EXCEED THE SPACE PROVIDED. Our studies have defined three systems that work together to mediate the polarized delivery of secretory 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, SedO, Sec15, Exo 70 and Exo84, termed the exocyst. It interacts with Sec4-GTP on the vesicles and Rho proteins on the plasma membrane. Fusion requires the SNAREs, Snc, Sso and Sec9 as well as Sec1, a protein that interacts with the assembled SNARE complex. This proposal focuses on the exocyst complex, its assembly and its interactions with other components of the secretory machinery. In specific: 1)?We will use genetic and biochemical approaches to probe the role of several Golgi-associated proteins, identified by affinity chromatography, in exocyst function. 2)'Using all purified components we will test the effects of Sec4, Rho1 and Rho3, in their different nucleotide states, on exocyst assembly. 3)?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. 4)?We will use a biochemical approach to identify the component of the plasma membrane that binds to the exocyst. 5)?We will determine which subunit of the exocyst interacts with Sec1 and determine the effects of the Sec1- exocyst complex on SNARE assembly in vitro. 6)-We will make use of the structural data provided by our collaborators to probe the biological functions of the structural features identified.
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