Chlamydomonas; NDUFAF3; assembly factor; complex I; mitochondrial respiratory chain; Electron Transport Complex I; Plant Proteins; Ferredoxins; Ferredoxins/metabolism; Chlamydomonas reinhardtii/metabolism; Chlamydomonas reinhardtii/genetics; Electron Transport Complex I/metabolism; Electron Transport Complex I/genetics; Plant Proteins/metabolism; Plant Proteins/genetics; Chlamydomonas reinhardtii; Physiology; Genetics; Plant Science; Cell Biology
Abstract :
[en] The mitochondrial NADH:ubiquinone oxidoreductase, or complex I, is composed of a hydrophobic arm comprising the P module and a hydrophilic arm comprising the N and Q modules. The assembly of complex I is well characterized in humans and is catalyzed by a series of assembly factors that join the Q, P, and N modules sequentially. The complex I of protists and plants, however, contains additional ancestral features, namely a ferredoxin bridge that connects the matrix and the membrane arms and a γ carbonic anhydrase domain, whose mechanisms of assembly are unknown. In this work, a strain where the complex I assembly factor NDUFAF3 has been tagged with a 3×FLAG at the C-terminus is investigated in the green microalga Chlamydomonas reinhardtii. Like its human homolog, NDUFAF3 interacts strongly with the classical subunits of the Q and P modules, but also with the γ carbonic anhydrase domain and C1-FDX, a subunit of the ferredoxin bridge. The predicted structural positioning of NDUFAF3 within the Q module suggests a role in the formation of this bridge. In contrast, subunits of the N module are only loosely associated with NDUFAF3. We further demonstrate that the N module is attached at a later stage of assembly, suggesting that Chlamydomonas complex I assembles in a human-like sequence. This contrasts with what is documented in Angiosperms, where the N and Q modules are attached together before anchoring to the P module. Altogether, these results highlight a conserved and ancestral role of NDUFAF3 in complex I manufacture.
Disciplines :
Microbiology
Author, co-author :
Salinas-Giegé, Thalia ; Institut de biologie moléculaire des plantes, CNRS, Université de Strasbourg, Strasbourg, France
Ticoras, Mitchell ; Genetics and Physiology of Microalgae, InBioS/Phytosystems Research Unit, University of Liège, Liège, Belgium ; Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA
Waltz, Florent ; Biozentrum, University of Basel, Basel, Switzerland
Coosemans, Nadine ; Université de Liège - ULiège > Département des sciences de la vie
Fanara, Steven ; Functional Genomics and Plant Molecular Imaging, InBioS/Phytosystems Research Unit, University of Liège, Liège, Belgium
Chicher, Johana ; Plateforme Protéomique Strasbourg-Esplanade, Institut de Biologie Moléculaire et Cellulaire, UAR1589 du CNRS, Strasbourg Cedex, France
Hammann, Philippe ; Plateforme Protéomique Strasbourg-Esplanade, Institut de Biologie Moléculaire et Cellulaire, UAR1589 du CNRS, Strasbourg Cedex, France
Hamel, Patrice P ; Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA
Remacle, Claire ; Université de Liège - ULiège > Département des sciences de la vie > Génétique et physiologie des microalgues
Language :
English
Title :
NDUFAF3 is Involved in the Assembly of the Q/P Modules of Respiratory Complex I in the Green Microalga Chlamydomonas reinhardtii.
ANR - Agence Nationale de la Recherche SNF - Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung AvH - Alexander von Humboldt-Stiftung
Funding text :
This work was supported by FNRS\u2010FWO EOS Project (30829584 [to C.R.]), FNRS CDR (J.0175.20 and J.0149.23 [to C.R.]), Action de Recherche Concert\u00E9e from the University of Li\u00E8ge (DARKMET ARC grant 17/2108 [to C.R.]). F.W. acknowledges the Alexander von Humboldt (AvH) foundation for the Humboldt Research Fellowship for Postdocs and the Swiss National Science Foundation (SNSF) for the Swiss Postdoctoral Fellowship (project 210561). M.T. acknowledges funding from the Department of Molecular Genetics at The Ohio State University (Berl Oakley award), Erasmus+ mobility funds (to C.R. and P.H.), and financial support from STILSA (Students in Life Sciences Abroad). T.S.\u2010G. acknowledges the Agence Nationale de la Recherche (ANR) grant (ARAMIS, ANR\u201021\u2010CE12\u20100012, ANR\u201010\u2010IDEX\u20100002, ANR\u201020\u2010SFRI\u20100012, ANR\u201017\u2010EURE\u20100023 and ANR\u201011\u2010EQPX\u20100022). The mass spectrometer purchase was supported by the Interdisciplinary Thematic Institute IMCBio+, as part of the ITI 2021\u20132028 program of the University of Strasbourg, CNRS, and Inserm, under the framework of the French Investments of the France 2030 Program. Further funding support was from the R\u00E9gion Grand Est CPER2021\u20132027 (ImaProGen Project) and the Strasbourg Eurometropole. Funding:Funding: This work was supported by FNRS-FWO EOS Project (30829584 [to C.R.]), FNRS CDR (J.0175.20 and J.0149.23 [to C.R.]), Action de Recherche Concert\u00E9e from the University of Li\u00E8ge (DARKMET ARC grant 17/2108 [to C.R.]). F.W. acknowledges the Alexander von Humboldt (AvH) foundation for the Humboldt Research Fellowship for Postdocs and the Swiss National Science Foundation (SNSF) for the Swiss Postdoctoral Fellowship (project 210561). M.T. acknowledges funding from the Department of Molecular Genetics at The Ohio State University (Berl Oakley award), Erasmus+ mobility funds (to C.R. and P.H.), and financial support from STILSA (Students in Life Sciences Abroad). T.S.-G. acknowledges the Agence Nationale de la Recherche (ANR) grant (ARAMIS, ANR-21-CE12-0012, ANR-10-IDEX-0002, ANR-20-SFRI-0012, ANR-17-EURE-0023 and ANR-11-EQPX-0022). The mass spectrometer purchase was supported by the Interdisciplinary Thematic Institute IMCBio+, as part of the ITI 2021\u20132028 program of the University of Strasbourg, CNRS, and Inserm, under the framework of the French Investments of the France 2030 Program. Further funding support was from the R\u00E9gion Grand Est CPER2021\u20132027 (ImaProGen Project) and the Strasbourg Eurometropole. In memory of Dr. S. Massoz, who died at Age 34 and pioneered this research. M. Radoux is acknowledged for technical support. Dr. E. Meyer (Martin-Luther-University Halle-Wittenberg, Germany) is acknowledged for the kind gift of the antibodies against Arabidopsis Nad1. We thank G. Herinckx and Dr. D. Vertommen from UCLouvain (Belgium) for the pilot proteomics experiments done on BN-PAGE. We thank V. Cognat from the IBMP bioinformatics core facility for her assistance with the protein functional analyses.
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