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Mitochondrial respiration in algae : from mitochondrial
genome to regulation of photosynthesis.
ULg, Liège Claire RemacleRené MatagneMarie LapailleDenis BaurainSimon MassozVeronique LarosaBenjamin Bailleul
Franck Fabrice
UCL, LouvainMarc BoutryHervé Degand
UNAM, MexicoDiego Gonzalez-HalphenMyriam Vazquez-AcevedoAlexa Villavicencio-QueijeiroHéctor Astudillo
IBPC, ParisGiovanni FinazziJean Alric
Münster 11/06/2013
Energy production Respiration and Photosynthesis
PSIICyt
b6fPSI
2H2O O2+ 4H+
PQ
PQH2
4H+
8H+
CF1Fo
4H+
14H+
14H+3 ATP
2NADPH
Glycolysis
Pyruvate
ATP + NADH
3ATP
ChloroplastLumen
Sugar
Calvin
cycle
Starch
IIIIIVF1Fo
NAD(P)H
O2
Q/QH2
4H+ 2H+ 4H+10H+
Mitochondria Cytoplasm
NADH
Krebs
cycle Acetyl-CoA
GTP
3 ATP
II
Succinate
Summary
Part I :Chlamydomonas as a model organism to
study Complex I
Part II :
ATP synthase evolution in eukaryotes : Relation structure / function
Part III :
Interaction between respiration and
photosynthesis
WTdum25
nd1
dum20
nd1
dum17
nd6
dum22
nd4
dum24
nd4/nd5
dum23
nd5
-950 kDa
-700 kDa
?
Mitochondria : a common feature in eukaryotes
Proto-eucaryote
αααα-proteobacteria
NucleusPrimary
endosymbiosis
Mitochondria : a common feature in eukaryotes
Chlamydomonas reinhardtii
Merchant et al., 2007, Sciences
Chlamydomonas : mitochondrial respiratory-chain
3ATP
IIIIIVNDA
O2
Q/QH2
4H+ 2H+ 4H+10H+
Mitochondria
NADH
Aox
O2
II
NAD(P)H
Succinate
F1Fo
I
II
III
IV
F1Fo
Aox
NDA
050-7001(*3)
0100 (2)01(*2)
10H+ / 3ATP (?)
~1700 (V2)
0>17
~2001>14
6
~500 (III2)110
0~20004
4>10005>40
H+/eMw (kDa)Mt-encoded
Subunits
Chlamydomonas reinhardtii : genetic tools
Mitochondrion
Random mutagenesis (Ra)Site-directed mutagenesis (SD)
Nucleus
Random insertional mutagenesis (KO) : resistance to hygromycinRNA interference strategy (KD)
Matagne et al., 1989Remacle et al., 2006, PNAS
Berthold et al., 2002, ProtistFuhrmann et al., 2001, J. Cell. Sc.
Chlamydomonas mitochondrial/nuclear/chlorplastic genome :
Transmission mode
Boynton et al., 1987Matagne et al., 1993
Chlamydomonas : mitochondrial respiratory-chain
3ATP
IIIIIVNDA
O2
Q/QH2
4H+ 2H+ 4H+10H+
Mitochondria
NADH
Aox
O2
II
NAD(P)H
Succinate
F1Fo
I
II
III
IV
F1Fo
Aox
NDAJans et al., 2008; Lecler et
al., 2013
Mathy et al., 2010
Vazquez-Acevedo et al, 2006; Lapaile et al., 2010
(a,b)
Matagne et al., 1989, Remacle et al., 2010
Dorthu et al, 1992, Matagneet al., 1989, Duby et al,
1999
Cardol et al., 2005
Remacle et al., 2001(a,b), 2006; Cardol et al., 2002, 2004, 2006, 2008, 2011
Nukd
Nukd
Nukd
Mtra
Nukd, ko
Mtra
Mtsd,ra
Nuko,kd
Mutants
050-7001-3*
0100 (2)02*
3ATP
10H+
~1700 (V2)
017
~2001>14
6
~500 (III2)110
0~20004
4>10005>40
H+/eMw (kDa)Mt-encoded
Subunits
darkness
CI WT
Isolation of mitochondrial complex I mutants
1 kbdum23 dum17 dum20
dum25
dum
YeastAnimalsChlamy
3ATP
IIIIIVNDA
O2
Q/QH2
4H+ 2H+ 4H+10H+
Mitochondria
NADH
Aox
O2
II
NAD(P)H
Succinate
F1Fo
Dark
YeastAnimalsPlants
� ND3, ND4L, ND7 and ND9 are encoded in the nucleus
Study of complex I nucleus-encoded subunits
Strategy : RNA interference Principle Expression of double-stranded RNA
Specific degradation of endogenous RNA
Construction of an inactivation vector
Co-transformation :Arginine auxotrophy (ARG7)
Selection of Arg+
Analysis of 100 Arg+ transformants by PCR
– Rate of Co-transformation is ~50 %
Complex I activity and assembly
– ~5% clones defectives
E2 E2P E1I 320 pbE1
801 pb gene fragment 480 pb cDNA fragment
ARG7
Study of nucleus-encoded subunits : RNA interference
12%
CI activity
-1T 3' UTR nd5dum5
Mitochondrial mutants
0délétion cob + nd4 + 3' nd5dum24
0délétion cob + 3' nd4dum22
0-6 NT nd1 (codons 199-200) dum25
0-1T nd5 (codons 145-146) dum23*
0-1T nd1 (codon 243 )dum20
0-1T nd6 (codons 143-144) dum17
0Co-Nd3
Nuclear mutants
0Inactivation of Nd4L expression Co-Nd4L
0Co-Nd7 Inactivation de Nd7 expression
0Co-Nd9 Inactivation of Nd9 expression
Inactivation of Nd3 expression
Assembly of complex I in the mutants
0-69 NT nd4dum28
0nuo9 Insertion of HygR. Loss of Nd9
50%nuop4 Insertion of HygR in NUOP4 gene
0nuob10 Insertion of HygR (Nuob10/PSSW)
GenerationGeneration of of wallwall--lessless strainstrain
Purification of Purification of mitochondriamitochondria
SolubilizationSolubilization by by mildmild--detergentdetergent
BlueBlue--Native PAGENative PAGE
ComplexComplex enzyme enzyme detectiondetection
Complex I assembly in mutants
Purification of mitochondria from wall-less cells
Separation of solubilized mitochondrial complexes by BN-PAGE
Separation of complex components by 2D SDS-PAGE
Identification of proteins by mass spectrometry
- V
- I 950 kDa
- III
- IV
Genomic and proteomic characterization of complexI
Van Lis et al., 2003 Plant physiol. 132:318-30
Cardol et al 2004 BBA Bioenergetics 1658:212-24
Cardol et al 2005 Plant Physiol 137: 447-459
Chlamydomonas : mitochondrial respiratory-chain
WT dum25
nd1dum20
nd1
dum17
nd6
dum22
nd4
dum24
nd4/nd5
dum23
nd5
-950 kDa
-700 kDa
Complex I modular assembly deduced from mutant analysis
PDSWPDSW
Modular Evolution of Complex I - part I : to prokaryote
Modular Evolution of Complex I - Role of core subunits
Hunte et al, 2010, Hinchcliffe and Sazanov., 2006, Science; Efremov and Sazanov, 2011, Nature
14 subunits in bacteria !
Root
Adapté de Gabaldon et al., 2005 J. Mol. Biol.
Modular Evolution of Complex I - part II : to eukaryotes
2005 :
Conserved subunits :
32 Mammals/Fungi/Plants
�More than 10 lineages
specific subunits ?
(Cardol et al. 2005 Plant PhysGabaldon et al 2005 J. Mol Biol)
Modular Evolution of Complex I - part II : to eukaryotes
����Genomic data
Klodmann et al., 2010Bridges et al., 2010Gawryluk and Gray, 2010
����Proteic data
Identification of orthologs to lineage-specific subunits
1/ % of amino acid identities/similarities
Psi (Position specific iterative) BLAST (Altschul et al., 1997)
-profile on initial multiple alignement
-position specific scoring matrix
-iterative BLAST search
-the results of each "iteration" is used to refine
2/ protein size (+/- 50%)
3/Hydropathy profiles (Kite and Doolittle scale, Protscale)
4/ Putative transmembrane helices ?
5/ Structural motifs ?
Cardol et al., 2011, BBA
Identification of orthologs to lineage-specific subunits
Cardol et al., 2011, BBA
Modular Evolution of Complex I - part II : to eukaryotes
Part I : conclusions
1) Use of non mammalian model systems to study human diseases caused by complex I deficiency
2) newly-identified conserved components (e.g. SGDH, AGGG, KFYI)
probably play yet to elucidate conserved functions
3) x-ray cartography performed on Yarrowia complex I (Hunte et al. 2010)
highly relevant for understanding complex I from other sources.
4) machinery required for its assembly is well conserved among eukaryotes�Could be elucidated in non mammalian model systems
5) complexification of mitochondrial complex I did not occur progressively
during speciation of eukaryotic lineages
Paul D. Boyer John E. Walker
Pu J, Karplus M PNAS 2008;105:1192-1197
The Nobel Prize in Chemistry 1997"for their elucidation of the enzymatic mechanism underlyingthe synthesis of adenosine triphosphate (ATP)"
Part II : Structure and function of Complex V
Chlamydomonas
RNA interference : ATP2 (subunit ββββ)
Témoin ATP2
Wt ATP2 Wt ATP2
Light Dark
Lapaille et al., 2010, BBA
3ATP
IIIIIVNDA
O2
Q/QH2
4H+ 2H+ 4H+10H+
Mitochondria
NADH
Aox
O2
II
NAD(P)H
Succinate
F1Fo
Evolution of Complex V : Chlorophyceae
Yeast (~mammal)) Polytomella (~Chlamydomonas)
α, β, γ, α, β, γ, α, β, γ, α, β, γ, OSCP, δδδδ,a, b , c
Mammals(H.sapiens, B. taurus, 17)
Fungi(S. cerevisiae, 20)
εεεε, d, f, g, IF1, A6L
Red algaeRhodophytes
(Cyanidioschyzon merolae, 13)
Land plants
Green algaeChlorophytes
Trebouxyophyceae(Chlorella vulgaris, 14)
Chlorophyceae(C. reinhardtii, V. carteri,
Polytomella, 17
S. obliquus, 7)
Bryophytes(Physcomitrella patens, 15)
Embryophytes(A. thaliana, O. sativa, 15)
Prasinophyceae(O. tauri, M. pusilla, 14)
Bacterial core
Ancestral Eukaryotic-core
Complex V : Complexification in eukaryotes
In most cases :dispensable subunits
c c c c
δ
α β α β
γ
a
OSCP
b
F6IF1
d
A6L
ε
eg
Evolution of Complex V : Chlorophyceae
c c c c
δ
α β α β
γ
a
OSCP
b
fIF1
d
A6L
ε
eg
Evolution of Complex V : Chlorophyceae
c c c c
δ
α β α β
γ
a
OSCP
b
fIF1
d
A6L
ε
eg
Evolution of Complex V : Chlorophyceae
c c c c
δ
α β α β
γ
a
OSCP
b
fIF1
d
A6L
ε
eg
Transfer of Mitochondrial genes to
the nucleus
Loss of conserved subunits
Recruitement of ASAs
i) relocation of previously extant
proteins to the mitochondria,
ii) acquisition of novel genes by
lateral gene transfer
No sequence or pattern similarities in
existing DB
RNA interference : ASA7
Lapaille et al., 2010, Mol. Biol. Evol.
Chlamydomonas 3ATP
IIIIIVNDA
O2
Q/QH2
4H+ 2H+ 4H+10H+
Mitochondria
NADH
Aox
O2
II
NAD(P)H
Succinate
F1Fo
RNA interference : ASA7
Lapaille et al., 2010, Mol. Biol. Evol.
Chlamydomonas 3ATP
IIIIIVNDA
O2
Q/QH2
4H+ 2H+ 4H+10H+
Mitochondria
NADH
Aox
O2
II
NAD(P)H
Succinate
F1Fo
� ASA7 subunit stabilizes the dimeric form
�ASA7 confer resistance to natural
inhibitor present in soil-environment
stator composition and mitochondrial structure
Tubular ; >10 subunits
Crêtes lamellaires ; b, d, f, e, g
Discoidal ; 13 subunits
?
Bag-like tubulard,f,g,A6L
?
Asa1-9 lamellar
b2
Balabaskaran Nina, PloS Biology2010, 8(7), e1000418
Part II : Conclusions and perspectives
Transfer of mt genes coding for stator subunits to nucleus has led to recruitment of new subunit in various lineages
New subunits are responsible for the various dimeric structure of ATP synthase
Dimeric enzyme bends the inner membrane and are maybe responsible for cristae structure
Cristae structure imposes physical constraints (electric field) for the activity of ATP synthase
Part III. Energy production : Respiration and Photosynthesis
PSIICyt
b6fPSI
2H2O O2+ 4H+
PQ
PQH2
4H+
8H+
CF1Fo
4H+
14H+
14H+3 ATP
2NADPH
Glycolysis
Pyruvate
ATP + NADH
3ATP
ChloroplastLumen
Sugar
Calvin
cycle
Starch
IIIIIVF1Fo
NAD(P)H
O2
Q/QH2
4H+ 2H+ 4H+10H+
Cytoplasm
NADH
Krebs
cycle Acetyl-CoA
GTP
Extra NADPHATP
NDH
3 ATP
0 to 3 ATP
1 NADH
-1.5 ATP
-1 NADPH
1.3 ATP
1 NADPH
ATP
shortage
Shuttles
Shuttle
NAD(P)H
NDA
Aox
O2
Involvement of respiration in the light
Oxygen ‘uptake’
2PQPSII
2H2O O2+ 4H+
4H+
Fluo
0
50
100
150
200
250
300
0,E+00 2,E-05 4,E-05 6,E-05 8,E-05
µmoles O2 s-1 µg chla-1
rET
R-P
SII
Photo
synth
esis
(F
luo)
Net oxygen evolution
Inhibition studies
Photo
synth
esis
Respir
ation
IIIIIV
O2
Q/QH2
4H+ 2H+ 4H+
NADHAox
O2
SHAMAA
Low light
High light
3
1.8
1.2
0
ATP/NADH
max
Respiration
Photo
synth
esis I-
III,IV-
I/III,IV-
WT
Cardol et al., 2003, Plant Physol.
Involvement of respiration in the light Use of mutants
IIIIIV
O2
Q/QH2
4H+ 2H+ 4H+
NADHAox
O2
NDA
Part III. Energy production : Respiration and Photosynthesis
PSIICyt
b6fPSI
2H2O O2+ 4H+
PQ
PQH2
4H+
8H+
CF1Fo
4H+
14H+
14H+3 ATP
2NADPH
Glycolysis
Pyruvate
ATP + NADH
3ATP
ChloroplastLumen
Sugar
Calvin
cycle
Starch
IIIIIVF1Fo
NAD(P)H
O2
Q/QH2
4H+ 2H+ 4H+10H+
Cytoplasm
NADH
Krebs
cycle Acetyl-CoA
GTP
Extra NADPHATP
NDH
3 ATP
NAD(P)H
NDA
Aox
O2
ATP shortage ATP + NADH
Pasteur effect
Calvin
cycle
PSII
Cyt
b6fPSI
H2O O2+ 4H+
PQ
PQH2
4H+
8H+
CF1Fo
4H+
14H+
14H+ATP
NADPH
Chloroplast
NDH
Lumen
NADPH
Statetransition
STT
LP
LP
L-P
L-P
Mitochondrial deficiency
compensated by chloroplastic
state transition
Wolman, 2001, Embo reports, Cardol et al., 2003, Plant Physol.,Iwai et al. 2010, Nature
0
50
100
150
200
250
300
660 680 700 720 740
Re
lati
ve
flu
ore
sc
en
ce
in
ten
sit
y
I III IV
Wild type
untreated
'light 1'
N2
PSII
PSI
Cyclicelectron flow
0
50
100
150
200
250
300
660 680 700 720 740
dum 22
I III IV
untreated
'light 1'
N2
nm
Calvin
cycle
PSII
Cyt
b6fPSI
H2O O2+ 4H+
PQ
PQH2
4H+
8H+
CF1Fo
4H+
14H+
14H+ATP
NADPH
Chloroplast
NDH
Lumen
NADPH
No Statetransition
STT
LP
Cyclicelectron flow
3ATP
IIIIIVF1Fo
O2
Q/QH2
4H+ 2H+ 4H+10H+
NADH
Krebs
cycle Acetyl-CoA
GTP
3 ATP
NAD(P)H
NDA
Aox
O2
ATP shortage
Mitochondrial deficiency
compensated by chloroplastic
state transition
Depege et al., 2005, SciencesCardol et al., 2009, Plant Physol.Lapaille et al., 2010, BBA bioenergetics
Conclusion part III
Respiration can be an effective electron sink for photosynthetic electrons
State transition/cyclic electron flow are critical when mitochondrial respiration is impaired
Photosynthetic cyclic electron flow and respiration sustain ATP for carbon fixation
Future work…
Chloroplast acquired through primary endosymbiosis
1st
Heterotroph ancestor
Cyanobacteria
Photosynthetic ancestor
Noyau
Mitochondrion Primaryendosymbiosis
Chloroplast acquired through secondary endosymbiosis
2nd
2nd, 3rd
HeterotrophAlga
Chloroplast
Secondaryendosymbiosis
2nd, 3rd