Membrane trafficking occurs in all eukaryotic cells as a mechanism to maintain membrane balance and organellar identity. The complexity of protein trafficking is underscored by multiple and selective pathways. During this decade there has been an explosion in the identification of proteins involved in protein transport. The approach taken in this proposal is to use combined biochemical and genetic methods in Saccharomyces cerevisiae to understand sorting and transport. Specifically, we have analyzed the trafficking of a mutant plasma membrane protein (Pma1) which fails to arrive at the cell surface. Mutant Pma1 is delivered instead to the endocytic/vacuolar pathway where it is degraded. A collection of sop (suppressor of pma1) mutants has been isolated which prevents vacuolar degradation by allowing mutant Pma1 to move to the plasma membrane. Identification of the corresponding genes has revealed novel SOP genes which are believed to regulate sorting and trafficking in the endosomal system.
In Specific Aim 1, cell fractionation and indirect immunofluorescence experiments are proposed to dissect the mechanism of action of the newly-discovered SOP genes by determining whether mutant Pma1 travels from Golgi to cell surface, or endosome to surface in different sop mutants. Detection of mutant Pma1 movement from endosome to plasma membrane will help to establish the existence of a novel traffic pathway. Experiments in Specific Aim 2 focus on the role of VPS8 (identified as one of the sop mutants) in the regulation of endosomal traffic.
Specific Aim 3 addresses the hypothesis that there is a novel quality control mechanism which recognizes and directs mutant proteins to the endosomal/vacuolar pathway for degradation. Experiments will test whether vacuolar delivery of mutant Pma1 is signal-mediated, and whether the novel Sop proteins are involved in a mechanism by which proteins destined for the plasma membrane are sorted from proteins destined for the endosomal/vacuolar pathway. The work described in this proposal has implications for understanding intracellular protein trafficking in health and disease.
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