2/28/08
“A
molecular
Caliper
Mechanism
for
Determining
Very
Long‐Chain
Fa<y
Acid
Length”
Vladimir
Denic
and
Jonathan
S.
Weissman
Presented
by:
Myrthe
Braam
Bas;aan
Bijl
Agon
Hyseni
Tessa
Quax
Supervised
by:
Toon
de
Kroon
Very
long‐chain
faIy
acids
• VLCFA’s
are
hydrocarbon
chains
ranging
from
20
to
36
carbons
• Different
chain
length
enables
essen;al
roles
like:
– Stabiliza;on
of
high
curved
cellular
membranes
– Permeability
barrier
of
the
skin
– Signaling
• Defects
in
VLCFA
metabolism
are
associated
with
human
peroxisomal
disorders
VLCFA
synthesis
I
• VLCFA’s
are
synthesized
by
the
elonga;on
of
shorter
faIy
acids
produced
in
the
cytosol
by
FAS
(faIy
acid
synthase)
• VLCFA
biosynthe;c
machinery
is
embedded
in
the
ER
membrane
1
2/28/08
VLCFA
synthesis
II
VLCFA
synthesis
occurs
in
a
four‐step
biochemical
cycle
VLCFA
synthesis
III
• Several
enzymes
required
for
the
elonga;on
cycle
have
been
iden;fied
in
yeast.
• Elops
are
required
for
the
first
step
in
the
cycle
and
determine
the
length
of
cellular
VLCFA
products
• The
dehydratase
ac;ng
during
the
thirth
step
of
the
cycle
has
not
been
found
yet.
Research
ques;on
How
do
Elops
instruct
the
components
of
the
elonga;on
cycle
to
extend
shorter
FA’s
in
two‐carbon
addi;on
steps
to
yield
VLCFA
products
of
defined
lengths?
2
2/28/08
Results
in
summary
In
this
ar;cle
it
is
shown
that:
• Phs1p
is
the
missing
component
of
the
four‐step
elonga;on
cycle
• Elops
are
a
novel
family
of
condensing
enzymes
determining
the
VLCFA
length
by
a
caliper‐like
mechanism
• It
is
possible
to
recons;tute
the
complete
elonga;on
cycle
in
vitro
from
purified
components
• New
VLCFA’s
can
be
produced
by
ra;onal
design
of
Elops
Results:
Phs1p
• Phs1p
depleted
membranes
were
strongly
compromised
for
elonga;on
• The
first
two
steps
of
the
elonga;on
cycle
were
not
compromised
by
Phs1p
deple;on
Phs1p is responsible for the third step in the elongation cycle
Results:
Elops
• The
role
of
Elops
Fen1p
and
Sur4p
is
explored
in
vitro
• Proteoliposomes
with
Fen1p
or
Sur4p
catalyzed
forma;on
of
the
3‐keto
product
Fen1p and Sur4p catalyze the first step in the elongation cycle
3
2/28/08
Results:
Elops
II
• Elops
contain
a
HXXHH
mo;f
which
is
essen;al
for
func;on
in
vivo
• The
HXXHH
mo;f
is
not
required
for
incorpora;on
into
a
mul;protein
complex
3D
3C
Elops use a novel mechanism to initiate two-carbon elongation
Observa;ons
• ER
membranes
containing
a
single
Elop
can
convert
substrates
of
different
length
into
the
same
end
product
• ER
membranes
with
different
Elops
can
convert
the
same‐length
substrate
into
VLCFAs
with
different
length
Conclusion:
• Elops
are
able
to
specify
VLCFA
length
in
absolute
terms
4
2/28/08
Chain
lenght
specifica;on
Previously
shown:
• Δsur4p
cells:
lacking
C26;
overexpression
of
Fen1p
in
these
cells
fails
to
restore
C26
• Δfen1
cells:
amount
of
C26
is
increased
by
overexpression
of
Sur4p
• Only
condensa;on
of
short
substrates
possible:
follow
up
Proteoliposome
crea;on
• • • • Flag
tag
enzymes
and
purify
with
beads
Keep
proteins
in
detergent
to
form
micells
Add
synthe;c
phospholipids
Wash
away
detergent
with
biobeads
Remark:
• 4
proteins,
all
in
right
orienta;on
Chain
length
specifica;on
Incuba;on
of
proteoliposomes
with
C18‐CoA
and
radioac;ve
labelled
Malonyl‐CoA
Conclusion:
Sur4p
is
needed
for
elonga;on
of
substrate
5
2/28/08
Chain
length
specifica;on
Ques;on:
How
is
Sur4p
able
to
support
elonga;on
of
shorter
substrates?
• Sur4p
specifies
VLCFA
lenght
by
termina;ng
a
processive
elonga;on
mechanism
following
the
produc;on
of
the
C26
end
product.
Next:
Explora;on
of
the
u;lity
of
the
recons;tuted
system
for
studying
other
elops:
• Subs;tu;on
of
Fen1p
for
Sur4p
• Evalua;on
of
TbElo1p,
a
T.
Brucei
Elop
homologue
required
for
synthesis
of
C10
Chain
length
specifica;on
Conclusion:
Enzymes
2,3
and
4
provide
versa;le
cataly;c
plahorm
for
Elops
Chain
length
specifica;on
Ques;on:
How
do
individual
elops
specify
the
length
of
a
VLCFA
molecule?
6
2/28/08
Determina;on
of
chain
length
output
Conclusion:
Sur4p
started
behaving
like
Fen1p
Engineering
chain
length
output
K266
K266
Engineering
chain
length
output
7
2/28/08
Engineering
chain
length
output
Proposed
model
Discussion
• Polymeriza;on:
DNA,
RNA,
pep;des
• VLCFA
synthesis
requires
other
strategy
• Length
determina;on
within
machinery
itself
This
study
shows:
• Caliper‐like
mechanism
to
measure
VLCFA
length
• Fixed
site
within
Elop
• Subject
to
muta;onal
sliding
8
2/28/08
Discussion
• Condensa;on
steps
occur
at
cytosolic
side
• Lysine
residues
near
luminal
end
of
Elop
specify
acyl
length
• Termina;on
when
end
of
acylgroup
reaches
Lysine
Ques;ons
9