The overall goal of this research is to develop a mechanistic understanding of membrane protein sorting and assembly in the yeast Saccharomyces cerevisiae. Recent results indicate that the vacuole is the default compartment for membrane proteins that enter the yeast secretory pathway. To explain these results the vacuolar default model has been proposed. This model states that membrane proteins of the ER and Golgi contain retention signals preventing their transport to the vacuole, whereas plasma membrane proteins have positive sorting information, which ensures that they are not transported to the vacuole. The retention signal on the yeast Golgi membrane protein DPAP A has been characterized, and this information will be used to design genetic screens to identify components of the yeast Golgi retention apparatus.
Specific aims #1 and 2 are focused on the identification of components required for retention of membrane proteins in the yeast Golgi, and the molecular genetic and biochemical characterization of these components. Yeast mutants will be isolated that fail to retain a Golgi membrane protein, and the GAL4 two-hybrid genetic system will be used to identify genes encoding proteins that directly interact with the DPAP A Golgi retention signal. The genes identified will be sequenced, disruption alleles constructed to test the in vivo function of the components, and the Golgi retention machinery biochemically characterized.
Specific aim #3 will test the second part of the vacuolar default model by investigating the nature of the targeting information that directs the FUS1-encoded plasma membrane protein to the yeast cell surface. Gene fusions between the FUS1 gene and the gene encoding the yeast Golgi membrane protein, KEX1, will be constructed to map any region of the FUS1 protein capable of redirecting KEX1 protein lacking its Golgi retention signal to the yeast plasma membrane. A second focus of this proposal is the mechanistic analysis of the assembly and targeting of a multisubunit membrane protein complex, the yeast vacuolar membrane H+-ATPase. With a majority of the vacuolar H+- ATPase subunit genes cloned, the tools are now available to begin to address issues of the biosynthesis of this complex vacuolar membrane protein.
Specific aim #4 is designed to isolate and characterize vacuolar H+-ATPase deficient yeast mutants (vma), clone and characterize the VMA genes, and to investigate the function of the VMA proteins. We will focus on the VMA6, VMA21, and VMA22 genes and their encoded proteins, since these factors are absolutely required for the assembly of both the membrane and peripheral sectors of the yeast vacuolar H+- ATPase complex. The final specific aim (#5) is focused on investigating the biosynthesis, assembly, and targeting of the yeast vacuolar H+-ATPase membrane complex. The roles of individual VMA proteins in the assembly and function of the membrane sector of the vacuolar H+-ATPase will be assessed. The long-term objective of the studies with the vacuolar H+- ATPase is to develop a mechanistic understanding of the pathway of assembly for this very complex membrane protein.
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