"Defining the Cytoplasmic Degradation Pathway for Mitochondrial Proteins"
This proposal aims to characterize a recently identified mitochondrial protein turnover pathway, defined by hsp90- regulated stability of mitochondrial proteins in connection with the ubiquitin proteasome system. This pathway is active in multiple cell types and appears to regulate turnover of wild-type proteins encoded by both nuclear and mitochondrial genomes. Inhibition of hsp90-dependent mitochondrial protein degradation promotes assembly of respiratory chain complexes, cellular differentiation, and apoptotic cell death. Similar pathways appear to exist in lower eukaryotes, including yeast, motivating my attempt to initially identify components of this protein degradation pathway via a genetic screen in this organism. The power of yeast genetics will be used for a systematic and rapid interrogation of a large number of genes to isolate the ones involved in the regulation of mitochondrial proteome stability in conjunction with hsp90. The genetic and protein interaction network of hsp90 has been recently constructed in yeast but the effects on mitochondrial function and mass have not been addressed. In the initial phase I will examine the consequences of hsp90 and proteasome inhibition on mitochondrial mass, function and protein turnover. Next, genes regulating this process will be isolated in a screen for mutants failing to exhibit an increase in mitochondrial mass in response to hsp90 inhibition. The Saccharomyces cerevisiae Gene Deletion Collection and a subset of essential gene temperature sensitive mutants will be used. Last, human homologues of the genes identified in the yeast screen will be tested for equivalent roles in mammalian cells.