摘要：

The exocyst is an evolutionarily conserved octameric protein complex implicated in tethering of post-Golgi secretory vesicles to the plasma membrane prior to SNARE-mediated membrane fusion. How the exocyst mediates vesicle tethering and how this tethering complex itself is targeted to sites of exocytosis are not fully understood. Here, using the budding yeast as a model system, I characterized the function of the exocyst subunit Exo70 by a combination of genetic, biochemical and cell biological approaches. My studies have revealed important roles of Exo70 in both exocytosis and actin organization. Firstly, I found that mutations in Exo70 selectively block exocytosis of a major class of post-Golgi secretory vesicles, which carry materials for plasma membrane expansion and cell wall remodeling, and are therefore essential for cell growth. Furthermore, the secretion defect in the mutants is most pronounced during the early stage of cell cycle, which affects daughter cell growth primarily at early budding stage. Secondly, I found that Exo70 functions in concert with Sec3 to target the exocyst to the plasma membrane, which is a pivotal step for vesicle tethering. Exo70 interacts with phosphatidylinositol 4,5-bisphosphate through its C-terminal positively charged residues, which is critical for the membrane targeting of Exo70. Disrupting the membrane targeting of Exo70 or Sec3 alone does not block the membrane targeting of the exocyst. However, when the membrane targeting functions of Exo70 and Sec3 are both disrupted, the exocyst complex can no longer be anchored to the plasma membrane, which leads to defects in exocytosis. Finally, Exo70 also functions in regulating Arp2/3 mediated actin polymerization. Exo70 interacts with the Arc40 subunit of the Arp2/3 complex and synergizes with the WASP homolog Las17 for Arp2/3-dependent nucleation of branched actin filaments. Overall, the multifunction of Exo70 suggests that it may contribute to the coordination of exocytosis and actin dynamics during polarized cell growth, and is likely a target for spatial and temporal regulation of exocytosis. ^

展开