehyde in O. 05M cacodylate buffer plus O. ttM
pH 7.2
for 2.5 hours at 4°C.
Subsequently,
the
washed and post-fixed in 1% osmium tetroxide
in the
in
~.~~
sucro~;c,
cell~5
(0504)
Ir'en.!
for
2
hours, dehydrated through a graded ethanol series and cmbeddcd
29
in Spurr's epexy resin (Spurr 1969). Pale gold sections were
collected on formvar-coated copper grids, and stained with 2%
uranyl acetate in methanol, followed by lead citrate.
For immunolocalization of FCPC,
pale gold sections of
tissue fixed and embedded as described above were picked up on
formvar-coated nickel grids and subjected to the following
labelling regime: 0.58M (saturated) sodium meta-periodate, 45
min; PBS,
10 min; 1% bovine serum albumin (BSA)
min; anti-FCPC antiserum,
in PBS,
15
diluted 1:500 in BSA plus PBS,30
min; undiluted prote in A-gold in BSA plus PBS,
45 min. The
sections were stained as described above.
For localization of photosystem I, early stationary phase
cells of
E. tricornutum were harvested after 2-3 hours ln the
light period,
fixed in 1% glutaraldehyde in C.1M cacodylate
buffer plus O.4M sucrose,
washed
sucrose.
in
buffer
containing
for 1 hour at
decreasing
4°C,
and
concentrations
of
The cells were dehydrated in 25% ethanol at -4°C,
then in 50%,
were
pH 7.2,
75%,
infilt~ated
and 95% ethanol at -18°C.
Finally,
cells
and embedded in Lowicryl K4M resin according
to the following protocol: 95% ethanol:resin, 1:1, overnight;
95%
ethanol: resin,
1: 2 ,
2
times
for
2
hours
each,
th en
2
times
for
2
hours
each,
then
in
fresh
overnight;
resin
alone,
overnight.
Cells
were
resuspended
resin
and
transferred te BEEM capsules, centrifuged to concentrate the
cells, and polyrnerized at -18°C under ultraviolet light at 360
nm for 24 hours, followed by another 48 hours at 20°C. Pale
30
l
J
gold sections were floated face-down on drops of the following
solutions: 1% BSA in PBS, 30 min; anti-Ps I antibody, dilutcd
1:500 in BSA plus PBS,
each;
30 min; 1% BSA in PBS,
undiluted prote in A-gold
in BSA plus
4 times, 3 min
PBS,
~o
min.
sections were stained with 4% uranyl acetate followed by le<ld
citrate.
The following controls were performed for both studies:
1)
replacing antisera by non-immune serum, diluted 1:500
BSA plus PBS,
in
2) incubation on a drop of PBS in place of the
antisera. AIl viewing was done on a Philips EM 410 e1ectron
microscope operating at an accelerating voltage of 80 kV.
2.5 Quantitation of Gold Label
The thylakoids of most chromophyte algae are
appressed and arranged in bands of three.
1005e 1 y-
This association
results in a ratio of appressed to non-appressed membranes of
2:1.
For anti-FCPC,
gold particles were assigned to ono of
three classes. Class 1 consisted of gold partjcles
overJyjn~
either of the two outermost membranes, or lying wjthin
~
?
gold particle diameters outside a thylakoid tripl et. 1'hcso
particles were scored as
labell ing antigen
on unappressecl
membranes. Class 2 contained gold particles overlapping any of
the 4 central membranes, or lying in the lumen of the centrol
thylakoid;
these
membranes.
Class
were
3
entirely within the
regarded
consisted
of
as
gold
part icles
lumen of one of the outer
31
appro~;~-;(ld
labell ing
res j d j nq
thy1ay'ojd~;,
along with those whose edges were barely touching the cxternal
0r internaI membranes. Half the gold particles in this latter
class were subsequently assigned to appressed membranes, wi th
the other hal f allocated to unappressed membranes. A total of
1047 gold particles were counted.
For
anti-PS
I,
assignment
of
gùld
particles
was
complicated by the inability to resolve thylakoid membranes
clearly in non-osmicated tissue. Hence, a thylakoid triplet
was assumed to consist of 3 equally spaced thylakoids. Gold
particles
labelling
unappressed membranes
(class
1)
were
counted as described above. Those lying directly in the ce'1tre
of a triplet, or slightly off-centre, were considered to be
labell ing appressed membranes (class 2). Class 3 consisted of
gold particles that the authors deemed by visual impression to
be lying over the lumens of the two outer thylakoids
band.
in a
Finally, gold particles that were difficult to assign
clearly to ei ther class 2 or 3 were grouped into a fourth
class. Subsequently, the total gold particles in this latter
class were divided equally between classes 2 and 3. Finally,
half the revised
count
of gold particles
in
class
3 was
assigned to appressed membranes (class 2) and the other half
was assigned to non-appressed membranes (class 1) to give the
final
labelling distribution. A total of 734 gold particles
were
counted.
statistical
significance
of
the
distributions was determined by chi-square analysis.
32
observed
2.6 cytochemical staininq for PS l
Photosystem
l
activity
was
determined
the
by
photooxidation of 3,3'-diaminobenzidine (DAB) as described by
McKay and Gibbs (1990).
Late logarithmic phase cells of
~.
tricornutum were collected at hour 8 of the light cycle, and
fixed
in
2%
paraformaldehyde
in
O.lM
phosphate
buffer
(containing 0.2M sucrose), pH 7.4, for 20 min at 4°C in the
dark.
After
rinsing
in
buffer
containing
decreasing
concentrations of sucrose, cells were incubated in a solution
containing 1 mg/ml DAB in O.lM phosphate buffer (pH 7.4) for
1 hour at 20°C. The incubation was perforrned either in the
dark
for
control
cells,
or
under
a bank of
cool
white
fluorescent lights generating a photon fluence rate of 230
jLEm- 2s- 1 • Cells were subsequent1y washed in buffer, and postfixed in 1% oSO, in O.lM cacodylate buffer (pH 7.4) for 1 hour
at 4°C in the dark, washed again,
dehydrated
in a grùded
ethanol series and ernbedded in Spurr resin (Spurr 1969). Pale
gold sections were mounted on forrnvar-coated copper grids, and
viewed unstained as described above.
33
..,
3. Results
li
Figures
1
through
chloroplast morphology
3
in
show
representative
images
Phaeodactylum tricornutum.
of
The
thylakoids are organized into extended bands or lamellae which
traverse much of the length of the chloroplast.
A
single
band, the girdle lamella, encircles the rim of the chloroplast
(Fig.
1).
appressed
Each band characteristically consists of three
This
thylakoids.
gives
a
constant
appressed to non-appressed membranes of 2:1.
ratio
of
It should be
noted that the thylakoids are not tightly appressed as they
are in the grana of higher plants and green algae, but are
separated by a 2 to 3 nm space.
Occasionally, bands of two ta
four loosely appressed thylakoids are observed. For example,
at the arrow in Fig. l, the uppermost thylakoid terminates and
the band
continues as a
thylakoid pair.
However,
in the
labelling studies these uncommon two or four thylakoid bands
were not
included in counts of
gold
particles.
Embedded
within the chloropJast is a large central pyrenoid (Figs. 13) ,
a
region
of
concentrated
ribulose
bisphosphate
carboxylasejoxygenase (McKay and Gibbs 1991). The pyrenoid is
surrounded by
a
layer
of
electron-dense
material
and
is
bisected by a loosely appressed pair of thylakoids.
Figure 2 shows a section through the chloroplast of a
Spurr-embedded, late logari thmic phase cell of E. tricornutum.
This secti,",n has been labelled wi th hamologous antibody to the
1
34
L
1
maj or fueoxanthin-ehlorophyll gJg light harvesting protC' i 11
(FCPC), followed by protein A-gold (4-5 nm). Gold particlC's
are loealized over the thylakoid bands,
with very few
80011
over the pyrenoid or ehloroplast stroma. Most of the part lclc~. .
lie direetly over or toueh a thylakoid membrane.
Both tho
appressed and non-appressed membranes are labelled.
pyrenoid
thylakoids
are
also
labelled.
Cell
1'h0
seeUons
ineubated in PBS alone (not shawn), or in pre-immune serum
(Fig. 3) in place of the antiserum are negligibly labellecl.
Quanti tative evaluation of the go Id partiele di stribut i on
indieates that 68.1% of the gold partieles were assoeintocl
with appressed membranes, whereas 31.9% of the gold
particlc~
labelled antigen loeated on unappressed membranes (Fig. 6). In
arriving at these
figures,
gold partieles present
lumens of the two outer thylakoids in a
band
in tho
(16%
t
0
t'Il(>
total) were counted in a separate category, and half the totol
was assigned to appressed membranes, while the other hall
assigned to
un~ppressed
WBH
membranes. The observed values do not
deviate signifieantly (0.25 < E < 0.50) from the expected qold
partiele distribution of 66.6% on appressed versus 31.1% on
unappressed membranes which would result if Fepe werc pro:;('nt
in equal abundance on both types of membranes.
The distribution of PS l
in R.
tricornutum has
bccn
determined using an antibody raised against purified fiS 1
,r:articles from maize,
followed by protein A-gold
(5-6 nm)
(Fig. 4). In the interest of preserving antigenici ty,
36
0:;m1 um
T
,
"
FicJ.
4.
Chloroplast of a
E.
tricornutum cell embedded in
Lowicryl resin, and labelled with an anti-PS l antibody from
rnaize. Gold particles label the white thylakoid bands almost
exclusively. The stroma (2) and cell cytoplasm (Q) are free of
Jabel. Bar =
Fig.
and
5.
0.2~m;
X 92,500
Chloroplast section labelled with pre-immune serum
prote in A-gold.
observed. Bar =
Very
0.2~m;
few gold particles
X 92,500
37
(arrows)
are
1
t
1
c
tetroxide was not used as a post-fixative. Hence, membranes
are not clearly discernible, and thylakoid triplets appear as
white bands on a darker background (chloroplast stroma). As
seen in Fig. 4, labelling is specific to the chloroplast and
most
of the gold particles lie over the thylakoid bands,
including those that traverse the pyrenoid (not shown). Very
few gold particles were observed in sections incubated in preimmune serum (Fig. 5) or in PBS alone (not shown). Assignment
of gold particles to appressed and non-appressed membranes was
dependent on their relative positions within a thylakoid band
(see Materials and Methods).
that
60.5%
of
the
gold
The data in Figure 6 indicate
particles
were
associated
with
appressed membranes, and 39.5% wi th unappressed membranes. The
labelling of the unappressed membranes by antiserum to PS l is
slightly, but significantly (E < 0.001), higher than the 33.3%
expected
if
PS
l
were
to
get
equally
distributed
amongst
both
membrane types.
In
order
a
further
indication
of
PS
l
distribution, we employed a cytochemical procedure in which PS
l location could be determined through the photooxidation of
DAB to an electron dense deposit. Figure 7 shows that the DAB
reaction product is associated with aIl the thylakoids of the
chloroplast.
stained
by
In addition,
virtue
of
mitochondrial membranes are also
their
cytochrome
oxidase
activity.
Control cells, which had been incubated with DAB in the dark,
exhibited staining of mitochondria and peroxisomes, but the
(
38
Fig.
6.
Observed
thylakoids of
~.
distributions
of
gold
particles
over
tricornutum labelled with either anti-FCPC or
anti-maize PS land protein A-gold. The dotted lines represent
the
predicted distributions
membranes
on
appressed
(66.6% and 33.3% respecti vely)
and
unappresseLl
assuming that the
antigens were present in equal proportions on both types of
membrane.
39
.. Appressed
[J Unappressed
80
70
-------------------------------
60
t/)
Cl)
U
50
c.
40
t:as
"C
'0
C)
30
0~
20
-------------
..... 'o'.:.. ,",":..... .
,..
~ .' .......,.... "
..
. ....
,~ .... ..:, .-;..
.. ..../..... ..'
~"..
.. ,.
:
, ,
. ....
.
,
:
10
.'
',,;
0
FCPC
PSI
,
----
..
..
.'
....
. .
...
~
Longitudinal section through a cell of R. tricornubljTI
Fig. 7.
incubated in high light in the presence of DAB. Dense reaction
product
is
associated with
tlle thylakoid
lamellae nf
the
chloroplast (g), particularly with the two outer thylakoids.
In addition, the cristae and envelope of a mitochondrion (m)
also exhibit staining. Bar
Fig. 8.
the
Control cell of
dark.
peroxisome
=
E.
0.4~m;
X 41,000
tricornutum incubated with DAR in
The
mitochondrion
(m)
is
(12).
No reaction product
stained,
High1y
m~gnified
is
the
is observed associr1tec1
with thylakoids of the chiorop1ast Cg). Bar
Fig. 9.
as
=
0.4~m;
X 4],000
view of thylakoids stained with DAB.
The dense deposit has a punctate appearance, and appears ta be
1arge1y associated with the outer thylakoids of each band
(arrawheads).
The
internaI
thy1akoid
(white arrows) but ta a 1esser degree.
Bar
=
O.l~m;
X 115,000
40
also
g,
shows
staining
plastoglabulus.
-
•
chloroplasts
were
essentially
unstained
(Fig.
8).
Cl
o~~('
inspection of the pattern of DAB reaction product deposi tian
indicates that
heavily
the two outer thylakoids of
stained with
a
rather
punctate
each
appearance
reaction product (Fig. 9). The central thylakoid also
staining,
but to a rnuch lesser degree
thylakoids.
41
band
than
of
"rC'
tlw
exhibit~~
the outer two
4. Discussion
Although it
green
algae
apparatus
is well-established for higher plants and
that
the
components
of
the
photosynthetic
are non-randomly distributed among appressed and
unappressed membranes of grana and stroma thylakoids, i t
not
known
whether a similar
arrangement
is
is true for other
groups of algae. In this report, we present immunocytochemical
evidence
indicating
that
the
light-harvesting complex of
fucoxanthin-chlorophyll
Q/.Q
P. tricornutum, believed to be
associated with PS II (Manodori and Grossman 1990), is equally
distributed among both appressed and unappressed membranes,
whereas PS l
Importantly,
is
slightly more concentra ted in the
both complexes
are
present
latter.
on both types
of
membrane; thus, the extreme lateral heterogenei ty observed in
higher plants
is
not
present in
this
organisme
In this
respect, our results for E. tricornutum are similar to those
reported for another chromophyte alga, Cryptomonas rufescens
(Lichtlé et al. 1991).
chlorophyll Y.Q
However, in ç. rufescens, the 19 kDa
light-harvest.ing
protein is
slightly more
concentrated on the appressed membranes.
A potential difficulty with our results stems from the
methodol.:>gy used
in assigning gold particles to particular
membranes. Since the antibody-protein A-gold conjugate has a
definite length (maximum 18 nrn for 5 nm gold), i t is possible
that a gold particle labell ing a particular antigen will be
42
l
situated at a distance
FCPC,
from
the antigen.
For
eXi1mp 1 e,
or
t
a gold particle residing entirely within the lUn1e'n
0
one of the outer thylakoids in a lamella could be labell i
t
I1<'J
antigen on either the appressed or non-appressed mcmbrélnCt-;.
Such gold particles were placed into a separate category, ilnd
subsequently, the final count was divided equally bCtwc01l the
two membrane types. This procedure, however,
would tond ta
skew the labelling distribution in favor of the unapprcnt;cd
membranes.
This problem is compounded for PS 1.
In orc!0r t
0
obtain labelling of Phaeodactylum by the heterologous ùnt i maize PS l
antibody, the tissue was not fixed in osmium, ,1nd
was embedded iT'l Lowicryl resin. This protocol docs not ill) ow
for precise resolution of membranes. Thus, thylakoid ] ame') ) (10
appeared
as
chloroplast
white bands
stroma.
We
on
the
assumed
dark
background
0
that they comprised
f
the>
thrcc
equal }.y spaced thylakoids. Gold particles labell ing thyli'lko i d
lamellae were assigned ta categories on the basis of vi ralrll
impression
category
(see Materials and Methods).
(class
4)
was
created
As such,
an c·ytrrl
for gold parti cl es
vJllO~;C'
localization on either appressed membranes or within the outer
lumens could not be definitively determined. Dividing thcr:c
gold particles equally between appressed membranes and outer
thylakoid lumens, and subsequently, assign ing ha l f the 1 um(~n;:ll
count ta appressed membranes and the ether hal [ t_o un;)pprr·:':;(',l
membranes
final
intreduces a
distribution.
further margin of ambiqu i ty
Completely disregarding
"
43
gol cl
ta the'
pa rt j c J (~
from ambiguous categories (i. e.,
counts
lumenal counts
and
"class 4" counts, equalling 33% of the total counts) does not
al ter the final resul t for PS l distribution,
degree of statistical significance. Thus,
concentrated
membranes,
on unappressed
and the
PS
than on
(38%)
observed distribution
except for the
I is still more
appressed
(62%)
is significantly
different from the expected distribution (O. 025 < P < O. 05).
Eliminating
however,
lumenal
results
appressed
counts
in
membranes
for
71.7%
of
versus
FCPC
(16%
go Id
28.3%
of the
particles
labelling
total),
labelling
unappressed
membranes. These values are statistically different from the
expected 66.6% on the appressed membranes and 33.3% on the
non-appressed membranes
preferential
(0.001
< P < 0.005),
and suggest a
association of Fepe wi th appressed membranes.
However, the differences from an equal distribution are small
and we conclude that FCPC is essentially equally distributed
on both types of membranes.
Photosystem
l
activity,
and
by
extension,
its
localization, was also demonstrated through the photooxidat ion
of
DAB.
product
Our
results
demonstrate that
often appeared in patches in
outer thylakoids.
more stain.
the
dense
both the central
However, the outer thylakoids
It is tempting to
reaction
and
contained
speculate that this result
implies that a larger proportion of PS l is located on the
outer
membranes,
but
it
may
simply
be
that
the
outer
thylakoids are more accessible ta DAB than are the inner ones.
(
44
The reason for the punctate apprearance of reaction producr
i:~
uncertain, but it is not likely to be a secondary effcct
of
DAB polymer migration, since the polymer
j
s bel ieved to rem.l in
close to the site of its formation (Frederick 1987). Pcrh,lp:;
this result reflects a non-homogeneous distribution of PS 1
wi thln
the plane of
a single membrane,
al though
the '101 li
particle labelling did not support this.
In
thylé1koid~;
higher plants, the organization of
l nto
grana and stroma apparently serves to optimize photosynthct il'
efficiency by physically segregating PS II and PS l
(nennctt
In this scheme, when the plant is subj ectcd t(, 1 i qht
1983).
that is
preferentially absorbed by PS II, a
mobile [orm or
grana-resident LHe-II becomes phosphoryldted
(Mul1ett 1(81)
and migrates
out
to
the
stroma
thylakoids
whcre
it
C,ln
transfer excess excitation energy to PS I. These VJêlve 1 cnqth
dependent changes in the distribution of excitation encrrJY
between the photosystems are known as state transi tiom;.
The resul ts presented here indicate a sl ight prefcrcncl!
of PS l
is,
for unappressed membranes, but a significant ,lmount
nevertheless,
Furthermore,
if the
indicative of
PS II
these complexes are
These
results,
assaciated
wi th
PS II-associated
localization,
appressed
mernhrél nQ~;.
FCPC may be
taken a:-;
it appears that both of
equally distributed on bath membrrl ncs.
along with
the
fact
that
the division
of
thylakoids into grana and stroma is not present in djFltomr;,
indicate that
in these organisms,
45
PS
land
PS II are
not
•
segregated to the ext:ent seen in green plants. Thus,
(
doubtful
that
photosynthetic
efficiency
in
it
diatoms
is
is
controlled in a manner analogous to higher plants. Indeed,
state transitions in diatoms have not, to our knowledge, been
found,
and it
is possible that FCPC rnay normally transfer
excitation energy to both photosystems (Owens 1986). There is
evidence,
can
however,
respond
that the diatorn cylindrotheca fusifarmis
ta variations
altering its PS II/PS l
in
ratio,
arnbient
dS
light
intensity
by
weIl as by modulating the
size of its light-harvesting complexes (smith and Melis 1988) .
Further research should clarify the rneans by which optimal
photosynthetic efficiency in diatorns is attained.
(
46
l
CHAPTER
47
III
--
---
------------
1. Introduction
is
It
establ ished
weIl
mitochondria)
that
chloroplasts
(and
are not autonomous organelles. Although they
possess discrete genomes capable of replication, as weIl as a
protein
synthesis
apparatus,
most
organelle
proteins
are
encoded in the nuclear genome, and synthesized on cytoplasmic
ribosomes
before
becoming
localized
in
mitochondria
or
chloroplasts (for reviews, see Chua and Schmidt 1979; Schmidt
and Mishkind 1986; Lubben et al. 1988). This phenomenon gives
rise to a question of considerable current interest: how are
nuclear-coded plastid proteins transported from their site of
synthesis
into the
chloroplast?
certainly,
this
is not
a
trivial question, because a thylakoid lumen prote in of green
algae and higher plants, for example, must cross a total of
three membranes to reach
membranes
of
the
thylakoid membrane.
classes
of
membranes
algae
of
i ts functional
chloroplast
envelope,
This problem is
whose
chloroplast
location:
as
weIl
compounded
chloroplasts
endoplasmic
are
as
the
in certain
limited
reticulum
the two
by
(CER)
two
in
addition to the two membranes of the plastid envelope (Gibbs
1981). Thus,
a thylakoid lumen prote in would have to cross
five membranes to reach its destination.
In 1977, Dobberstein et al. showed that the small subuni t
(SSU) of the Cal vin cycle enzyme ribulose 1, 5-bisphosphate
carboxylasejoxygenase
(RuBisco)
48
was
first synthesized as a
larger
precursor
of
molecular
weight
approximdte 1y
')(llHl
greater than that of the mature SSU. They proposcd thi1t tllC'
extra sequence in the precursor (preSSU)
functioned
ln the
transport of the prote in into the chloroplast. In add i tian,
since the preSSU entered chloroplasts and was subsequent 1 y
processed to its mature size aIl in the absence of protoin
1~78:
synthesis (Chua and Schmidt
Highfield and Ellis 1978),
it seemed that transport was a post-translational event.
Close
( J rn C))
examination of preS SU by Schmidt et al.
revealed the presence of an N-terminal sequence of am i no .le id:;
that is
removed during processing
sequence was
implicated
as
being
of preSSU to
responsible
SSIJ.
[or
'l'h i [;
prote i n
transport, and was appropriately named "transit sequence" (Tf;)
(Chua and Schmidt 1979). A number of other chloroplast prote in
precursors from a variety of species were shown to sha rc th
i:;
structural organization (Schmidt and Mishkind 1986), nnd th0
structure and composition of TS's has since been the subjoct
of many studies
1979;
(Schmidt and Mishkind 1986: Schmidt et
della-Cioppa et al.
Heijne et al.
1989).
1987;
Keegstra et al.
The importance
1989;
transport
von
of the TS in protei n
transport is underscored by the observation that its
abolishes
~1.
into the chloroplast
rcmov~J
(Mishkind ct
1985). Deletion and mutation analysis indicated that
il
1.
seq\l(>nc(>:~
at the N- and c-termini of the TS were essential for trdn!;port
to occur (Reiss et al. 1987; Smeekens et al. 1989), while th0
central portion, although highly conserved, was not
49
critic~l
.
:~
Furthermore,
the
TS
was
considered
to be
sufficient
for
directing import of proteins into chloroplasts on the basis of
experlments showing that chimeric precursors, consisting of
different TS's fused to foreign proteins, entered homologous
and heterologous chloroplasts,
processed to
their mature size
and were at least partially
(Lubben and Keegstra
1986;
Lubben et al. 1988). However, disparate results have arisen to
challenge this notion (e.g. Lubben et al.
1989; Smeekens et
al. 1989), and it remains possible that certain sequences in
the mature prote in may act in concert with the TS to effect
import by conferring a suitable three-dimensional conformation
on the precursor.
The entire process of protein import consists of three
discrete steps: binding of the precursor to the chloroplast
envelope,
translocation
across
the
membrane(s),
proteolytic processing of the precursor to
and
remove the TS.
Initially, binding of precursors to the envelope was thought
to be an energy-independent process (Pfisterer et al.
1982;
Cline et al. 1985), but a recent study has yielded results ta
the contrary (Olsen et al. 1989). Available evidence indicates
that binding results from the interaction of precursors with
membrane-localized proteinaceous receptors (Cline et al. 1985;
Bitsch and Kloppstech 1986; Cornwell and Keegstra 1987; Pain
et al. 1988), and that
~hese
receptors may be located in areas
of close envelope membrane appression, or contact sites (Pain
et al. 1988). However, to date, an envelope receptor has yet
(
50
to be purified.
The
process
of
translocation
is
definitely
C'llorqy-
dependent (Grossman et al. 1980; Pain and Blobel 1987; Thcq ot
al. 1989) and does not require a membrane potential, as
case for mitochondria. Unfortunately, the actual
translocation
remains
largely
unknown,
tho
15
mcch~ni~m 01
al thollqh
proh' i n
phosphorylation may be invol ved (e. g. SolI and Blichiln,lIl
Once the proteins are transferred to the chloroplast
1 <)1) 1) .
Gtrom~,
or during the transfer process itself, they undergIJ enzymlltic
processing to their mature size, possibly via intenncd Llte'
steps (Robinson and Ellis 1984; Mishkind et al. 1985;
al.
1986; Reiss et al.
order to
ot
M~rkH
1987). Much work is sti 11 ncot!ot!
characterize the
TS
sequences
invo] v(ld
in
in
r;uch
processing.
The chloroplasts of most groups of algae are limitcd by
four membranes rather than the two surrounding highcr plnnt
and green algal chloroplasts. In addition to the inncr two
membranes making up the chloroplast envelope êlre two
O\lh'l
membranes comprising the chloroplast endoplafjmic rc\t i ('III
(CER) (Gibbs
1981).
The outermost membrane of the
shawn to be studded with ribosomes
arranged
CEH
in
(Gibbs 1970). Moreover, in aIl species of algae
\lm
hilf; b00n
poly!';ome~;
possc~~~;
i nq
CER, there exists a network of vesicles and tubules cill10d th0
periplastidal reticulum lying between the CER dnù env 0 10fJP
r.1embranes (Gibbs
(1979)
1981).
These characteristics have l cù Gi bb:.
to prop0se that the polysomes on the outer
51
CER
mcmbrilnp
arc
engaged
in
the
synthesis
of
nuclear-coded
plastid
proteins, and that the periplastidal vesicles are involved in
the transport of these proteins into the chloroplast. Thus,
nuelear-coded proteins would be passed into the lumen of the
CER
in
a
co-translational
fashion,
analogous
to
that
of
secretory proteins (Ellis et al. 1980). Vesicles containing
these
proteins
membrane
and
would
then
pineh
subsequently
fuse
off
from
the
inner CER
with
the
outer
envelope
membrane, thereby depositing the proteins in the lumen of the
envelope.
Finally,
the proteins would
traverse
envelope membrane in an unknown fashion.
the
support
for this
hypothesis has come from a variety of morphological
1981)
and
evidenee.
experimental
(Smith-Johanssen
Interestingly,
Grossman
et
and
al.
inner
(Gibbs
Gibbs
1972)
(1991)
have
determined that the N-termini of fueoxanthin-ehlorophyll g!Q
proteins
of
the
diatom
Phaeodactylum
trico:rn'ltum
sequences that resemble the signal sequences
of
contain
secretory
proteins rather than the transit sequences of nuclear-coded
plastid proteins of higher plants and green algae.
We
have
employed
the
technique
of
immunoeleetron
microscopy in an attempt ta determine whether periplastidal
vesicles
are
involved
fueoxanthin-chlorophy1l
in
the
transport
prote in
gjç
complex
chloroplast of Phaeodactylum tricornutum.
that the labelling of the periplastidal
uncommon.
52
of
the
into
major
th~
The resul ts show
vesicles was very
2. Materials and Methods
2.1 Cell Culture
Phaeodactylum tricornutum Bohlin was obtained From the
Culture Collection of Algae at the university of Texas (UTEX
646) and cultured in f/2 medium as described previously (soc
Materials and Methods, p. 28).
2.2 Fixation and Embedding
For routine ul trastructural observation, cells were ri xcd
in
glutaraldehyde
and
osmium
according
to
the
procedure
described previously (see Materials and Methods, p. 29), and
embedded
in
Spurr's
epoxy
resin
(Spurr
1969).
immunolabelling experi.ments, fixation was carried out
For
for
ùfJ
routine observation, or according to the following reqimcn:
early stationary phase cells of
~.
tricornutum were hélrve:-;tcd
2-3 hours into the light period, and fixed for 60 min at
in
1%
glutaraldehyde
containing
O. 4M
in
sucrose.
containing decreasing
were
dehydra~ed
O.IM
Af ter
cacodylate
several
buffer,
washes
concentrations of sucrose,
in a graded ethanol series, and
pli
in
~oc
7.2,
bu f fer
the cc
l]:j
in[iltr~t0~
and embedded in Epon resin.
2.3 Immunolabelling
Antiserum raised against a mixture of the 19 and 19. ':) ).:Di1
polypeptides of the major fucoxanthin-chlorophyll Q/ç liqhtharvesting
complex
(FCPC)
of
53
f.
tricornutum
was
kindly
provided by A.
Grossman
(Oept.
of Plant Biology,
Carnegie
Institute of Washington, Stanford, CA). Antibodies were used
at dilutions of 1:100, 1:250 or 1:500. Labelling and staining
of
spurr-embedded
tissue
was
performed
as
described
in
Materials and Methods, p. 30. Preparation of the protein A-gold
used in the se labelling experiments is outlined on pp.28-29.
For the labelling of the Epon-embedded cells, grids containing
pale gold-coloured sections were floated section-side down on
drops of the following solutions: phosphate buffered saline
(PBS) , 10 min: 1% bovine serum albumin (BSA) in PBS, 15 mini
antiserum in BSA plus PBS,
anti-rabbit-gold
30 min;
PBS rinse,
2 min;
goat
(GAR-gold, E.Y. Labs, San Mateo, CA, USA),
diluted 1:30 in BSA plus PBS, 30 min. Controls were performed
by incubating sections on drops of PBS alone or on drops of
pre-immune serum, diluted 1:500 in BSA plus PBS in place of
the antiserum. AlI sections were stained with uranyl acetate
and lead citrate and viewed on a Philips EM 410 transmission
electron microscope operating at a voltage of 80 kV.
(
54
1
3. Results and Discussion
Figures
chloroplast
1
through
morphology
4
illustrate
that
are
features
pertinent
of
to
diatom
import
of
proteins into chloroplasts. The chloruplasts of diatoms ilrp
surrounded by four membranes. The outer two membranes campI" i
~;e
the chloroplast endoplasmic reticulum, whereas the inner two
membranes make up the chloroplast envelope
network
of
vesicles
is
reticu1um,
chloroplast
vesic1es
and
observed
envelope
are
tubules,
in
the
(Fig.
invariab1y
named
space
1) .
located
In
(F igs.
1-'1).
periplil~;t
the
between
the
the
i<l,ll
{lnd
CEI<
thc~;p
Phaeodactwm,
a10ng
side
1\
tlw
0 [
ch1orop1ast c10sest to the central region of the ccli. 'l'he
periplastidal reticulum has been jmplicated in the
tr(1n~;port
of proteins into chloroplasts (Gibbs 1979).
surroundi~g
the
reveals that the lumen of the
CEI~
Closer inspection of the four membranes
chloroplast
(;.'1gs.
2-4)
often appears to be dilated compared to that of the envclopC'.
Ribosomes
may
be
seen
associated
wi th
the
outermost
membrane (Figs. 2 and 5). It has been suggested (Gibbs
CEl<
107~)
that these ribosomes are engaged in the synthesis of nuclcarcoded plastid proteins
in a
fashion
analogous
to thflt
of
secretory protein synthesis on rough endoplasmic reticulum jn
the cytoplasm (Ellis et al. 1980). The arrows in Figure 1 show
that
the outer
CER membrane
il; continuous wi th
the
outer
membrane of the nuclear envelopE! 1 in accordance VIi th prov j
..
55
ou~:
1
1
Vigo 1. Cross-section through a cell of f. tricornutum showing
feiltures pertinent to transport of proteins into chloroplasts.
The chloroplast is surrounded by four membranes - two of the
chloroplast
endoplasmic
reticulum
(cer)
and
two
of
the
chloroplast envelope (ce) . Vesicles of periplastidal reticulum
(c.g.
small
chloroplast
arrows)
are
ER
chloroplast
and
seen
mitochondria; QY, pyrenoid. Bar
56
in
=
the
spa ce
envelope
0.3~m;
between
the
membranes.
m,
X 69,500
Fig. 2. Highly rnagnif ied view of the membranes surrounding the
chloroplast in
E.
tricornutum. Ribosomes (small arrows)
are
SGen associated with the outer membrane of the chloroplast
cndoplasmic reticulum (cer). The lumen of the chloroplast ER
is dilated compared ta that of the chloraplast envelope (ce).
QU, nucleus. Bar =
0.2~m;
X 102,500
Fig. 3. Longitudinal section through a cell of P. tricornutum.
The arrows designate areas of continuity between the outer
membranes of the chloroplast endoplasmic reticulum (cer) and
nuclear envelope. The arrowheads point to infoldings of the
inner CER membrane. nu, nucleus. Bar
FiCJ.
4.
section
tricornutum.
through
The
=
0.3J,Lm: X 76,000
the chloroplast of
arrowheads
point
to
what
cell
of
E.
appear
to
be
a
vesicles pinching off from the inner chloroplast endoplasmic
rGticulurn
(cer)
pyrenoid. Bar
=
membrane.
O.2~rn:
ce,
X 102,500
57
chloroplast
envelope:
RY,
observations in diatoms (see Gibbs 1981).
Figures 3 and 4 show what appear to be infoldings of the
inner
CER membrane.
These membrane
protrusions
have
been
observed previously in the Crysophyte alga Ochromonas danicà
(Gibbs
1979),
and
she
suggested
that
they
represent
pinching off of periplastidal vesicles from the
the
inner CER
membrane. These vesicles would purportedly enclose nuclearcoded proteins that had been deposited in the lumen of the CER
after being synthesized on the ribosomes associated with the
outer CER membrane. Subsequently, the vesicles would fuse with
the outer membrane of the chloroplast envelope, and deposit
the proteins
into the lumen of the envelope.
Finally,
the
proteins would cross the innermost membrane of the envelope
before reaching their functional destination.
Electron microscopic immunocytochemistry has shown i tsel f
a
useful
technique
in
localizing
subcellular cornpartments.
antigens
In this regard,
to
various
we have employed
this technique in an attempt to localize two proteins of the
fucoxanthin-chlorophyll g/ç light-harvesting complex (FCPC) of
E.
tricornutum
within
the
periplastidal
vesicles.
These
proteins have been previously shown to be nuclear-coded in
Phaeodactylum
(Fawley
and
Grossrnan
1986).
Success
in
localizing thern in the periplastidal vesicles would provide
cornpelling evidence for a
role of the vesicles
in protein
transport.
Figure 5 shows a cross-section of a cell of
58
E.
r
(
Fig. 5. Cross-section through a cell of
E.
tricornutum fixed
in glutaraldehyde, embedded in Epon resin, and labelled with
anti-FCPC antiserum (1:500 dilution), followed by GAR-gold (10
nm).
Labelling is specifie to the chloroplast (ç).
No qold
particles are observed over the periplastidal reticulum (QI).
Note
the
well-preserved
ribosomes
periplastidal compartment. Bar
=
Fig. 6. Epon-embedded section of
anti-FCPC
antiserum
(1:250
(arrows)
0.3~m;
E.
bordering
X 58,000
tricornutum labelled with
dilution)
and
GAR-gold.
labelling is seen over the periplastidal compartment (ru:).
=
0.2~m;
the
No
I3él
r
X 47,500
Fig. 7. spurr-embedded section of E. tricornutum labelled wi th
anti-FCPC antiserum (1:500 dilution), followed by protein Agold (4-5 nm). The periplastidal reticulum (N) is unlabell cd.
Bar
=
O.l~m;
X 107,500
Fig. 8. spurr-embedded section of E. tricornutum labelled wi th
1:100 anti-FCPC antiserum, and prote in A-gold (4-5 nm). Two
gold particles (arrows) are situated over the periplélstj Ùill
reticulum
(l2J;:).
These
gold
background labelling. Bar
=
particles
O.l~m;
59
probably
X 107,500
rcprCf;cnt
1
tricornutum that has been fixed in glutaraldehyde ùnd embc'ddcd
in Epon resin.
This section has been labelled with
rilbllÏ t
antiserum to the 19 and 19.5 kDa proteins of the FCPC of l'.
tricQrnutum,
rabbit)
label
followed by a
secondary antibody
complexed to 10 nm gold particles
is
clearly
predominantly
seen
lying
associated
over
wi th
the
the
(GAR-gold).
chloropli1st
Goltl
,
l
ilnd
c'
"
1 .. 111ellcll. . ,
thylakoid
including those traversing the pyrenoid.
.. nt i-
(goùt
Few gold pa rt icI c:;
'.
are observed in the cell' s cytoplasm or pyrenoid matrix,
01'
in
sections incubated in PBS or pre-immune serum in plùce of thC'
antibody (not shown). Importantly, no gold particl cs
lying over the periplastidal compartment
fact,
i1
Fig.
(l2..I,
rc !,('cn
')).
1 Il
lack of significant labelling over this reC"Jlon Wc)! . . )
consistent observation
(Figs.
6-8).
Two gold pa rticle~; ,lI"C'
seen lying over periplastidal vesicles in Figure 8, but th('!;(·
may be reflecting background labelling.
As with aIl negative results,
the se results
~;holild
hl'
interpreted with caution. The absence of any lobcllinq of th,·
periplastidal vesicles by anti-FCPC cannot be com;tr1l0d
cl!;
proof that they are not involved in the transport of nuclodrcoded pIast id proteins into the chloroplast. It is
that FCPC (and other chloroplast proteins)
Po~;!;
i Il 1
(>
i5 present in th!'
vesicles, but that its concentration i5 too low to ùllow lor
detection
by
the
immunolabell ing
possible to create conditions in E. tricornutum
were being synthesized in large quantities,
60
r fit
techn igue.
j
vif' rr·
n It/h i ch 1 CI'(:
locdlizdtion of
PCPC in the vesicles using immunoelectron microscopy might be
feasible.
Al ternati vely,
arrestation of the import process
itself might also enable use of the immunolabelling ter.hnique.
This
feat
has
been accomplished for
the transport
of the
enzyme polyphenol oxidase into the chloroplasts of a number of
plant species using a fungal toxin, tentoxin (e.g. Vaughn and
Duke 1981, 1984). However, whethar or not tentoxin, or other
compounds
will
be
found
that
exert
a
similar
effect
in
Phaeodactylum is not known. smith-Johanssen and Gibbs (1972)
have shown that growing the chrysophyte Ochromonas danica in
the presence of chloramphenicol (an inhibitor of chloroplast
protein
synthesis)
hypertrophy,
caused
the
periplastidal
vesicles
presumably because transport of nu
to
~lear-coded
proteins into the organelle was slowed down. This effect was
not observed in Phaeodactylum (unpublished observation) .
An alternative explanation for the lack of periplastidal
vesicle labelling is that these vesicles are not involved in
protein transport after aIl. Rather, i t is possible that a fter
entering the CER lumen, the plastid prote in crosses th€ inner
membrane of the CER and the two membranes of the chloroplast
envelope simultaneously
membranes
search,
no
elucidation
are
fused.
su ch
of
at a
Unfortunately,
contact
the
contact site where aIl
means
sites
by
were
which
after
an
observed.
three
exhaust:ve
Thus,
nuclear-coded
the
plastid
proteins enter the chloroplast of algae possessing CER will
have to await the results of future studies.
61
Conclusions
From
the
data
presented
here,
it
appeùrs
th,1t
thl'
extensive lateral heterogeneity of photosynthetic comploxC'!;
observed in higher plants is not characteristic of diatomG, ln
Phaeodactylum,
proteins of the
light-harvesting
distributed
complex,
equally
fucoxanthin-chlorophy 1 l
associated
amongst
with
appressed
PS
'1/1'
Il,
<11'('
uni1rrrC'r;~;C'd
and
membranes.
Photosystem l
is also present on both type!;
membranes,
but wi th a sl ight enrichment on the
membranes.
The distributions of these complexes
01
\.lnnppre~;!;l'd
must
h,w('
important consequences on the regulation of photo:.ynthf't i ('
efficiency in these organisrns. This is an intr i CJlll I1q proh 1pm
since
in
their
natural
aquatic
environrnents,
diiltom!;
rll ('
subj ect to wide-ranging fluctuations in ambient l ight qU,II i ty
and quantity.
It will be interesting to see how the othol-
complexes of the photosynthetic apparatus are distributod.
The role of the periplastidal reticulurn in the
of proteins into chloroplasts remains uncertain.
tr"n~;p()l't
Under the'
experimental conditions used,
the imrnunolabell i ne; techn i qU('
did
resolution
not
provide
sufficient
te
unequivocdlly
implicate the periplastidal vesicles in the irnport of
into
the
chloroplast
experimental
of
conditions,
E.
tricornuturn.
in
concert
biochemical techniques, should shed
1 ight
FCI'C
Manipulcltion
wi th
the
on the
li' ,f'
qLlQ~;t
fi
f
i nI) rd
the mechanism of protein transport in algae possess lnq CEP,
62
01
,,~;
weIl as on the raIe of the periplastidal reticulum in this
transport.
63
r
Literature cited
Akerlund H-E, Andersson B, Albertsson P-A (1976) Isolation ot
photosystem II enriched membrane vesicles From sp i n,wh
chloroplasts by phase partition.
Biochim Biophys Act,l
449:525-535
Allred DR,
Staehelin LA
cytochrome
complexes
b 6/f
of
Lateral distribution of tl1C'
(1985)
and
coupling
chIoroplast
factor
thylakoid
syntheti:l~>C'
ATP
membranes.
Pl ant
Physiol 78:199-202
Allred DR,
Staehelin LA
cytochrome
b 6 /f
Spatial organization of the
(1986)
complex
within
chloroplast
thy1<ü;o id
membranes. Biochim Biophys Acta 849:94-103
Anderson
JM
Consequences
(1981)
photosystem 1 and 2
of
spatial
separation
in thylakoid membranes of
01
hi qhe r
plant chloroplasts. FEBS Lett 124:1-10
Anderson JM (1982) Distribution of the cytochromes of np i n,wh
chloroplasts between the appressed membranes of
stacks and stroma-exposed thylakoid regions.
qriH1,J
FEUS Ictt
138:62-66
Anderson
JM,
Andersson
photosynthetic
B
(1982)
membranes:
The
lateral
architecture
and
of
tra n:CiVe n;p
organization. Trends Biochem Sci 7:288-292
Anderson
JM,
Melis
photosystems
in
A
(1983)
separate
Localization
regions
of
of
membranes. Proc Natl Acad Sei USA 80:745-749
64
differcnt
chl oropJ
(l~;t
Andersson B, Anderson JM (1980)
dist.ribut.ion
thylakoid
of
Lateral heterogeneity in the
chlorophyll-protein complexes
membr.anes
of
spinach
chloroplasts.
of
the
Biochim
Biophys Acta 593:427-440
Armand PA, Arntzen CJ (1977) Localizatian and characterization
of photo system II in grana and stroma lamellae.
Plant
Physiol 59:398-404
Armand
PA,
Staehelin
LA,
Arntzen
CJ
(1977)
relationship of photosystem l, photosystem II,
Spatial
and the
light-harvesting complex in chloroplast membranes. J Cell
Biol 73:400-418
Bennett J
(1983) Regulation of photosynthesis by reversible
phosphorylation of the light-harvesting chlorophyll g/b
protein. Biochem J 212:1-13
Berkaloff C, Duval JC, Hauswirth N, Rousseau B (1983)
Freeze
fracture study of thylakoids of Fucus serratus. J Phycol
19:96-100
Bitsch
A,
Kloppstech
K
(1986)
Transport
of
protein
into
chloroplasts. Reconstitution of the binding capacity for
nuclear-coded precursor proteins n
envelopes with detergents. Eur J
r
~~
~r
solubilization of
Biol 40:160-166
Callahan FE, Wergin WP, Nelson N, Edelmall M, Mattoo AK (1989)
Distribution of thylakoid proteins between stromal and
granal
lamellae
photosystem
in
Spirodela:
Dual
II components. Plant Physiol
65
location
91:629-635
of
f
Chua NH, Schmidt GW (1978) Post-translational trilI1Spol·t· intll
intact chlorop1asts of a precursor to the
it
~;ubun
SIllc111
of ribulose-1, 5-bisphosphate carboxylase. Proc Natl ACdd
Sci USA 75:6110-6114
Chu a
NH,
Schmidt
GW
(1979)
Transport
of
81:~Gl-~H
mitochondria and chloroplasts. J Cell Biol
Cl ine
K
(1988)
Light-harvesting
chlorophyll
intu
protC'.În:;
\
prut (' in.
!.l/!;J
Membrane insertion, proteolytic processing, asscmb 1y i
11
t ()
LHCII, and localization to appressed membranes oeclln; ln
chlorop1ast lysates. Plant Physiol 86:1120-1126
Cl ine K,
Werner-Washburne M,
Lubben TH,
Keegst ra
K
(1 q~: t»)
Precursors to two nuclear-coded chloroplilst prote i Il::
III nt!
to the outer envelope membrane before being unportc'd
1 r1t-n
chloroplasts. J Biol Chem 260:3691-3696
Cornwell KL,
Keegstra K (1987)
Evidence that
il
chloropl.\::t
surface protein is associated with a spec i t iL
b i ncll nq
site for the precursor to the slnall subunlt of rihlll():;(·1,5-bisphosphate carboxylase. Plant Phys iol Rt}:
'n~()- n~
Cox RP, Andersson B (1981) Lateral and transvcrr;c orqan
of cytochromes in the
chloroplast thyl ako id
t)
I:;.lt l 'lIl
mpml>r
111('.
Biochem Biophys Res Commun 103:1336-1342
Dahl in C (1989) Immunogold local ization of LIIC 11 proto in': in
chloroplasts with different degrees of
gril 11,1
i
IV)
(l'Ji;
1)
!;tdd:
induced by SAN-9789. Physiol Plant 76:438-444
Della-Cioppa
G,
Kishore
GM,
Beachy
R11,
rralcy
PT
Protein trafficking in plant cells. Plant Phy!jlO)
968
66
(lI!:
'J(,',-
Di Paolo ML, DaI Belin-Peruffo A, Bassi R (1990) Immunological
studies
on
chlorophyll-a/b
distribution
in
thylakoid
proteins
membrane
and
their
domains.
Planta
181: 275-286
Dobberstein B, Blobel G, Chua NH (1977) In vitro synthesis and
processing of a putative precursor for the srnall subuni t
of ribulose-1, 5-bisphosphate carboxylase of Chlamydomonas
reinhardtii.
Dwarte
DM,
proc Natl Acad sci USA 74:1082-1085
Vesk
M
(1982)
Freeze-fracture
thylakoid
ultrastructure of representative members of "chlorophyll
Cil
algae. Micron 13:325-326
Dwarte D, Vesk M (1983) A freeze-fracture study of cryptomonad
thylakoids. Protoplasma 117:130-141
Ellis RJ, Sr"ith SM, Barraclough R (1980) Synthesis, transport
and assembly of ch1oroplast proteins.
Genome
Expre~sion
Organization and
In Leaver CJ
in Plants,
(ed.)
Plenum
Publishing Corp., New York, pp. 321-335
Fawley
MW,
Grossman
harvesting
AR
complex
(1986)
of
Polypeptides
the
tricornutum are synthesized
j
diatom
of
a
light-
Phaeodactylum
n the cytoplasm of the cell
as precursors. Plant Physiol 81:149-155
Freder ick SE (1987) DAB procedures.
In Vaughn KC (ed.) CRC
Handbook of Plant Cytochemistry, Vol.
Raton,
Ghirardi ML,
pp. 3-23
Melis A (1983)
Localization
electron transport cornponents
sheath
l, CRC Press, Boca
of photosynthetic
in rnesophyll
and bundle-
chloroplasts of Zea rnays. Arch Biochem Biophys
224:19-28
67
Gibbs
SP
The compara t ; 'Je ul trastructurC'
(1970)
0
the ,11 '1,11
t
chloroplast. Ann NY Aead Sei 175:454-47]
Gibbs
SP
The
( 1979)
synthesized
route
proteins
of
entry
into
of
cytoplilsm 1 <"'"<11 1 y
chloroplasts
or
alfJ<lC'
possessing chloroplast ER. J Cell Sci 35:253-266
Gibbs
SP
The
(1981)
chloroplast
rot i CUIUlll:
endoplasmic
structure, function, and evolutionary siqniric<1l1cc. Int
Rev Cytol 72: 49-99
Goodchild
Anderson
DJ,
Immunocytochemical
Andersson
JM,
( \ (HI'''l)
B
localization of the cytochroIno h/l
compi ex of chloroplast thylakoid membranes. CC' li ni 01 1nt
Rep 9:715-721
Goodc!"lild
Andersson
DJ,
Immunocytoehemical
associated
w i th
Anderson
B,
localization
the
oxygen
,lM
(\')H'.b)
po 1ypr'pt i dl",
01
evoJ v inq
(lI
photosynthesis. Eur J Cell Biol 36: 294 -298
Grevby
C,
Axelsson L, Sundqvist C
plastid
differentiatlon
in
(1989)
the
Liqht- i
brown
alqû
nd('pc~nd('nt
1
n<1 ri
Il
pL":('
poIYl1cptid(,:;
1)';
L,'m
d
Saccharina (Phaeophyceae). Phycologia 28: 37 5-J81
Grossman A, Bartlett S, Chua NH (1980) Energy-dcpendcnt
of
cytoplasmically
chloroplasts.
Grossman A,
proteins
Nature 285: 625-628
Manodori A,
of
chlorophyll
Q,
synthesized
Snyder
diatoms:
0
their
(1991)
Liqht-h,lrvr':,t 1 nq
relat j on:,h i p
tu
12 binding proteins of hiqher pl,mt"-:;
their mode of transport
into
(submi tted)
68
plastids. Mo l
Con
t
h('
<1
rl' 1
(;('n('t
Hall
(
DO,
Edge H,
Kalina M (1971)
photoreduction
in
the
The site of
larnellae
of
ferricyanide
isolated
spinach
chloroplasts: a cytochemical study. J Cell Sci 9:289-303
Highfield PE, Ellis RJ (1978) Synthesis and transport of the
srnall
subunit
of
chloroplast
ribulose
bisphosphate
carboxylase. Nature 271:420-424
Hinshaw .JE, Miller KR (19139) Local ization of l ight-harvesting
complex I I to the occluded surfaces of photosynthetic
membranes. J Cell Biol 109:1725-1731
Izawa S (1980) Acceptors and donors for electron transport. In
San Pietro A (ed.) Methods in Enzyrnology, Vol. 69 Part C,
Photosynthesis and Nitrogen Fixation, Academic Press, New
York, pp. 413-434
Keegstra K, Olsen I.J, Theg SM (1989) Chloroplastic precursors
and their transport across the enve10pe nembranes. Annu
Rev Plant Physiol Plant Mol Biol 40:471-501
Lichtle C,
Duval JC,
Hauswirth N,
Spilar A
(1986)
Freeze
fracture study of thylakoid organization of Cryptomonas
rufescens
(Cryptophyceae)
according
to
illumination
conditjons. Photobiochem Photobiophys 11:159-171
Lichtle C, McKay RML, Gibbs SP (199.L) Irnmunogold loca li zation
of
PSI
and
thylakoids
PSII
of
light-harvesting
Cryptomonas
complexes
rufescens
in
the
(Cryptophyceae).
Planta (subrnitted)
Lubben
TH,
Gatenby
M,
Ahlquist
P,
Keegstra
K
(1989)
Chloroplast irnport characteristics of chirneric proteins.
Plant Mol Biol 12:13-18
69
Lubben TH, Keegstra K
(1986)
impo~"t
Efficient in vitro
ot
cytosolic heat shock protein into pea chloropldsü-;.
,l
l't'Ol'
Natl Acad Sei USA 83: 5502-5506
Lubben TH, Theg SM,
Keegstra K (1988)
Transport 01
pt"oh>in:.
into chlorop1asts. Photosyn Res 17:173-194
Ludwig M, Gibbs SP (1989) Localization of phycocrythrln dt tilt'
lumenal surface of the thylakoid membrùne in
Rl1ot!olllol1d:;
lens. J Cell Biol 108:875-884
Maenpaa
Pt
Andersson
sensitivity
to
B,
Sundby
C
(1987)
photoinhibition
DltlC'rClll'C'
betwcen
III
II
111
rh('
appressed and non-appressed thylakoid reg ion~;.
l'Efl~;
I.e' tt-
215: 31-36
Manodori At Grossman AR (1990) Sequence homology b(·twcC'n Ilqht
harvesting
polypeptides
Phaeodactyl'Jm
Current
of
tricornutum.
Research
in
plants
In
and
Baltschc[UÜ:y
Photosynthesis,
Il 1,1 t" (llll
tll0
Vol.
M
(r'd.)
1\111.,.;('1"
~,
Academie Publishers, Amsterdam, pp. 511-S44
Marks DB,
site
Keller BJ,
Hoober JK (1986) A secondùry pro('(".'.1
in the precursor of the small subunit of
rIlllllo'.r.
bisphosphate carboxylase of ChlamydomoJE!fi rr' i nhd nll
1. Plant Physiol
I1rJ
1
1 y-
81:702-704
Marty D (1977) Cytophys.i.olog ie vegetale - loce1} i
Zdt
1on
III t" t",j-
structurale des sites d'activite des photosy:,tpmf':; 1 (·1
II dans les chloroplastes in si tu.
C. R. Acad
~;c
(1',)
r
J '.)
285D: 27-30
McKay RML, Gibbs SP (1990)
pyrenoid
of
Phycoerythrin is ab:.wnt
Porphyr idium
cruentum:
implications. Planta 180:249-256
70
f rom
rh,·
photo~;yntlJ,.·t 1 ('
McKay
RML,
Gibbs
SP
(1991)
Composition
cytochemical
pyrenoids:
and
function
of
immunocytochemical
and
approaches. Can J Bot, in press
McLdchlan J
(1973) Growth media - marine. In Stein JR (ed.)
Handbook of Phycological Methods.
Culture Methods and
Growth Measurements. cambridge Univ. Press, New York, pp.
25-51
Melis A, Homann PH (1978) A selective effect of Mg 2+ on the
photochemistry
at
one
type
of
photosystem
II
of
chloroplasts.
Svensson
P,
Albertsson
reaction
Arch
center
Biochem
in
Biophys
190:523-530
Melis
A,
organization
of
the
PA
ch1oroplast
(1986)
The
thy1akoid
domain
membrane.
Localization of photo system l and of the cytochrome b 6 -f
complex. Biochim Biophys Acta 850:402-412
Mi 11er KR (1980) A chloroplast membrane lacking photosystem I.
Changes in unstacked membrane regions.
Biochim Bicphys
Acta 592:143-152
Miller KR, Staehelin LA (1976) Analysis of the thylakoid outer
surface: coupling factor is 1imi ted to unstacked membrane
regions. J Ce11 Biol 68:30-47
Mishkind
ML,
Wessler
detE'~minants
maturation
SR,
Schmidt
GW
in transit sequences:
by
vascular
plant
(1985)
Functional
import and partial
chloroplasts
of
the
ribulose-l,5 bisphosphate carboxylase small subunit of
Chlamydomonas. J Cell Biol 100:226-234
71
Morrissey
PJ,
McCauley
Melis
SW,
A
Dirferentl,ll
(1986)
detergent-solubilization of integral thylakoid
complexes
in
spinach
chloroplasts.
L~élt
Local
photosystem II, cytochrome be, -f complex élnd
lllelllbr,\Iw
ion
01
photm;y~;t l'Ill
1. Eur J 8iochem 160:389-393
Mullet JE
(1983) The amino acid sequence
segment
which
stacking)
regulates
0
membrane
in chloroplasts. J
the pol ypC'pt i dl'
f
adhcs 1 on
2~8:q941-004R
Biol Chem
Mullet JE, Burke JJ, Arntzen CJ (1980)
(q 1',1 nd
Chlorophyll-protc>ln~;
nI
photo system 1. Plant Physiol 55:814-822
Murphy
The
(1986)
DJ
photosynthetic
molecular
membranes
of
organization
higher
o l t 111'
plants.
BI CH"h 1 III
Biophys Acta 864:33-94
Nir
l,
Pease
OC
Chloroplast
(1973)
orgélnizeltlcn
dnd
tl1f>
ultrastructural localization of photosystC'lll!; r dnd 1 [. ,J
Ultrastruc Res 42:534-550
Olive
J,
Wollman
Local ization
FA,
of
Bennoun
the
core
P,
and
ReCOllvreur
peripheral
photo system l in the thylakoid membranes of
M
(j')f\
antenll<l0
Il
() 1
~bJ.0Jl1ydomon()~;
reinhardtii. Biol Cell 48:81-84
Olsen
LJ,
Theg
required
SM,
for
Selman
the
BR,
binding
Keegstra
of
K (1 f'JB'1)
precursor
AT\'
prote' i 1)',
l',
tn
chloroplasts. J Biol Chem 264:6724-672'1
Owens
TG
(1986)
Light-harvesting
tunction
in
the
(J i ;ltOlll
Phaeodactylum tricornutum. II. Distribution of cxci Lit i UI)
energy between the photosystems. Plant Physjol
72
BO:'J'l'J-ïl)(,
Pain
D,
Biobei G
(1987)
protein
import
requires a chloroplast ATPase.
into chioroplasts
Proc Natl Acad Sci USA
84:3288-3292
Pa in
D,
Kanwar
Blobel
YS,
G
(1988)
Identi f ication
of
a
for protein irnport into chloroplasts and i ts
receptor
localization to envelope contact zones. Nature 331:232237
Park RB, Sane PV (1971) Distribution of function and structure
in chloroplast lamellae. Ann Rev Plant Physiol 22: 395-4JO
Pt isterer J,
Lachmann
proteins
into
P,
Kloppstech K
chioroplasts:
(1982)
binding
of
Transport of
nuclear-coded
chioroplast proteins to the chioroplast envelope. Eur J
Biochem 126:143-]48
Reiss B, Wasmann CC, Bohnert HJ (1987) Regions in the transit
peptide of SSU essential for transport into chlC'roplasts.
Mol Gen Genet 209:116-121
Rhiel
E,
Kunz
J,
Wehrmeyer
W (1989)
Immunocytochemical
localization of phycoerythrin-545 and of a chlorophyll
S!/ç
light-harvesting
complex
in
Cryptomonas
maculata
(Cryptophyceae). Bot Acta 102:46-53
Rhiel E, Morschel E, Wehrmeyer W (1985) Correlation of pigment
depri vat ion and ul trastructural organization of thylakoid
membranes
in
Cryptomonas
waculata
foilowing
nutrient
deficiency. Protoplasma 129:62-73
Robinson
C,
Ellis
chloroplasts:
RJ
(1984)
Transport
of
proteins
the precursor of smail subuni t
into
ribulose
bisphosphate carboxylase is processed to the mature size
in two steps. Eur J Biochem 142:343-346
73
Roth
J
(1982)
The
qual i tati ve
protein
and
A-gold
quanti tati ve
(pAg)
techn i que
approdch
for
,1
,lnt i qL'n
local ization on thin sections. In Bullock GH, l'ct nI:;;. l'
(eds. )
Techniques
in
Immunocytochemist ry ,
Vol.
1,
Academie Press, New York, pp. 127-133
Sane PV,
Goodchild DJ,
Park RB
Charact0r 1 ~,lt ion 01
(1970)
chloroplast photosystems l
and II separilted by
d
110n-
detergent method. Biochirn Biophys Acta 21G:162-1ï8
Schmidt GW, Devillers-Thiery A, Desruisseaux H, BIobel \', Chu.)
NH (1979) NH 2-terminal amino acid sequences of preL'u r!~()1 ",
and
mature
forms
of
the
ribulose-1,5-bisphosph~tv
carboxylase small subuni t from Chlamydomonas
r~ LnJl~\ (dt
1i .
J Cell Biol 83:615-622
Schmidt GW, Mishkind ML (1986) The transport of prote i n~; i 11\
U
chloroplasts. Ann Rev Biochem 55:879-912
Seibert
DeWit
M,
M,
Staehelin
LA
(1987)
local ization of the 0z-evol v ing apparatcls to mult i me t- i ('
(tetrameric) particles on the lumenal sur(clce of 1 n'('I.('etched photosynthetic membranes.
J
Cell Biol
1U'):?;J',j-
2265
Shaw
PJ,
Henwood
JA
(1985)
Immuno-gold
10c,1l
ÎZdt
Ion
01
cytochrome f, light-harvesting complex, A'1'P synthd!;Û rlnrl
ribulose 1, 5-bisphosphate
carboxylase/oxygena~~c.
P! dnt d
165:333-339
Simpson DJ,
of
Bassi R, Hinz UG (1987) Cell-specUic 0:.:prr,!:<.ion
LHCII
reaction
Wettstein
and
the
centres
D,
Chua
organisation
in
NH
of
chloroplast
(eds.)
the
thy la~:o 1 cJs.
Plant fv'Iolecu),lr
Plenum Publishing Corp., New York, pp.
74
photo~;yntl)('t 1 C
91-]04
1n
von
llio!(JCJ'I,
simpson DJ, Bassi R, Vallon 0, Hoyer-Hansen G (1988) Location
and
orç;anization
of
the
photosynthetic
reaction
Grass G,
DO
Hall
chlorophyll-proteins
centres
(eds.)
in
higher plants.
Photocatalytic
of
In
Production of
Energy-Rich Compounds, Elsevier Appl ied Science, pp. 195209
simpson DJ, Vallon 0, von Wettstein D (1989) Freeze-fracture
studies on barley plastid membranes VIII. In viridis- 115 ,
a
mutant
evolution
cytochrome
completely
enhancer
b-559
lacking
1
photosystem
(OEEl)
accumulate
and
in
the
II,
oxygen
a-subunit
appressed
of
thylakoids.
Biochim Biophys Acta 975:164-174
Simpson DJ, von Wettstein D (1989) The structure and function
of the thylakoid membrane. Carlsberg Res Commun 54:55-65
Slot JW, Geuze HJ (1981) Sizing of protein A-colloidal gold
probes for immunoelectron microscopy. J Cell Biol 90:533536
Smeekens S, Geerts D, Bauerle C, Weisbeek P (1989) Essential
function in chloroplast recognition of the ferredoxin
transit peptide processing region. Mol Gen Genet 216: 178182
smith BM, Melis A (1988) Photochemical apparatus organization
in
the
diatom
Cylindrotheca
fusiformis:
photosystem
stoichiometry and excitation distribution in cells grown
under high and low irradiance.
Plant Cell Physiol 29:
761-769
Smi th-Johanssen H, Gibbs SP (1972) Effects of chloramphenicol
on
chloroplast
and
mitochondrial
ultrastructure
Ochromonas danica. J Celi Biol 52:598-614
75
in
SolI
J,
Buchanan BB
Phosphorylation
(1983)
of
ch 1 orop l ,1~,t
ribulose bisphosphate carboxylase/oxygenase s\ll<ll 1 subun 1 t
by an envelope-bound protein kinase in si tlJ.
a Rio 1 Che!11
258:6686-6689
Spear-Bernstein L,
Miller KR
(1985)
Are the photosyntl1C't iL'
membranes of cryptophyte 3.lgae inside out? Protopl,l!;m<l
129:1-9
Sprague
SG,
Camm
EL,
Reconstitution
of
Green
BR,
Staehelin
light-harvesting
LA
(I<)B ' ))
complex0!.-;
.,ntl
photosystem II cores into galactolipid and phosphol
1\11<1
liposomes. J Cell Biol 100:552-557
Spurr AR (1969)
A low-viscosity epoxy resin embcddinq medllll11
for electron microscopy. J Ultrastruct Res 26: 11-4 1
Staehel in LA (1976) Reversible particle movements
<1f;::;O('
i ,ltf'd
with unstacking and restacking of chlorapl élst mcmbr .111(':;
in vitro. J
Cell Biol 71:136-158
Staehelin LA (1986) Chloroplast structure and suprùmolccuLl1
organizatian of photosynthetic membranes. In Pi rson 1\,
Zimmermann
MH
(eds.)
Encyclopedia
of Pl<lnt
fJhy~;j()I()qy
(New Series), Vol. 19, Springer-Verlag, Berl in, pp.
Staehelin LA,
Arntzen CJ
membrane function:
(1983)
Regulation
of
1-;:,1
chlornpl,,:;t
protein phosphoryldt ion chtlnqc:; tllf'
spatial organization of membrane components . .]
Cc 1 1 1\
i (J 1
97:1327-1337
Svensson
P,
Albertsson
P-A
enriched photosystem II
(1989)
Preparation
membrane
vesiclcs
detergent method. Photosyn Res 20:249-259
76
of
by
hlCfhly
r1
n()l1-
Sweeney BM (1981)
Freeze-fractured chloroplast membranes of
Gonyaulax polyedra (Pyrrophyta). J Phycol 17:95-101
'rheg SM,
Bauerle C, Olsen lJ, Selman BR,
Internal
ATP
is
the
only energy
Keegstra K (1989)
requirement
for the
translocation of precursor proteins across chloroplastic
membranes. J Biol Chem 264:6730-6736
Thornber JP (1986) Biochemical characterization and structure
of
pigment-proteins
Pirson A,
Physiology
of
photosynthetic
Zimmermann MH
(New
(eds.)
Series),
organisms.
Encyclopedia of
Vol.
19,
In
Plant
springer-verlag,
Berlin, pp. 98-142
'rschopp J (1984) Ul trastructure of the membrane attack complex
of complement. Heterogeneity of the complex caused by
different
degree
of
C9
polymerization.
J
Biol
Chem
259:7857-7863
Vallon 0, Wollman FA, Olive J (1985) Distribution of intrinsic
and extrinsic
between
subunits
appressed
thylakoid membrane:
and
of the
PS
II
protein
complex
non-appressed
regions
of
an immunocytochemical
study.
the
FEBS
Lett 183:245-250
Vallon 0, Wollman FA, Olive J
the
main
protein
(1986) Lateral distribution of
complexes
of
the
photosynthetic
apparatus in Chlamydomonas reinhardtii and in spinach: an
immunocytochemical study using intact thylakoid membranes
and a PS II enriched membrane preparation. Photobiochem
Photobiophys 12:203-220
77
,
if
Vaughn KC (1987) Photosynthetic partial reactions. In Vauqhn
RC (ed.) CRC Handbook of Plant Cytochemistry Vol 1 T:
Other cytochemical staining procedures, CRe Press, Boen
Raton, pp. 37-43
Vaughn KC, Duke sa (1981) Tentoxin-induced 105s of
polyphenol oxidase. Physiol Plant 53:421-428
p1a~tidic
Vaughn RC, Duke sa (1984) Tentoxin stops the proccssing 01
polyphenol oxidase into an active protein. Phys iol 1'1,1 nt
60:257-261
Vaughn RC,
Vierling E,
Duke sa,
Alberte RS
( 1<:lB -3)
Immunocytochemical and cytochemical localization 01
photosystems l and II. Plant Physiol 73:203-207
Von Heijne G, Steppuhn J, Herrmann RG (1989) Domain structure'
of mi tochondrial and chloroplast targeting pcptidcf;. Eu)'
J Biochem 180:535-545
Webber AN, Platt-Aloia KA, Heath RL, Thomsor WW (1 <:l8B) ')'IH'
marginal regions of thylakoid membranes: a parti~1
characterization by polyoxyethylene sorbitan monolaur,ltc
(Tween 20) solubilization of spinach thylako ids. l'hy~; i 01
Plant 72:288-297
Ultrastructural
Wrischer
M
(1978)
diaminobenzidine photooxidation in
Protoplasma 97:85-92
n!
locali7.ation
etiochl oropl d~',t~;.
Wrischer M (1988) Cytochemical localization of the activity of
photosystem II in bean etiochloroplasts. Acta Bot Cro~t
47:25-28
78
localization
or
UI trastructural
(1989 )
Wrischer
M
photosynthetic activi ty in thylakoids during chloropl ,l~,t
development in maize. Planta 177: 18-23
'79