Golgi Targeting and Retention of Yeast Kex2 Protease

酵母 Kex2 蛋白酶的高尔基体靶向和保留

• 批准号: 7458655
• 负责人: ROBERTA S. FULLER
• 金额: $ 28.35万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7458655
• 关键词: Acanthocytosis; Antibodies; Biochemical; Biochemical Genetics; Biogenesis; Biological Assay; Cell membrane; Cells; Chorea; Clathrin; Clathrin Adaptors; Clathrin-Coated Vesicles; Coated vesicle; Cohen syndrome; Data; Defect; Disease; Employee Strikes; Endopeptidases; Enzymes; Event; Exhibits; Genes; Goals; Golgi Targeting; Grant; Hereditary Disease; Homologous Gene; Human; Integral Membrane Protein; Localized; Measures; Membrane; Membrane Transport Proteins; Molecular; Mutation; Nerve Degeneration; Neurodevelopmental Disorder; Pathway interactions; Peptide Hydrolases; Process; Proteins; Publishing; Reaction; Recruitment Activity; Recycling; Regulation; Role; Site; Sorting - Cell Movement; System; Transcription Factor AP-1; Transport Vesicles; Vacuolar Protein Sorting; Vesicle; Work; Yeasts; base; intracellular protein transport; late endosome; protein localization location; receptor; reconstitution; temperature sensitive mutant; trans-Golgi Network

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 Strong preliminary data, both published and unpublished, form the basis for the proposed work.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

描述（由申请人提供）：通过TGN和内体隔室之间的动态回收来实现反式高尔基网络（TGN）的跨膜蛋白的稳态定位。这些转运途径将加工酶（例如Kex2蛋白酶和脂肪蛋白）定位到其作用部位，影响溶酶体/液泡生物发生以及调节质膜转运蛋白和受体的水平。这些途径的基本重要性由它们的进化保护强调。定义TGN-粘体运输途径的需求是生物化学上的最后一个赠款时期，开始开发快速，定量的测定，以无细胞的重构这些运输途径。在这个赠款期间，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的关键途径和在任何其他系统中尚未重组的蛋白质定位中的关键途径。我们的总体目标是定义囊泡萌芽，货物排序和调节的机制，以及在此步骤中起作用的网状蛋白涂层囊泡的形成中。强有力的初步数据均已发布和未发表，构成了拟议工作的基础。该提案的具体目的是：1。严格确定反应中是否有涂有网状蛋白的囊泡中间体，以净化和表征该中间体并确定其分子组成，并设计囊泡萌芽测定法，从而为货物招募提供直接的测定法。 2。为了阐明GGA1/2p与AP1适配器在囊泡形成和货物选择中的作用，并确定它们的募集方式。 3。确定如何将货物蛋白募集到TGN-PVC传输囊泡以及它们如何与适配器或囊泡外套的其他组件相互作用。