Sec4 is GTP binding protein of the ras superfamily that plays a key role in the regulation of the final stage of the yeast secretory pathway. Sec4 undergoes an obligatory cycle of GTP binding, hydrolysis and exchange that is coupled to a cycle of membrane attachment and dissociation. To understand the role of Sec4 in vesicular traffic control we will address the interactions of Sec4 with the accessory proteins that control each step of this cycle. 1) The hydrolysis of GTP by Sec4 is stimulated by interaction with a Sec4 specific GAP. The features of Sec4 that confer specificity will be identified by competition studies with Sec4/Ypt1 chimeras. We will generate GAP mutants and study their phenotype. We will localize GAP and identify associated proteins. 2) GDI is an evolutionarily conserved accessory protein that inhibits the dissociation of GDP from proteins of the Sec4/Ypt1/rab family and will release them from membranes in a nucleotide specific fashion. We have identified a yeast GDI activity and have cloned a candidate gene. We will address its role in the Sec4 cycle through gene disruption and gene product depletion studies. We will study the interaction with Sec4/Ras chimeras to define the structural elements of Sec4 that confer the ability to bind GDI. 3) We have identified a novel exchange protein termed Dss4. We will define the regions of Sec4 that confer the specificity of interaction with Dss4 through studies on Sec4/Ypt1 chimeras. We will determine the intracellular location of Dss4 and identify associated proteins. We will determine if Dss4 can effect the dissociation of GDI from Sec4 and address the nucleotide dependence of this reaction. 4) We will use a """"""""tagged"""""""" Sec4 construction to isolate a possible membrane receptor. Sec4 associated membrane proteins will be cloned and their role in secretion addressed through gene disruption and gene product depletion studies.
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