Functional Reconstitution of Yeast Exocytosis

描述(申请人提供)：膜融合是参与不同细胞事件的基本过程，如受精和神经分泌。生物膜融合依赖于蛋白质来驱动膜的合并，并可能通过体内特定的脂质几何结构来促进。一系列完整的膜蛋白统称为SNARS，介导细胞内运输小泡的融合。虽然陷阱以特定的方式配对，以提供驱动核聚变的机械能量，但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试验来检查胞吐过程中膜融合的机制细节。SNARES和调节蛋白将在体内被操纵，它们对膜融合的具体影响可以在体外直接分析。对分泌过程中执行和调节膜融合的动态蛋白质-蛋白质相互作用的分子评价可能会导致重要的治疗靶点。我们首先研究Sec1p在酵母胞吐中的调节作用。Sec1p主要与酵母t-SNARE复合体结合，直接刺激膜融合。我们将研究Sec1p如何与t-SNARE复合体和完全组装的三元SNARE复合体结合，以及这种结合的功能后果。我们还将通过比较神经元Sec1(n-Sec1)和其酵母对应的Sec1p来确定Sec1p刺激的机制基础。接下来，我们将剖析质膜T-SNARE复合体在体外和体内的功能。Sso1p N末端调节域(NRD)在体外是非必需的，但在体内是必需的。我们将确定Sso1p N末端调节域的功能，验证Sso1p NRD为Sso1p核心H3结构域提供伴侣功能的假设。此外，我们还研究了Sso1p多碱质膜旁区域的功能，这是所有质膜陷阱中的一个保守序列。第三，我们分析了由芽胞形成特异性t-SNARE轻链Spo20p驱动的膜融合，它需要在双层中添加磷脂酸才能有效融合。我们通过检测每个t-SNARE复合体的脂质需求和结构稳定性，探索了t-SNARE轻链Sec9p和Spo20p之间差异的机制基础。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的质膜囊泡将与现有的合成蛋白脂质体一起用于研究与天然膜的融合，以揭示与SNARE突变体在体外和体内融合的差异。

DESCRIPTION (provided by applicant): 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 Sec1p in yeast exocytosis. Sec1p binds primarily to the yeast t-SNARE complex and directly stimulates membrane fusion. We will investigate how Sec1p 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 Sec1p stimulation by comparing neuronal Sec1 (n-Sec1) with its yeast counterpart Sec1p. Next, we will dissect plasma membrane t-SNARE complex function in vitro and in vivo. The Sso1p N-terminal regulatory domain (NRD) is dispensable in vitro but required in vivo. We will determine the function of the Sso1p N-terminal regulatory domain testing the hypothesis that the Sso1p NRD serves a chaperone function for the Sso1p core H3 domain. Additionally, we examine the function of the Sso1p 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.