Golgi Targeting and Retention of Yeast Kex2 Protease

描述（由申请人提供）：反式高尔基网络（TGN）跨膜蛋白的稳态定位是通过TGN和内体室之间的动态循环来实现的。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被它们的进化守恒所强调。由于需要从生物化学角度定义tgn -内体运输途径，我们在上一个资助期开始开发快速、定量的方法来检测这些运输途径的无细胞重建。在此资助期内，我们将特别关注从TGN到晚期内体的囊泡运输，这是溶酶体生物发生和加工蛋白质定位的关键途径，在任何其他系统中都没有重建。我们的总体目标将是确定在这一步骤中起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。本建议的具体目标是：1。严格确定反应中是否存在网格蛋白包覆的囊泡中间体，对该中间体进行纯化和表征，确定其分子组成，并设计囊泡出芽试验，为货物招募提供直接试验。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招募到TGN-PVC运输囊泡中，以及它们如何与适配器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 Strong preliminary data, both published and unpublished, form the basis for the proposed work.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.