Rab7 and Accessory Protein Function in Late Endocytosis

Rab7 和辅助蛋白在晚期内吞作用中的功能

• 批准号: 9982161
• 负责人: Angela Wandinger-Ness
• 金额: $ 69.5万
• 依托单位: University of New Mexico Health Sciences Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9982161&HistoricalAwards=false
• 关键词: Rab7; Accessory; Protein; Function; Late

This project is a continuation of studies initiated with support from NSF CAREER Award MCB9507206. The overall objective is to investigate the molecular mechanisms underlying late endocytic membrane transport. Endocytosis is the process whereby eukaryotic cells internalize materials from their environment and transfer them to appropriate intracellular compartments. Endocytosis allows cells to sample their environment, to receive specific signals from other cells, and to remove materials from the environment. The process involves the involution of a portion of the plasma membrane of the cell surrounding the material to be internalized, followed by a pinching-off of this membrane to form the endosome. The endosome then moves from the periphery of the cell to the interior, where it may fuse with other membrane-enclosed compartments, such as lysosomes, to deliver its cargo. Detailed analyses of endosome morphology and numerous studies tracing the movement of molecules along both the exo- and endocytic pathways have engendered a great appreciation for the complexity of both the organelles and the pathways, which includes the function of effector proteins such as G-proteins. In particular, the small G-protein, rab7, has formed the focal point for ongoing studies in this laboratory because of its unique localization to the late endosome and its demonstrated importance in regulating late endocytic membrane transport. During the previous CAREER award several key observations were made that are relevant to the current project. First, rab7 was shown to be uniquely required for transport from early to late endosomes and to have no function in subsequent transport from late endosomes to lysosomes. Therefore, rab7 is an ideal 'molecular handle' for further dissection of this important transport step. As proof of principle, several rab7 interacting partners, likely to be further components of the transport machinery, have already been identified by genetic and biochemical means. Most notably, rab7 was shown to interact with a phosphatidyl inositol 3-kinase whose activity was critical for transport from early to late endosomes. An additional requirement for a phosphatidyl inositol 3-kinase activity in transport from late endosomes to lysosomes was also observed, consistent with the idea that rab proteins serve as specific activators of more general downstream effectors controlling membrane fusion. Second, a clarification of the pathways interconnecting the Golgi and endosomes was achieved. The information gained about the routes whereby newly synthesized lysosomal proteins and internalized molecules flux to and through the endocytic pathway is relevant to the proposed studies on endosome dynamics. Third, rab7-positive endosomes (visualized with green fluorescent protein-rab7 chimeras) were found to translocate along microtubules implicating microtubule motors as components of the transport machinery. The reagents, technologies and aggregate experience gained through CAREER award funding place the laboratory in a unique position to conduct the current project. Three specific aims are proposed that will further clarify the protein components regulating late endocytic membrane transport. These are: 1, examination of the role of the vps34 phosphatidyl inositol 3-kinase, which was observed to interact with rab7 in a rab7-conformation-dependent manner, in endocytic transport; 2, analysis of the functions of two novel rab7-interacting proteins, previously identified in a yeast two-hybrid assay, using an established in vitro transport assay based on sequential cleavage of VSV G protein; and 3, monitoring of endosome dynamics in real time using green fluorescent protein-rab7 chimeras. Completion of this project will advance knowledge of how molecules flux through endosomes and what regulates these processes. Such information serves as a paradigm for the molecular mechanisms underlying all membrane transport processes, including those involved in virus entry, antigen processing and presentation, and lysosomal function.

这个项目是在NSF职业奖MCB9507206的支持下发起的研究的继续。总体目标是研究晚期内细胞膜转运的分子机制。内吞作用是真核细胞将环境中的物质内化并转移到适当的细胞内隔间的过程。内吞作用允许细胞对其环境进行采样，接收来自其他细胞的特定信号，并将物质从环境中移除。这一过程包括将被内化的物质周围的细胞质膜的一部分内化，然后掐掉这一膜以形成内吞体体。然后，内体从细胞的外围移动到内部，在那里它可能会与其他被膜包裹的隔室融合，如溶酶体，以运送货物。对内体形态的详细分析和大量追踪分子沿胞外和胞内途径运动的研究，使人们对细胞器和途径的复杂性产生了极大的认识，其中包括G蛋白等效应蛋白的功能。特别是小G蛋白Rab7，由于其在晚期内吞体内的独特定位以及在调节晚期内细胞膜运输中的重要性，已成为该实验室正在进行的研究的焦点。在上一次职业奖期间，提出了几点与当前项目有关的重要意见。首先，Rab7被证明是从早期到晚期内小体运输所唯一需要的，而在随后从晚期内小体到溶酶体的运输中没有任何功能。因此，Rab7是进一步剖析这一重要运输步骤的理想分子把手。作为原则的证据，已经通过遗传和生化手段确定了几个与Rab7相互作用的伙伴，它们很可能是运输机械的进一步组成部分。最值得注意的是，Rab7被证明与磷脂酰肌醇3-激酶相互作用，该激酶的活性对于从早期到晚期的内吞体内运输至关重要。还观察到在从晚期内体到溶酶体的运输过程中对磷脂酰肌醇3-激酶活性的额外要求，这与RAB蛋白作为控制膜融合的更一般下游效应器的特定激活剂的想法一致。第二，明确了连接高尔基体和内体的途径。关于新合成的溶酶体蛋白和内化分子进入和通过内吞途径的途径，所获得的信息与拟议的内体动力学研究有关。第三，发现Rab7阳性的内体(用绿色荧光蛋白-Rab7嵌合体显示)沿着微管移位，这意味着微管马达是运输机械的组成部分。通过职业奖励资助获得的试剂、技术和积累的经验使实验室处于开展当前项目的独特地位。提出了三个特定的目标，以进一步阐明调节晚期内细胞膜运输的蛋白质成分。它们是：1，检测Vps34磷脂酰肌醇3-激酶，它被观察到以一种依赖于rab7构象的方式与rab7相互作用，在内胞转运中；2，分析两个新的与rab7相互作用的蛋白的功能，以前在酵母双杂交实验中，使用建立的基于VSV G蛋白的序列切割的体外转运实验；以及3，使用绿色荧光蛋白-rab7嵌合体实时监测内体动态。该项目的完成将促进人们对分子如何通过内体流动以及是什么调节这些过程的了解。这些信息为所有膜转运过程的分子机制提供了范例，包括那些涉及病毒进入、抗原处理和呈递以及溶酶体功能的分子机制。