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