Mitochondrial Complex I (CI), also known as NADH:ubiquinone oxidoreductase is the first and largest enzyme complex of the mitochondrial electron transport chain (ETC) and entry point for electrons from NADH. The fully assembled complex has a molecular weight of ~1 MDa and is L-shaped with a membrane arm embedded in the inner mitochondrial membrane and soluble arm protruding into the mitochondrial matrix. Together, eukaryotic CI is composed of more than 40 subunits: 14 core subunits conserved from bacteria in addition to 25-35 non-core or accessory subunits, plus 9 non-protein cofactors (1 flavin mononucleotide and 8 iron-sulfur clusters). Due to the obvious complexity of the holoenzyme, the assembly process requires proteins not included in the final complex that are collectively termed assembly or biogenesis factors. Unexpectedly, defects in CI are implicated in a number of severe human disorders including Leigh syndrome, lactic acidosis and stroke-like episodes (MELAS) syndrome, and Parkinson’s disease. However, the underlying genetic defects have been identified in only 60% of patients with CI deficiency, occurring in genes encoding a CI subunit or previously identified biogenesis factor. It is generally accepted that the causative mutations occurred in genes encoding novel CI biogenesis factors in the remaining 40% of patients.
Chlamydomonas reinhardtii is an established model system for the study of mitochondrial respiration. In contrast to other model systems, Chlamydomonas CI mutants are still viable but have a characteristic slow growth in the dark (SID) phenotype in respiratory conditions (dark plus acetate). In Chapter 2, with the goal of identifying novel genes encoding factors controlling mitochondrial CI biogenesis, an insertional mutagenic screen was previously performed in Chlamydomonas. Of more than 54,000 insertional mutants, 22 were SID, 13 of which were also reduced for CI activity and/or assembly and termed “amc” for assembly of mitochondrial complex I. Importantly, of these 13 amc mutants, 2 (amc5 and amc9) were found with lesions disrupting nuclear genes encoding CI subunits, thereby validating the efficacy of this approach. Intriguingly, each of the disrupted genes had homologues in humans and were null for their respective transcripts, providing an opportunity to model effects of mutations linked to human CI deficiency.
In Chapter 3, two additional mutants, amc1 and amc11 were significantly decreased for CI enzymatic activity due to a block in the assembly process resulting in accumulation of a 700 kDa subcomplex. These mutants were later found to be allelic, harboring insertions in exon 2 of Cre16.g688900, a predicted protein coding gene not previously affiliated with CI biology. These mutants were renamed amc1-1 and amc1-2, and the defective gene, Cre16.g688900, as AMC1, encoding a large protein of biased amino acid composition and lacked any conserved domains. As expected, the N-terminus of AMC1 was sufficient to target a heterologous reporter to yeast mitochondria. Notably, other large low complexity proteins have been implicated in chloroplast gene expression, and subsequent investigation of mitochondrial transcripts revealed a specific decrease in the nd4 transcript. Intriguingly, loss of ND4 results in the same assembly intermediate observed in amc1-1 and amc1-2 mutants. Taken together, these results are consistent with a function of AMC1 in mitochondrial gene expression, specifically in expression of the nd4 subunit, but the precise stage remains to be experimentally determined.
A fifth mutant obtained from this screen, amc12, was deficient in multiple respiratory enzyme complexes of the ETC but subsequently found to have a second, independently segregating mutation contributing to the SID phenotype. The single mutant (hereafter lcla1), which was genetically linked to the insertional cassette, was isolated and characterized in Chapter 4. Unusual among the collection of amc mutants, lcla1 was also severely affected for growth in high light, and instead resembled the isocitrate lyase (icl) mutant. This light sensitivity was exacerbated by introduction of an arginine auxotrophic marker in lcla1 and icl but not control strains, further suggestive of a common biological pathway. Since lipids are increased in the icl mutant and high light is a stress condition known to induce plastid lipid synthesis, triacylglycerol (TAGs) content was measured in lcla1. In normal growth conditions, cytoplasmic TAG-containing lipid bodies were increased in lcla1 due to a defect in lipid catabolism. Furthermore, lcla1 was resistant to treatment with cerulenin, a plastid fatty acid synthesis inhibitor, in high light suggesting the high light sensitivity was due to lipotoxicity.
The insertional cassette in lcla1 was mapped to Cre07.g329861, predicted to encode a large protein with numerous low complexity regions but without any conserved domains suggestive of a biological function. Introduction of an epitope tag facilitated detection of the encoded isoforms at the expected sizes and the gene renamed LCLA1 for Low Complexity protein in Lipid Accumulation. The N-terminus of one isoform was sufficient to direct a chimeric reporter to the ER in mixotrophic conditions (light plus acetate), the site of de novo peroxisome biogenesis. Shifting cultures to respiratory conditions, the reporter redistributed to puncta resembling peroxisomes. Due to these observations and the compartmentation of glyoxylate cycle and fatty acid oxidation enzymes in peroxisomes, an intriguing possibility is for a role of LCLA1 in peroxisome biogenesis. Indeed, only ~1/3 of the proteins involved in peroxisome biogenesis are conserved in Chlamydomonas. While the precise function of LCLA1 remains to be elucidated, here we provide support for a role in lipid metabolism, indirectly through peroxisome function.
In summary, from an insertional mutagenic screen for mitochondrial CI deficiency, two nuclear mutants encoding subunits of CI were obtained as well as two additional mutants disrupted for novel genes. The first, AMC1, was shown to encode a novel CI biogenesis factor controlling expression of the mitochondrial-encoded nd4 transcript. The second, LCLA1, encoding at least two isoforms that participate in lipid homeostasis, possibly through peroxisome biogenesis, an organelle central to lipid metabolism and acetate assimilation. Interestingly, from this screen we have identified two novel genes encoding large, low complexity proteins participating in diverse biological processes.