TRAFFICKING PATHWAYS TO THE CELL SURFACE IN YEAST

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DESCRIPTION: Membrane recycling occurs in all eukaryotic cells as a mechanism to maintain membrane balance and organellar identify. In mammalian cells, a recycling pathway between the endosome and plasma membrane is taken by many internalized membrane proteins and lipids. However, relatively few of the components of the vesicular transport machinery by which transport through the endosomal pathway occurs is poorly understood. One major goal of this proposal is to use combined biochemical and genetic approaches in Saccharomyces cerevisiae to define an endosome-to-plasma membrane traffic pathway and to dissect the molecular mechanism of endosomal recycling. Our approach is to analyze the trafficking of a mutant plasma membrane protein (Pma1) which fails to arrive at the cell surface and is delivered instead to the endocytic/vasuolar pathway. A collection of suppressor of pma1 (sop) mutants have been isolated which re-route mutant Pma1 to the plasma membrane. In Specific Aim 1, cell fractionation and indirect immunofluorescence experiments are proposed to determine whether some sop mutants suppress pma1 by allowing mutant Pma1 to move to the cell surface after it has entered the endosomal system. Experiments in Specific Aim 2 focus on VPS8, identified as an sop mutant, and its role in regulation of endosomal traffic. Specific Aim 3 describes proposed work in three newly-discovered SOP genes which encode novel membrane protein which homology to mammalian receptors. Experiments will test whether these gene products, SOP3, SOP4, and SOP5, are involved in a mechanism by which proteins destined for plasma membrane delivery are sorted at the Golgi from proteins destined for the endosomal/vacuolar pathway. The work described in this proposal has implications for a number of disease states involving defects in intracellular protein trafficking.