Extended Synaptotagmins (E-Syts)是一类参与内质网-细胞质膜互作建立和维持的关键分子。我们在前期工作中发现E-Syt1的胞质域部分能够在钙离子的调控下转移磷脂，但E-Syt2和E-Syt3能否转移脂质还不清楚。在预实验中，我们成功地将全长E-Syt1重组到膜上并观察到了钙离子依赖的脂转移活性，同时还发现E-Syt2能够介导非钙离子依赖和钙离子依赖两种途径的脂转移。我们提出假设“内质网上的E-Syt1通过C2C结合钙离子和PI(4,5)P2来调节接触位点的形成，并通过C2A结合钙离子来调控脂转移；E-Syt2和E-Syt3通过C2C与质膜作用维系接触位点并介导非钙离子依赖的脂转移，通过C2A结合钙离子调控钙离子依赖的脂转移。”本项目将通过膜蛋白功能重组等方法对这一假设进行逐步论证，揭示不同E-Syts在内质网和细胞质膜互作中维持接触位点和转移脂质的分子机制。

The extended synaptotagmins (E-Syts) are key proteins involved in the endoplasmic reticulum-plasma membrane interactions. They tether the opposed membranes to form membrane contact sites. We have recently found that the cytosolic domain of E-Syt1 transfers glycerophospholipids between membrane bilayers in the presence of Ca2+. However, it’s still not known if E-Syt2 and E-Syt3 could mediate lipid transfer. In our preliminary studies, we successfully reconstituted the full-length E-Syt1 into the liposomes, and demonstrated that this reconstituted E-Syt1 could transfer lipid in a Ca2+ dependent manner. Unexpectedly, we discovered that both Ca2+-independent and Ca2+-dependent lipid transfer were mediated by E-Syt2. We hypothesize that E-Syt1 tethers the membranes through its C2C domain binding to Ca2+ and PI(4,5)P2, and uses its Ca2+ -bound C2A domain to further regulate lipid transport. We also hypothesize that E-Syt2 and E-Syt3 maintain the membrane contact sites and spontaneous lipid transfer through the C2C domain, while stimulate the lipid transfer by C2A in the presence of Ca2+. In this proposal, we will take advantage of our unique functional reconstitution and the microsomal vesicles based assay to disclose the function and molecular mechanisms of E-Syts in the endoplasmic reticulum- plasma membrane contact sites.