Protein and phosphatidylinositol kinases in yeast protein sorting

Citation

Stack, Jeffrey H. (1995) Protein and phosphatidylinositol kinases in yeast protein sorting. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ayzy-yn85. https://resolver.caltech.edu/CaltechETD:etd-10262007-081900

Abstract

The yeast vps mutants are defective in the delivery of proteins to the vacuole. The products of the VPS15 and VPS34 genes encode homologs of a serine/threonine protein kinase and a phosphatidylinositol 3-kinase (Ptdlns 3-kinase), respectively, that are required for the sorting of soluble vacuolar proteins. Mutations altering highly conserved residues in the catalytic domain of either protein result in the missorting and secretion of vacuolar hydrolases such as carboxypeptidase Y, suggesting that protein and lipid phosphorylation reactions are required for the vesicular transport of vacuolar proteins. Biochemical characterization of Vps34p has shown that, in addition to possessing Ptdlns 3-kinase activity, Vps34p undergoes an autophosphorylation reaction, indicating that it is a novel multiple specificity kinase able to phosphorylate both lipid and protein substrates.

The Vps15 protein kinase both functionally and physically interacts with the Vps34 PtdIns 3-kinase and the two proteins form a complex associated with the cytoplasmic face of an intracellular membrane fraction most likely corresponding to a late Golgi compartment. In addition to recruiting Vps34p to the membrane site of its phospholipid substrate, we have found that Vpsl5p is also required for the activation of Vps34p as Vps34p Ptdlns 3-kinase activity is extremely defective in vps15 mutant strains. Vpsl5p protein kinase activity appears to be responsible for the association with and subsequent activation of Vps34p because vpsl5 kinase domain mutations result in defects in Ptdlns 3-kinase activity and the mutant Vps15 proteins are unable to associate with Vps34p. Together, these results have demonstrated that a functional and stable complex between Vpsl5p and Vps34p is absolutely required for vacuolar protein sorting.

Use of a temperature-conditional allele of VPS34 that is for both protein sorting and Ptdlns 3-kinase activity has allowed us to demonstrate the direct involvement of Ptdlns 3-kinase in vacuolar protein sorting. Our findings with Vps34p suggest that the functions of mammalian phosphoinositide 3-kinase may include the regulation of membrane trafficking and have led us to propose that Ptdlns(3)P is involved in regulating intracellular protein sorting reactions in all eukaryotic cells.

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