Mitochondria, the “powerhouse” of the cell, produce energy through the mitochondrial respiratory chain, which comprises five major complexes. Complex I is the first and most complicated enzyme required in this process. With more than 40 subunits, one FMN molecule and eight Fe-S clusters, the assembly of mitochondrial Complex I is highly intricate. Complex I deficiency in humans causes severe metabolic defects leading to fatal diseases such as encephalomyopathy and Parkinson’s disease. In 60% of the patients presenting Complex I dysfunction, there is no molecular explanation and it is assumed that defects in yet-to-be discovered assembly factors are responsible for the Complex I deficiency. In this study, we present the identification of a novel factor required for the biogenesis of Complex I in a green alga.
Study of Complex I biogenesis in humans is very limited due to the lethality of patients with Complex I-related diseases and associated ethical concerns. Chlamydomonas reinhardtii, a unicellular photosynthetic alga,is an ideal model organism for the study of Complex I assembly and function. Chlamydomonas is genetically tractable and it’s Complex I subunit composition is similar to its human counterpart. Unlike mammalian systems, Complex I deficient mutants in Chlamydomonas are viable due to their photosynthetic ability. Importantly, Chlamydomonas is the only model system whose nuclear and mitochondrial genomes can be easily manipulated for the study of mitochondrial Complex I.
The major goal of the study presented in this thesis is to understand the biogenesis of mitochondrial Complex I using Chlamydomonas reinhardtii as a model. In Chapter 2, we describe a forward genetic screen, performed in Chlamydomonas reinhardtii, to identify novel loci controlling Complex I biogenesis. Six Complex I mutants amc8-to-amc13 (assembly of mitochondrial complex I) were isolated from this screen. In Chapter 3, we determine that the AMC9 locus is defined by the gene encoding a core 24 kDa Complex I subunit, NUO5. Conserved from bacteria to humans, this subunit is essential for the NADH oxidation activity of Complex I. In Chapter 4, we report the identification of a novel factor, AMC11, involved in Complex I biogenesis. We show that AMC11 is targeted to the mitochondria through its N-terminal sequence and that loss of AMC11 affects mitochondrial gene expression, resulting in a Complex I assembly defect. Finally, in Chapter 5, we describe the molecular characterization of additional amc mutants that display reduced levels of assembled Complex I. We describe that, although they maintain the structural integrity of Complex I, these mutants are associated with changes in mitochondrial transcript abundance. Overall, our study is the first to identify a novel Complex I biogenesis factor using a forward genetic screen.