Functionalizing the mitochondrial proteome

Mitochondria are small but complex organelles with a disproportionately large impact on human health. Changes in mitochondrial enzyme activities, respiratory capacity, genome sequence, and superoxide generation play important roles in the pathogenesis of heart failure, cancer, neurodegenerative disorders such as Parkinson's, Alzheimer's, and Huntington's disease, and in aging and longevity. The best current inventory of mammalian mitochondrial resident proteins consists of 1098 proteins (Pagliarini et al. 2008). Surprisingly, nearly 300 of these proteins are uncharacterized. This includes many that are highly conserved throughout eukarya, a strong indication that they perform a fundamentally important function. The genes that encode the mitochondrial proteome are heavily represented amongst known human disease genes, with about 20% of predicted human mitochondrial proteins implicated in one or more hereditary diseases (Andreoli et al. 2004, Elstner et al. 2008). Presumably, the quarter of the mitochondrial proteome that is uncharacterized contains many others that await discovery. Making this connection would be greatly facilitated by an understanding of the genetic connections, biochemical properties, and physiological functions of these proteins. Therefore, elucidating the functions of these uncharacterized, conserved mitochondrial proteins will not only explain important aspects of mitochondrial biology, but will also provide a framework for identifying new human disease genes. 

As a first step towards this goal, we used the yeast, Saccharomyces cerevisiae, as a model system in which to genetically and biochemically characterize a number of evolutionarily conserved but understudied mitochondrial protein families. A subset of these proteins, all of which had no previously described function, were analyzed by a combination of biochemical, metabolomic, and cell biological approaches to reveal novel roles in mitochondrial biology, some of which are described below.

 
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Mitochondrial Pyruvate Carrier

The fate of pyruvate is one of the most important metabolic decisions made by eukaryotic cells. Most normal, differentiated mammalian cells partition pyruvate primarily toward transport into mitochondria where it is oxidized for efficient ATP production. The partitioning of pyruvate in stem cells, cancer cells, and failing hearts, however, is different—away from mitochondrial oxidation. Our ability to understand the molecular basis for these metabolic distinctions has been hampered by the surprising fact that the mitochondrial pyruvate transporter had not been identified until now. We discovered a protein complex consisting of Mpc1 and Mpc2 that constitutes the major mitochondrial pyruvate transporter in yeast, Drosophila, and humans. Empowered by this discovery, we found that three families with children suffering from lactic acidosis and hyperpyruvatemia had causal mutations in MPC1.

 
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MitoFAS

We demonstrated that the mitochondrial acyl carrier protein (ACP), which has a well-known role in mitochondrial fatty acid synthesis (mitoFAS), plays an unexpected and evolutionarily conserved role in FeS biogenesis. Subsequent work, highlighted the role of mitoFAS as a nutrient-sensitive pathway that provides an elegant mechanism whereby acetyl-CoA regulates its own consumption via coordination of lipoic acid synthesis and tricarboxylic acid (TCA) cycle activity, iron-sulfur (FeS) cluster biogenesis, assembly of oxidative phosphorylation complexes, and mitochondrial translation.

 
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Mitochondrial Protein Quality Control

Msp1/ATAD1 is a AAA-ATPase protein that we found to reside on the mitochondrial outer membrane. Through a combination of genetics and biochemistry, we showed that it extracts proteins that mis-localize to the mitochondrial outer membrane, which we observed as a surprisingly common phenomenon. We demonstrated this function for the protein family in yeast, human cells, and knockout mice.

We found that Vms1 is required for the stress-responsive mitochondrial recruitment of Cdc48/VCP/p97 and Npl4, which play a role in protein extraction and degradation. Further studies led to the conclusion that Vms1 is a critical component of a previously unknown system for mitochondrial protein quality control, eliminating damaged or misfolded proteins that promote progressive mitochondrial dysfunction.

 
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Regulation of Electron Transport Chain Assembly

We found that both Sdh5 and Sdh8 are required for the assembly of the succinate dehydrogenase complex (Complex II) of the electron transport chain. As a result of these mechanistic functional studies, we were able to discover that familial mutations in human SDH5 (SDHAF2) cause a paraganglioma tumor syndrome. We also discovered a role for Rcf1 in the normal assembly of respiratory supercomplexes in yeast and mammals. Deletion of the RCF1 gene caused impaired respiration and elevated mitochondrial oxidative stress and damage.