Abstract 2217 Discrete, multi-modal activation of the exocyst coordinates polarized exocytosis

Cell shape is a critical determinant of cell function, and most cells undergo dramatic shape changes. The molecular mechanisms that control shape change are highly conserved in all eukaryotes. Asymmetric cell growth is primarily mediated through polarized exocytosis, where post-Golgi vesicles are transported to specific locations on the plasma membrane. Subsequent fusion of these vesicles promotes membrane expansion in that direction. Decision-making about where and when exocytosis should occur is regulated by a host of protein partners which, in part, establish the identity of the vesicle and the target membrane. Central to tethering is a highly conserved protein complex called the exocyst. The hetero-octameric exocyst complex binds to proteins and lipids on the surface of the plasma membrane, on the vesicle, and in the cytoplasm. These interactions confirm membrane identity and promote SNARE assembly. However, the mechanism by which the exocyst mediates and links tethering and fusion comprises a major knowledge gap. Interactions between the exocyst and Rho GTPases on the plasma membrane allosterically activate the exocyst complex and promote exocytosis. Our laboratory has recently discovered a second, discrete mechanism of exocyst activation whereby interactions between the exocyst and a second cytoplasmic tether, the Tomosyn/Lgl homolog Sro7, promote a different mechanism of allosteric activation within the exocyst complex. Based on these findings, we hypothesize that multi-modal activation mechanisms of the exocyst complex work in concert to verify the identities of post-Golgi vesicles and the plasma membrane and to promote exocytosis. Direct comparison of GOF mutants which mimic Sro7- and Rho/Cdc42-activation of the exocyst reveal distinct mechanisms and outcomes. We propose a model by which these two activation pathways reside within the same tethering complex but remain insulated from one another. This work leads to an interesting hypothesis that to synchronize tethering and fusion, the exocyst undergoes specific conformational changes depending on which protein it binds to. These conformational changes allosterically activate the exocyst in distinct ways to facilitate the next steps of exocytosis, by increasing the exocyst binding affinity for a different protein partner. To continue this work, we are identifying GOF mutants in each subunit of the exocyst that mimic discrete activation mechanisms. Using gain-of-function screens, we have identified and characterized the distinct outcome of activation for the Sec10 subunit of the exocyst complex that does not appear to interact with either the Rho/Sro7 activator or the Snc/Sec4 output. Our results suggest that Sec10 activation can mimic either Rho-mediated activation or Sro7-mediated activation.