Functional Reconstitution of Yeast Exocytosis

膜融合是参与受精和神经分泌等多种细胞活动的基本过程。 生物膜融合依赖于蛋白质来驱动膜的合并，并可能由特定的脂质促进。 活体内的几何图形。一个统称为SNARS的完整膜蛋白家族介导了 细胞内运输小泡。虽然陷阱以特定的方式配对以提供机械能来驱动融合， 协调这一事件的监管因素在空间和时间上的微妙相互作用仍然难以捉摸。我们使用一种 与纯化的酵母SNARE蛋白和脂类、酵母遗传学和细胞生物学相结合的体外融合分析 检查胞吐过程中膜融合的机制细节。圈套和调节蛋白将是 在体内操作，其对膜融合的具体影响可以在体外直接分析。一种分子 评价分泌过程中执行和调节膜融合的动态蛋白质-蛋白质相互作用 可能会导致重要的治疗靶点。 我们首先研究Seclp在酵母胞吐中的调节作用。Seclp主要与酵母t-SNARE结合 复杂，并直接刺激膜融合。我们将调查Seclp如何绑定到t-SNARE复合体和 完全组装的三元陷阱复合体和这种绑定的功能后果。我们还将确定 神经元型SECL(n-SECL)与酵母菌SECLP刺激的机制基础比较下一首, 我们将在体外和体内解剖质膜T-SNARE复合体的功能。Ssolp N端调控 结构域(NRD)在体外是非必需的，但在体内是必需的。我们将确定Ssolp N端子的功能 调节域测试假设Ssolp NRD为Ssolp核心H3提供伴侣功能 域。此外，我们还研究了Ssolp多碱结合膜区的功能，这是一个在 全是质膜陷阱。第三，我们分析了产孢量特定的t-SNARE光驱动的膜融合。 链Spo20p，需要在双层中添加磷脂酸才能有效融合。我们探索了 T-SNARE轻链Sec9p和Spo20p差异的机理基础 每个t-SNARE复合体的要求和结构稳定性。最后，我们比较了圈套介导的融合和 纯化的细胞器和合成脂质体。纯化的分泌囊泡和倒置的质膜囊泡将被 与现有的合成蛋白脂质体一起用于研究与天然膜的融合，以努力揭示 体内外与SNARE突变体的融合。

Membrane fusion is a fundamental process involved in diverse cellular events such as fertilization and neurosecretion. Biological membrane fusion relies on proteins to drive membrane merger and is likely facilitated by specific lipid geometries in vivo. A family of integral membrane proteins collectively known as SNAREs mediates the fusion of intracellular transport vesicles. While SNAREs pair in specific ways to provide the mechanical energy to drive fusion, the delicate interplay of regulatory elements that orchestrate this event in space and time remain elusive. We use a combination of in vitro fusion assays with purified yeast SNARE proteins and lipids, yeast genetics, and cell biology to examine mechanistic details of membrane fusion during exocytosis. SNAREs and regulatory proteins will be manipulated in vivo and their specific effects on membrane fusion can be directly analyzed in vitro. A molecular appreciation of the dynamic protein-protein interactions that execute and regulate membrane fusion during secretion could potentially lead to important therapeutic targets. We begin by examining the regulatory role of Seclp in yeast exocytosis. Seclp binds primarily to the yeast t-SNARE complex and directly stimulates membrane fusion. We will investigatehow Seclp binds to the t-SNARE complex and fully assembled ternary SNARE complex and the functional consequences of this binding. We will also determine the mechanistic basis for Seclp stimulation by comparing neuronal Seel (n-Secl) with its yeast counterpart Seclp. Next, we will dissect plasma membrane t-SNARE complex function in vitro and in vivo. The Ssolp N-terminal regulatory domain (NRD) is dispensable in vitro but required in vivo. We will determine the function of the Ssolp N-terminal regulatory domain testing the hypothesis that the Ssolp NRD serves a chaperone function for the Ssolp core H3 domain. Additionally, we examine the function of the Ssolp polybasic juxtamembrane region, a conserved sequence in all plasma membrane SNAREs. Third, we analyze membrane fusion driven by the sporulation specific t-SNARE light chain Spo20p, which requires the addition of phosphatidic acid to the bilayer for efficient fusion. We explore the mechanistic basis for the difference between the t-SNARE light chains Sec9p and Spo20p by examining lipid requirements and structural stability of each t-SNARE complex. Finally, we compare SNARE-mediated fusion with purified organelles and synthetic liposomes. Purified secretory vesicles and inverted plasma membrane vesicles will be used with existing synthetic proteoliposomes to study fusion with native membranes in an effort to reveal differences in fusion with SNARE mutants in vitro and in vivo.