Our studies have defined three systems that work together to mediate the polarized delivery of secretor vesicles, their localized tethering to exocytic sites and their fusion with the plasma membrane. Delivery requires actin, a type V myosin, the GTPase Sec4 and its exchange protein Sec2. Tethering requires a complex containing Sec3, Sec5, Sec6, Sec8, SeclO, Secl5, Exo 70 and Exo84, termed the exocyst.It interacts with Sec4-GTP on the vesicles and Rhol on the plasma membrane. Fusion requires the SNAREs, Snc, Sso and Sec9 as well as Sec 1, a protein that interacts with the assembled SNARE complex. This proposal focuses on the exocyst complex, its assembly and its interactions with the secretory vesicles and plasma membrane. In specific: 1) We will use a biochemical approach to identify the component of the secretory vescie that is recognized by Secl 5 and may provide specificity to the tethering process. 2) We will determine if activation by Sec4 mediates the binding of Sec 15 to Sec 10 or the interaction of a Seci 5/Sec 10 subcomplex with Sec5 and we will reconstitute this assembly step in vitro. 3) We will analyze the process of exocyst assembly using a battery of approaches. We will identify the subcomplexes present in a sec4 mutant and test the effects of Rhol and Rho3 on assembly. 4) We will reconstitute vesicle tethering in vitro and test the requirements for Sec4-GTP and each of the subunits of the exocyst in the tethering reaction. 5) We will use a biochemical approach to identify the component of the plasma membrane that binds to the exocyst. 6) We will determine if Seci or Sso overexpression can bypass the deletion of any of the exocyst structural genes and determine the effects on the spatial regulation of surface growth. 7) We will test the role of the exocyst in catalyzing the assembly of the SNARE complex and look for interactions of specific subunits of the exocyst with component SNAREs.
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