Mitochondria are essential organelles within cells. They are positioned at the center of numerous cellular events including ATP generation, steroid and amino acid biosynthesis, fatty acid metabolism, Fe-S cluster assembly and apoptosis. Maintenance of 'normal'mitochondrial biogenesis is therefore critical to cell viability. One feature that has emerged as a key regulated step in mitochondrial physiology is the maintenance of organelle morphology. A number of proteins identified for their role in controlling organelle dynamics have been shown to also influence the transport, processing, and assembly of mitochondrial preproteins. One such protein is the mitochondrial inner membrane peptidase Pcp1p. In yeast, Pcp1p has been linked to the processing of the dynamin-related protein Mgm1p. Mgm1p processing is dependent not only on Pcp1p activity but also on a functional TIM23- PAM translocation complex suggesting a functional link between the two. These results form the basis of the hypothesis tested in our proposal. Our preliminary data shows that Pcp1p is a subunit of a large protein complex. The broad objective of this proposal is to identify subunits of this complex, while directly testing for a direct physical linkage between Pcp1p and subunits of the TIM23 translocon. Using Saccharomyces cerevisiae as our model system, we will utilize two independent methodologies to identify and characterize protein components of the Pcp1p complex. In a biochemical approach, sucrose gradient ultracentrifugation and Blue-Native PAGE analysis will be carried out to characterize the biophysical properties of this complex. Co-migration of Pcp1p with components of the TIM23 translocon will be measured by Western blot analysis. Proteins shown to co-purify with Pcp1p will be identified by MALDI-TOF. These biochemical methods will be complemented by classic genetics approaches designed to identify genes that code for proteins involved in the formation of the Pcp1p complex. We propose to generate temperature sensitive alleles of PCP1 to be used for the isolation of overexpression suppressors. Putative candidates identified using both of these methods will likely include protein subunits of the Pcp1p complex, accessory proteins involved in substrate recognition, scaffolding proteins involved in Pcp1p complex assembly, and/or protein subunits of the TIM23 translocon. Our results will help build a model for the functional organization of the yeast Pcp1p peptidase within the mitochondrial inner membrane and its interactions with the preprotein translocation machinery. In accordance with the mission of the R15 program, the simplicity of both the model system and the techniques used throughout the proposal will ensure that undergraduate students will be able to make significant contributions to this proposal.
It is becoming increasingly evident that mitochondrial function is controlled by the intersection of multiple pathways including those involved in directing mitochondrial dynamics and preprotein transport. This study's aim is to provide evidence for a physical link between components of the preprotein transport machinery and proteins involved in regulating organelle structure. Understanding the molecular nature of these interactions will provide us with a basis for the design of future treatments targeting various mitochondrial neuropathies in humans.
