Our long-term research objective is to understand the biogenesis of mitochondrial respiratory chain (MRC) complexes. The MRC is present in the inner mitochondrial membrane and is the main site for cellular respiration and energy production. It consists of five multi-protein complexes composed of ~90 subunits that assemble into supra-molecular structures called supercomplexes. MRC biogenesis is a complex, multi-step process facilitated by hundreds of uncharacterized factors. Despite insights gained through decades of research on mitochondrial bioenergetics, our understanding of MRC biogenesis is incomplete, and many fundamental questions remain unanswered. For example, what factors are required for the formation and stabilization of MRC complexes and supercomplexes? What role does the lipid milieu play in MRC complex assembly? We have two ongoing projects that will address these important questions using the tools of genomics, genetics, and biochemistry in human cells and the yeast Saccharomyces cerevisiae.
1) Discovery and Characterization of Novel MRC Biogenesis Genes
In order to systematically discover novel MRC biogenesis genes, we have used bioinformatic tools to identify a subset of uncharacterized mitochondrial proteins that are conserved between yeast and humans. We predict that many of these proteins are involved in fundamental functions of mitochondria including energy production by MRC. We will experimentally validate these candidate genes using the “nutrient-sensitized screen” that we have developed. The MRC biogenesis genes identified as screening hits can act at any step in the canonical pathway of MRC biogenesis including mitochondrial DNA maintenance and expression, addition of cofactors to the MRC apoenzymes, and formation of the supercomplex. We will assign novel MRC genes to these specific steps using a battery of biochemical and molecular assays. Identifying novel MRC genes will not only provide a protein parts-list to understand the fundamental pathway of MRC biogenesis, but will also be a valuable database for identifying potential mitochondrial disease-candidate genes.
2) Phospholipid:Protein Interactions in MRC Function and Formation
The large body of data obtained from biochemical and genetic studies have identified critical roles of mitochondrially synthesized phospholipids, cardiolipin, and phosphatidylethanolamine (PE) in MRC function and formation. Recently, we have discovered that a clinically used compound, meclizine, specifically inhibits the synthesis of non-mitochondrial PE and attenuates MRC activity, raising the intriguing possibility that non-mitochondrial PE can modulate MRC function. Using meclizine in combination with genetic mutants of phospholipid biosynthetic pathways, we will determine the contribution of non-mitochondrial phospholipids in MRC structure and activity. Understanding the biochemical basis of phospholipid:MRC protein interactions will allow manipulation of MRC function, which is clinically important since modulating MRC activity has been shown to be efficacious in the treatment of cardiovascular and neurodegenerative disorders.