Functional Reconstitution of Yeast Exocytosis

膜融合是一个基本的过程，涉及到不同的细胞事件，如受精和神经分泌。 生物膜融合依赖于蛋白质来驱动膜合并，并且可能由特定脂质促进 体内几何形状。一个被统称为SNARE的完整膜蛋白家族介导了 胞内转运囊泡。虽然SNARE以特定的方式配对以提供驱动聚变的机械能， 在空间和时间上协调这一事件的调控因素之间的微妙相互作用仍然难以捉摸。我们使用一个 使用纯化的酵母SNARE蛋白和脂质的体外融合测定、酵母遗传学和细胞生物学的组合， 研究胞吐作用中膜融合的机制细节。SNARE和调节蛋白将被 在体内操作，并且它们对膜融合的特异性作用可以在体外直接分析。分子 在分泌过程中执行和调节膜融合的动态蛋白质-蛋白质相互作用的评价 有可能成为重要的治疗靶点 我们开始通过检查Seclp在酵母胞吐中的调节作用。Seclp主要与酵母t-SNARE结合 复合物并直接刺激膜融合。我们将研究Seclp如何与t-SNARE复合物结合， 完全组装的三元SNARE复合物和这种结合的功能后果。我们还将确定 通过比较神经元Seel（n-Secl）与其酵母对应物Seclp，为Seclp刺激提供机制基础。下一个是 我们将在体外和体内解剖质膜t-SNARE复合物的功能。Ssolp N-末端调节 结构域（NRD）在体外是必需的，但在体内是必需的。我们将确定Ssolp N-末端的功能 调节域测试Ssolp NRD为Ssolp核心H3提供伴侣蛋白功能的假设 域此外，我们还研究了Ssolp多碱基跨膜区的功能，Ssolp多碱基跨膜区是一个保守序列， 所有的质膜陷阱。第三，我们分析了由孢子形成特异性t-SNARE光驱动的膜融合， 链Spo 20 p，需要向双层添加磷脂酸以实现有效融合。我们探索 通过检查脂质，t-SNARE轻链Sec 9 p和Spo 20 p之间差异的机制基础 每个t-SNARE复合物的要求和结构稳定性。最后，我们比较了SNARE介导的融合与 纯化的细胞器和合成的脂质体。纯化的分泌囊泡和倒置质膜囊泡将被 与现有的合成脂蛋白体一起研究与天然膜的融合，以揭示 在体外和体内与SNARE突变体融合。

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.