Asymmetric tethering by exocyst in vitro requires 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 tethering in vitro and 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.