Transcript Biophysics_lecture(17Nov06)

Biogenesis of energytransducing membranes in
chloroplasts and mitochondria
Prof. Patrice Hamel
Department of Plant Cellular and Molecular Biology
and
Molecular and Cellular Biochemistry
([email protected])
Mitochondria and chloroplasts are related
organelles involved in the conversion of energy
Mitochondria
Respiration
(NADH, ATP)
Chloroplasts
Photosynthesis
(NADPH, ATP)
Mitochondria and chloroplasts are related
organelles involved in the conversion of energy
nucleus
DNA
DNA
Mitochondria
Chloroplasts
Mitochondrial inner membrane
Thylakoid membrane
Specialized membranes containing multimeric complexes involved in electron transfer reaction
ATP
ADP + Pi
Fd
2NADP++ 2H+
4H+
stroma
FNR
2NADPH
PQ
PQ
lumen
4H+
2 H2O
4H+
+ O2
3 H+
ATP synthase
plastocyanin
PSI
PSII
cyt b6f
c-type cytochromes are essential
for photosynthesis
stroma
lumen
cyt f
cyt c6
plastocyanin
PSI
PSII
cyt b6f
What are c-type cytochromes?
CXXCH motif
Cys
Cys
heme
p-side: periplasmic space
Bacteria
p-side: intermembrane space p-side: thylakoid lumen
Mitochondria
Plastids
The cytochrome c biogenesis question
Bacteria
heme
apocyt c
p-side
p-side
Mitochondria
p-side
S S
holocyt c
Plastids
Biochemical requirements to complete
cytochrome c maturation
p-side
Heme transport
Biochemical requirements to complete
cytochrome c maturation
p-side
p-side
3+
S
S
Heme transport
2+
HS
HS
Reduction of heme
and cysteine thiols
Biochemical requirements to complete
cytochrome c maturation
p-side
p-side
3+
S
p-side
S
Heme transport
HS
2+
HS
HS
S S
HS
Reduction of heme
and cysteine thiols
Catalysis of thioether bond
linkage (heme lyase)
Beat Michigan!
Yeahh!
Chlamydomonas, green and cool!
F
N
CP
M
 Fresh water unicellular alga with compatible mating types (mt+ and mt-)
Can grow in the light (photosynthesis) or on a carbon source (respiration)
Nuclear genome entirely sequenced and annotated
Amenable to molecular genetic manipulation of the nuclear, chloroplast
and mitochondrial genomes
Chlamydomonas reinhardtii, a model to study
plastid cytochrome c biogenesis
Minimal (high light)
Acetate (low light)
 Chlamydomonas photosynthetic mutants are viable
WT
mutant
WT
mutant
Cytochrome f and cytochrome c6 function
in photosynthesis in Chlamydomonas
Minimal (high light)
Acetate (low light)
 Chlamydomonas photosynthetic mutants are viable
 Two c-type cytochromes function in photosynthesis:
membrane-bound cyt f and soluble cyt c6
WT
mutant
WT
mutant
PSI
stroma
b6f
thylakoid membrane
thylakoid
cyt f
ee-
Plastids
cyt c6
lumen
Biogenesis of holocytochrome f and holocytochrome c6
in Chlamydomonas plastids
Apocytochrome f is encoded
by the chloroplast genome
cytoplasm
stroma
apocytochrome f
thylakoid
lumen
thylakoid lumen
Biogenesis of holocytochrome f and holocytochrome c6
in Chlamydomonas plastids
Apocytochrome c6 is encoded
by the nuclear genome
cytoplasm
apocytochrome c6
stroma
apocytochrome f
thylakoid
lumen
thylakoid lumen
Biogenesis of holocytochrome f and holocytochrome c6
in Chlamydomonas plastids
heme is synthesized in the stroma
cytoplasm
apocytochrome c6
stroma
apocytochrome f
thylakoid
lumen
thylakoid lumen
heme
How are apoforms of cyt f and c6 converted
to their holoforms in the thylakoid lumen?
cytoplasm
stroma
apocyt f
lumen
apocyt c6
thylakoid lumen
cytoplasm
stroma
lumen
holocyt f holocyt c6
thylakoid lumen
Ccs mutants are photosynthetic deficient and do not
accumulate holoforms of plastid c-type cytochromes
Minimal (high light)
Acetate (low light)
Isolation of photosynthetic mutants that display a
dual deficiency in holocyt f and holocyt c6
WT
ccs
WT
ccs
ccs: cytochrome c synthesis
cytoplasm
apocyt c6
WT ccs
stroma
lumen
holocyt f holocyt c6
thylakoid lumen
apocyt f
WT ccs
Anti-cyt f
Anti-cyt c6
cyt f heme
stain
cyt c6 heme
stain
The ccs mutants are blocked at heme attachment
pre-apocyt c6

cytoplasm
pre-apocyt c6
i-apocyt c6
stroma

apocyt c6
X
holocyt c6

i-apocyt c6

apocyt c6
thylakoid lumen
thylakoid lumen
The ccs mutants are blocked at heme attachment
pre-apocyt c6

cytoplasm
pre-apocyt c6
i-apocyt c6
stroma

apocyt c6
X
holocyt c6
WT ccs
pulse

i-apocyt c6

apocyt c6
thylakoid lumen
thylakoid lumen
The ccs mutants are blocked at heme attachment
cytoplasm
pre-apocyt c6
pre-apocyt c6

i-apocyt c6
stroma


apocyt c6
X

apocyt c6
thylakoid lumen
thylakoid lumen
holocyt c6
WT ccs
pulse
ccs
0 10
30
i-apocyt c6
60’ chase
The ccs mutants are blocked at heme attachment
cytoplasm
pre-apocyt c6
pre-apocyt c6

i-apocyt c6
stroma


apocyt c6
X

apocyt c6
thylakoid lumen
thylakoid lumen
holocyt c6
WT ccs
pulse
i-apocyt c6
ccs
0 10
30
60’ chase
The ccs mutants synthesize the apoforms of
cyt c6 and cyt f but fail to convert them to their
holoforms because of a block at the heme
attachment step
Seven loci are required for the maturation
of plastid c-type cytochromes
Minimal (high light)
WT
ccs
Acetate (low light)
WT
ccs
Plastid
Nucleus
ccsA CCS1
CCS2
CCS3
CCS4
CCS5
CCS6
A CcsA-Ccs1 complex involved
in heme delivery?
heme
stroma
H
CcsA
36 kD
H
H
H
Ccs1
WxW
lumen
Ccs1 is part of a ~ 200 kDa complex
whose accumulation is dependent upon
the presence of CcsA
Blue Native
PAGE
Anti-Ccs1
A candidate cytochrome c assembly complex
heme
Plastid
Nucleus
ccsA
CCS1
CCS2
CCS3
CCS4
CCS5
CCS6
stroma
H
H
CcsA
36 kD
Ccs1
Ccs1
36 kDa
H
WxW
Ccs5
6167
kDa kD
H
Ccs2
Ccs3
lumen
Ccs4
Ccs6
Purification of the CCS complex and resolutions of its constituents
Molecular cloning of other CCS genes
p-side
Ccs4 and Ccs5 are involved
in redox metabolism?
3+
S
2+
HS
S
HS
lumen
Reduction of heme
and cysteine thiols
ccs4 and ccs5 are rescued
by exogenous thiol compounds
WT
ccs5
0
cyt f heme
stain
a-cyt f
1 mM
0
1 mM
DTT
Complex I is the largest respiratory complex in
the mitochondrial inner membrane
Complex I is the largest respiratory complex in
the mitochondrial inner membrane
 > 40 subunits (FMN, FeS)
Dual genetic origin (5 to 9 subunits encoded in the mitochondrial genome
 Many human disease are caused by complex I deficiencies
and 50% of complex I defects have no molecular explanation
Chlamydomonas, a model system to study
complex I biogenesis
Acetate in the dark
NADH
complex I
WT
ATP
Complex I
mutant
UQ
complex III
complex II
Cytochrome c
complex IV
ATP
ATP
Succinate
cob
nd4
nd5
cox1
nd2 nd6
nd1
rtl
5 mitochondria-encoded subunits and 37 nucleus-encoded subunits
O2
Isolation of nuclear mutants deficient
for complex I assembly
Transformation
with hph gene
Selection of transformants on
TAP + Hygromycin B plates
Chlamydomonas
wild type cells
Wild type strain
acetate in the
dark
acetate in the
light
Complex III mitochondrial mutant
Complex I nuclear mutant
Complex I mitochondrial mutant
Incubation at 25ºC, high light, 7-10 days
Isolation of nuclear mutants deficient
for complex I assembly
Acetate in the light
Acetate in the dark
WT
complex I mutant
complex I candidate
mutant
Biochemical verification of complex I assembly defect
(in gel-staining for complex I activity)
Molecular analysis of the mutant to identify the disrupted
gene
You said it already!!
Chamy is cool!
Beat Michigan!