Polarized exocytosis requires the proper localized delivery, docking and fusion of secretory vesicles with sites of active growth on the plasma membrane. Members of the Tomosyn/Lgl/Sro7 family play important roles in vesicle trafficking and cell polarity in eukaryotic cells. The yeast homolog, Sro7, is thought to act as a downstream effector of the Sec4 Rab GTPase to promote SNARE assembly during Golgi to cell surface vesicle transport. Here we report the identification of a Sec4 binding site on the surface of Sro7 that is contained within a cleft created by the junction of two adjacent β-propellers which form the core structure of Sro7. We combined in vitro results with in vivo suppression studies and in silico modeling to validate the Sro7-Sec4 docking interaction interface. Close examination of this docking model suggests a structural basis for the high substrate and nucleotide selectivity in effector binding by Sro7. Analysis of the surface variation within the homologous interaction site on Tomosyn-1 and Lgl-1 structural models suggests a possible conserved Rab GTPase effector function in Tomosyn vertebrate homologs. Additionally, overexpression of either Sro7 or the Exocyst complex component Rab effector, Sec15, results in the formation of a cluster of post-Golgi vesicles within the cell. We describe a novel assay that recapitulates post-Golgi vesicle clustering in vitro utilizing purified Sro7 and vesicles isolated from late secretory mutants. We made use of this assay to analyze the effects of Sro7 mutants in which conserved charged patches on Sro7 were mutated to the reverse charge and found that one of the charge reversal mutant proteins, Sro7-R189D,R222D, had a specific defect in clustering vesicles both in vivo and in vitro. We show that this mutation acts by effecting a conformational change in Sro7 from a “closed” to “open” structure, suggesting a novel function for this conformational switch in Sro7 to coordinate Rab-dependent membrane tethering with regulation of SNARE assembly prior to membrane fusion.