Clathrin-mediated vesicular transport is important for normal growth and development of all eukaryotes. Clathrin is required for endocytosis of many nutrients and signal transducing receptors, sorting from the late Golgi to the lysosome, and maturation of regulated secretory granules. Studies in Dr. Lemmon's lab have taken advantage of the powerful molecular genetic methods available in yeast to analyze the function of clathrin. Previous studies have shown that disruption of the clathrin heavy chain gene (CHC1) is lethal in some yeast strains. This observation has permitted the identification of five multicopy suppressors (SCD) genes whose overexpression suppresses the lethality of clathrin-deficient yeast. The SCD genes encode components of the vesicular transport machinery, novel proteins, and known proteins that unexpectedly rescue cells lacking clathrin. The latter have suggested relationships between processes not previously predicted and have provided novel insights into the global effects of clatherin deficiency. This application proposes studies on the SCD5 and SCD3 genes. SCD5 encodes a novel protein with a post Golgi secretory function. Recent evidence suggests SCD5 may be a targeting subunit for type 1 protein phosphatase (PP1), a broad specificity phosphatase with diverse cellular functions. This indicates SCD5 may be involved in regulating a specific phosphorylation event in the late secretory pathway. A combination of genetic, cellular and biochemical approaches will be used to further define the function of SCD5, especially in relation to PP1. SCD3 encodes a member of the 14-3-3 protein family. The 14-3-3 proteins are widely expressed in eukaryotes and appear to function in multiple cellular processes. Dr. Lemmon's genetic analysis in yeast has provided evidence that 14-3-3 proteins may play a role in vesicular transport of regulate a process whose function is affected by alterations in vesicular transport. She will test whether 14-3-3 proteins directly influence membrane sorting and use genetic approaches to identify the players in the membrane associated pathways that require 14-3-3 function. The studies outlined in this proposal will provide insights into the function of SCD5, PP1, and 14-3-3 proteins. This will lead to a fuller understanding of the role of the SCD proteins in vesicular transport and their relationship to clathrin function.
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