Asymmetric tethering by exocystin vitrorequires a Rab GTPase, a v-SNARE and a Sac1-sensitive phosphoinositide lipid

Tethering factors play a critical role in deciphering the correct combination of vesicle and target membrane for subsequent fusion. The exocyst plays a central role in tethering post-Golgi vesicles to the plasma membrane, although the mechanism by which this occurs is poorly understood. We recently established an assay for measuring exocyst-mediated vesicle tetheringin vitroand we have adapted this assay to examine the ability of exocyst to tether vesicles in an asymmetric fashion. We demonstrate that exocyst differs from another post-Golgi vesicle tethering protein, Sro7, in that it is fully capable of tethering vesicles with functional Rab GTPase, Sec4, to vesicles lacking a functional Rab GTPase. Using this assay, we show that exocyst requires both the Rab and R-SNARE, Snc1, to be present on the same membrane surface. In contrast, using Sac1 phosphatase treatment, we demonstrate a likely role for phosphoinositides on the opposing Rab-deficient membrane. This suggests a specific model for exocyst orientation and its points of contact between membranes during heterotypic tethering of post-Golgi vesicles with the plasma membrane.