Golgi Targeting and Retention of Yeast Kex2 Protease

描述（由申请人提供）：跨高尔基体网络（TGN）的跨膜蛋白的稳态定位是通过TGN和内体区室之间的动态循环来实现的。这些转运途径将加工酶（例如 Kex2 蛋白酶和弗林蛋白酶）定位到其作用位点，影响溶酶体/液泡生物发生并调节质膜转运蛋白和受体的水平。这些途径的基本重要性通过它们的进化保守性得到了强调。由于需要从生化角度定义 TGN 内体转运途径，因此我们在上一个资助期内开始开发快速、定量的检测方法，以对这些转运途径进行无细胞重建。在此资助期内，我们将特别关注从 TGN 到晚期内体的囊泡转运，这是溶酶体生物发生和蛋白质定位处理中的关键途径，尚未在任何其他系统中重建。我们的总体目标是确定在此步骤中发挥作用的网格蛋白包被囊泡形成过程中的囊泡出芽、货物分类和调节以及囊泡分裂的机制。已发表和未发表的强有力的初步数据构成了拟议工作的基础。该提案的具体目标是： 1. 严格确定反应中是否存在网格蛋白包被的囊泡中间体，纯化和表征该中间体并确定其分子组成，并设计一种囊泡出芽测定法，为货物招募提供直接测定。 2. 阐明 Gga1/2p 与 AP1 接头在囊泡形成和货物选择中的作用，并确定它们如何被招募到膜上。 3. 确定货物蛋白如何被募集至 TGN-PVC 运输囊泡以及它们如何与接头或囊泡外壳的其他成分相互作用。

DESCRIPTION (provided by applicant): Steady-state localization of transmembrane proteins of the trans Golgi network (TGN) is achieved by dynamic recycling between TGN and endosomal compartments. These transport pathways localize processing enzymes (e.g. Kex2 protease and furin) to their sites of action, effect lysosomal/vacuolar biogenesis and regulate levels of plasma membrane transporters and receptors. The fundamental importance of these pathways is underscored by their evolutionary conservation. The need to define TGN-endosomal transport pathways biochemically led us in the last grant period to begin to develop rapid, quantitative assays for cell-free reconstitution of these transport pathways. In this grant period, we will specifically focus on vesicular transport from the TGN to the late endosome, a key pathway both in lysosomal biogenesis and in processing protein localization that has not been reconstituted in any other system. Our overall goals will be to define mechanisms of vesicle budding, cargo sorting and regulation and vesicle scission in the formation of the clathrin coated vesicles that function in this step. Strong preliminary data, both published and unpublished, form the basis for the proposed work. The Specific Aims of this proposal are: 1. To determine rigorously whether there is a clathrin-coated vesicle intermediate in the reaction, to purify and characterize this intermediate and determine its molecular composition and to devise a vesicle budding assay that provides a direct assay for cargo recruitment. 2. To elucidate the roles of Gga1/2p versus AP1 adaptors in vesicle formation and cargo selection and determine how they are recruited to membranes. 3. To determine how cargo proteins are recruited to TGN-PVC transport vesicles and how they interact with adaptors or other components of the vesicle coat.