Golgi Targeting and Retention of Yeast Kex2 Protease

描述(由申请人提供)：跨高尔基体网络(TGN)跨膜蛋白的稳态定位是通过TGN和内体隔间之间的动态循环实现的。这些转运途径将加工酶(如Kex2蛋白酶和Furin)定位到它们的作用部位，影响溶酶体/空泡的生物发生，并调节质膜转运蛋白和受体的水平。这些途径的根本重要性被它们的进化保守性所强调。由于需要从生物化学的角度定义TGN-内体转运通路，我们在上一次资助期间开始开发快速、定量的分析方法，用于这些转运通路的无细胞重建。在这个授权期，我们将特别关注囊泡从TGN到晚期内小体的运输，这是溶酶体生物发生和处理蛋白质定位的关键途径，在任何其他系统中都没有重新构建。我们的总体目标将是确定囊泡萌发、货物分类和调节以及囊泡破裂在这一步骤中起作用的笼蛋白涂层囊泡的形成机制。强劲的初步数据，包括已公布和未公布的数据，构成了拟议工作的基础。这项建议的具体目标是：1.严格确定反应中是否存在包被网状蛋白的囊泡中间体，纯化和表征该中间体并测定其分子组成，并设计一种囊泡萌发试验，为货物的募集提供直接检测。2.阐明GGa1/2p和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到TGN-PVC运输小泡中，以及它们如何与小泡外壳的适配器或其他成分相互作用。

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 Strong preliminary data, both published and unpublished, form the basis for the proposed work.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.