CGM - DepartmentRegulation and Compartmentalization of Cellular Functions - G. Dujardin's team
Control of the assembly of respiratory complexes
Principal Investigator: G. DUJARDIN, with C. Panozzo, J. Ostojic
Last update: 28-Oct-2011
Fig. 1 : The respiratory functions in S. cerevisiae
Mitochondria are organelles essential for cell life and death. The main function of mitochondria is respiration and the generation of ATP via the oxidative phosphorylative pathway. The respiratory chain is composed of five respiratory complexes located in the inner membrane (Fig. 1).
They are each composed of several different protein subunits (4 to 48) that are encoded either by the nuclear genome or by the mitochondrial DNA (download figure 2 : "Mitochondrial of S. cerevisiae, S. pombe and H. sapiens" 150 ko). Most of the proteins involved in the respiratory complex assembly are conserved through evolution. In man, defects of the mitochondrial respiratory complexes are responsible for many neuromuscular or generalized syndrome diseases. The same defect in yeast is not lethal as the cells are able to satisfy their energy requirement by fermentation. Thus, S. cerevisiae is an excellent model to study respiratory functions.
Our group studies the assembly of yeast respiratory complexes by combining genetic and biochemical approaches. Although the composition of these complexes is well known, the assembly mechanisms are still mysterious although about 20 assembly factors that are not themselves part of the complexes, have already been identified.
We have introduced mutations in the genes encoding either the respiratory complex subunits, in particular the complex III subunits, or the assembly factors. For several years we have particularly developed the study of the protein Oxa1p. (Fig. 3).
Fig. 3 : They control the insertion of proteins within the membranes and the translocation of hydrophilic domains through these membranes. In eukaryotic cells, Oxa1p is located in the inner membrane of mitochondria and is required for the co-translational insertion and assembly of several membrane subunits of respiratory complexes. The Oxa1 proteins from strict aerobic organisms are thus essential. In the yeast S. cerevisiae, the absence of Oxa1p leads to a complete respiratory deficiency but is not lethal, thanks to the ATP produced via the fermentation process.
We have shown that the respiratory deficiency is due to pleïotropic defects of respiratory complex assembly, the un-assembled subunits being rapidly degraded. In order to determine the role of the various domains of Oxa1p, we have introduced mutations in the various hydrophobic and hydrophilic domains of Oxa1p and analysed their consequences on respiratory complex assembly. Starting from these oxa1 mutants, we have isolated several extragenic suppressors. Preliminary studies suggest that they encode proteins controlling the mitochondrial regulatory or signalling processes, or mitochondrial proteins. The study of these suppressors is developed by combining the powerful techniques of yeast molecular genetic techniques and biochemical approaches.
In order to determine the assembly level and the functionality of the five respiratory complexes in the mutants, we are using several techniques. Cytochrome spectra on whole cells allow to rapidly determine which complex is affected (Fig. 4).
Fig. 4 : Analysis of cytochrome spectra
The assembly level of the various complexes is subsequently analysed in details on purified mitochondria (1) on Western blot (Fig. 5) that allow the analysis of the accumulation level of subunits for which antibodies are available, the un-assembled subunits being rapidly degraded; (2) on non-denaturating gels (Fig. 6) in order to visualize the assembled complexes or sub-complexes.
Fig. 5 : "Western Blot" analysis - Accumulation of complex V subunits in mitochondrial membrans
Fig. 6 : BN-PAGE (Blue Native Poly-Acrylamide Gel Electrophoresis)
In collaboration with M. Zivy and L. Negroni (Ferme du Moulon, Gif sur Yvette, France), we have developed a protocol allowing the identification of most subunits of yeast complexes by mass spectrometry. In parallel, the activity of these complexes can be quantified par spectrometry or directly revealed on non-denaturating gels.
The combination of these genetic and biochemical approaches should permit to better understand the assembly mechanisms of respiratory complexes, to discover new actors of this process and to determine their level of action.
Collaborations 2000-2007
N. Altamura et coll. (CNR, Bari, Italie)
M. Corral-Debrinski et C. Jacq (ENS, Paris, France) link
P. Hamel et S. Merchant (UCLA, Los Angeles, USA) link
L. Negroni et M. Zivy (Ferme du Moulon, Gif, France) link
J. Rossier et coll. (ESPCI, Paris, France) link
J. Velours (IBGC, Bordeaux, France) link
Publications since 2000
Glatigny, A., Mathieu, L., Herbert, CJ., Dujardin, G., Meunier, B., Mucchielli-Giorgi, MH. (2011) An in silico approach combined with in vivo experiments enables the identification of a new protein whose overexpression can compensate for specific respiratory defects in Saccharomyces cerevisiae. BMC Syst Biol, 5 (1) 173.
Vallières, C., Trouillard, M., Dujardin, G., Meunier, B. (2011) Studying the deleterious effect of the QoI resistance mutation G143A in the intron-containing cytochrome b gene and the by-pass mechanisms. Appl Environ Microbiol, 77 (6) 2088-93.
Mathieu, L., Marsy, S., Saint-Georges ,Y., Jacq, C., Dujardin, G. (2010) A transcriptome screen in yeast identifies a novel assembly factor for the mitochondrial complex III. Mitochondrion, 77 (6) 2088-93.
Mathieu, L., Bourens, M., Marsy, S., Hlavacek, O., Panozzo, C. and Dujardin, G. (2010) A mutational analysis reveals new functional interactions between domains of the Oxa1 protein in Saccharomyces cerevisiae. Mol Microbiol, 75 (2) 474-88.
Panozzo, C., Bourens, M., Nowacka, A. and Herbert, C.-J. (2010) Mutations in the C-terminus of the conserved NDR kinase, Cbk1p of Saccharomyces cerevisiae, make the protein independent of upstream activators. Mol Genet Genomics, 283 (2) 111-22.
Lelandais, G., Saint-Georges, Y., Geneix, C., Al-Shikhley, L., Dujardin, G. and Jacq, C. (2009) Spatio-temporal dynamics of yeast mitochondrial biogenesis: transcriptional and post-transcriptional mRNA oscillatory modules. PLoS Comput Biol 5 (6) e1000409.
Bourges, I., Mucchielli, M.-H., Herbert, C.-J., Guiard, B., Dujardin, G. and Meunier, B. (2009) Multiple Defects in the Respiratory Chain Lead to the Repression of Genes Encoding Components of the Respiratory Chain and TCA Cycle Enzymes. J Mol Biol, 23 (1) 23-42.
Nouet, C., Truan, G., Mathieu, L. and Dujardin, G. (2009) Functional Analysis of Yeast bcs1 Mutants Highlights the Role of Bcs1p-Specific Amino Acids in the AAA Domain. J Mol Biol, 388 (2) 252-61.
Bonnefoy, N., Fiumera, H.-L., Dujardin, G. and Fox, T.-D. (2009) Roles of Oxa1-related inner-membrane translocases in assembly of respiratory chain complexes. Biochim Biophys Acta, 1793 (1) 60-70.
Marsy, S., Frachon, P., Dujardin, G., Lombès, A. and Lemaire, C. (2008) Respiratory mutations lead to different pleiotropic effects on OXPHOS complexes in yeast and in human cells, FEBS Lett, 582 (23-24) 3489-93.
Lemaire, C. and Dujardin, G. (2008) Preparation of Respiratory Chain Complexes from Saccharomyces cerevisiae Wild-Type and Mutant Mitochondria : Activity Measurement and Subunit Composition Analysis. In Organelle Proteomics. Methods Mol Biol, 432, Pflieger, D. and Rossier, J. (Ed.), Humana Press
Rousset, S., Mozo, J., Dujardin, G., Emre, Y., Masscheleyn, S., Ricquier, D. and Cassard-Doulcier, A.-M. (2007) UCP2 is a mitochondrial transporter with an unusual very short half-life. FEBS Lett, 581 (2) 479-82
Nouet, C., Bourens, M.,Hlavacek, O., Marsy, S., Lemaire, C., and Dujardin G. (2007) Rmd9p controls the processing/stability of mitochondrial mRNAs and its overexpression compensates for a partial deficiency of Oxa1p in Saccharomyces cerevisiae. Genetics 175 (3) 1105-15.
Sellem, C. H., Marsy, S., Boivin, A., Lemaire, C. and Sainsard-Chanet, A. (2007) A mutation in the gene encoding cytochrome c(1) leads to a decreased ROS content and to a long-lived phenotype in the filamentous fungus Podospora anserina. Fungal Genet Biol, 44 (7) 648-58.
Lemaire, C., Negroni, L., Guillot, A., Marsy, S., Zivy, M., and Dujardin, G. (2005). LC-MS/MS identification of the mitochondrial respiratory complex subunits separated on one-dimensional BN-PAGE in Saccharomyces cerevisae wild type and mutant strains. Mol Cell Proteomics 4, S382.
Hlavacek, O., Bourens, M., Salone, V., Lachacinski, N., Lemaire, C., Dujardin, G. (2005) The transcriptional activator HAP4 is a high copy suppressor of an oxa1 mutation. Gene 354, 53-7.
Sellem, C.H., Lemaire, C., Lorin S., Dujardin, G., Sainsard, A. (2005) interactions between the oxa1 and rpm1 genes modulates respiratoty complex assembly and life span in Podospora anserina. Genetics, 169, 1379-1389
Lemaire, C., Guibet-Grandmougin, F., Angles, D., Dujardin, G., Bonnefoy, N. (2004) A yeast mitochondrial membrane methyl-transferase-like protein can compensate for oxa1 mutations . J. Biol. Chem, 279, 47464-47472
Fisher N, Castleden CK, Bourges I, Brasseur G, Dujardin G, Meunier B. (2004) Human disease-related mutations in cytochrome b studied in yeast. . J. Biol. Chem, 279, 12951-12958
Hamel, P., Saint-Georges, Y., de Pinto, B., Lachacinski, N., Altamura, N. and Dujardin G. (2004) Redundancy in the function of mitochondrial phosphate transport in Saccharomyces cerevisiae and Arabidopsis thaliana.Mol. Microb., 51, 307-317
Golik P., Bonnefoy N., T. Szczepanek, T., Saint-Georges Y., Lazowska J. The Rieske FeS protein encoded and synthesized within mitochondria complements a deficiency in the nuclear gene (2003) Proc. Natl Acad. Sci. USA, 100, (15):8844-8849.
Bernard, D., Gabilly, S., Dujardin, G., Merchant, S. and Hamel P. (2003) Overlapping specificities of the mitochondrial cytochrome c and c1 heme lyases. J. Biol. Chem., 278, 49732-49742
Sylvestre, J., Margeot, A., Jacq, C., Dujardin, G. and Corral-Debrinski, M. (2003). The role of the 3' untranslated region in mRNA sorting to the vicinity of mitochondria is conserved from yeast to human cells. Mol. Biol. Cell. , 14, 3848-3856
Saint-Georges, Y., Bonnefoy, N.,di Rago J-P, Chiron, S. and Dujardin G. A pathogenic cytochrome b mutation reveals new interactions between subunits of the mitochondrial bc1 complex. (2002) J. Biol. Chem., 277, 49397-49302
Pflieger, D., Le Caer, J-P., Lemaire, C., Bernard B.A., Dujardin, G., Rossier, J. (2002) Towards the systematic identification of mitochondrial proteins by LC-MS/MS. Anal. Chem., 74, 2400-2406. The complete data are available at our mitochondria site .
Saint-Georges, Y., Hamel, P., Lemaire, C. and Dujardin G.(2001) Role of positively charged transmembrane segments in the insertion and assembly of mitochondrial inner-membrane proteins. Proc. Natl. Acad. Sci. USA , 98, 13814-13819
Lemaire, C., Hamel, P., Velours, J. and Dujardin G. (2000) Absence of the mitochondrial AAA protease Yme1p restores F0-ATPase subunit accumulation in an oxa1 deletion mutant of Saccharomyces cerevisiae. J. Biol. Chem., 275, 23471-23475
Bonnefoy, N., Kermogant, M., Groundinsky, O., and Dujardin, G., (2000) The respiratory gene OXA1 has two fission yeast orthologues which together encode a function essential for cellular viability. Mol. Microbio., 35, 1135-1145.
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