PASK regulation
PASK activation model

Mitochondrial Proteome

Functional Annotation of 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 (Pagliarini, et al. 2008). 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 toward this goal, we bioinformatically identified many mitochondrial protein families that are pan-eukaryotic and unstudied. Using yeast mutants generated in our lab, we have analyzed the loss-of-function growth phenotypes for each. We have also determined the subcellular and sub-mitochondrial localization for each protein. Such higher throughput, standardized studies have then been used to guide specific hypotheses for a subset of proteins. These projects, all focused on proteins for which no function had ever been described, have progressed to various levels of understanding; four are published.

  1. Hao, et al (2009) Science (Hao, et al. 2009),(Hao, et al. 2009). We found that Sdh5 is necessary for the assembly of the succinate dehydrogenase complex (Complex II) of the electron transport chain, where it catalyzes the insertion of the obligate flavin-adenine dinucleotide cofactor into the catalytic subunit. 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.
  2. Heo, et al (2010) Mol Cell (Heo, et al. 2010),(Taylor, et al. 2011). Our understanding of mitochondrial protein quality control has lagged behind that of the cytosol and other organelles. 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. We are in the process of characterizing Vms1-/- mice generated in our lab.
  3. Chen, et al (2012) Cell Metabolism (Chen, et al. 2012). Mitochondria actively control the production and removal of reactive oxygen species. One key mechanism is the regulated organization of the electron transport chain complexes into large macromolecular assemblies (called supercomplexes), which are believed to facilitate efficient electron flow thereby limited reactive oxygen species production. The components and regulation of the supercomplex assembly system are almost completely unknown. We discovered that Rcf1 is required for 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. The identification of the function of this conserved protein family enables us, for the first time, to genetically probe the importance of respiratory supercomplexes in mitochondrial function and integrity.
  4. Bricker, et al (2012) Science (Bricker, et al. 2012). 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.

In summary, our goal to functionally annotate the mitochondrial proteome has enabled discovery of biochemical functions important for mitochondria, elucidation of the genetic basis of two human diseases and catalysis of future studies with a direct impact on common human diseases. In addition to the four projects described above, we are actively pursuing the functions of a number of additional protein families. We are only scratching the surface, however. The majority of the protein families we identified initially await a concerted effort and we are confident that important discoveries will follow.