Rab7 and Accessory Protein Function in Late Endocytosis

该项目是在NSF CAREER Award MCB 9507206支持下启动的研究的延续。 总体目标是研究晚期内吞膜转运的分子机制。内吞作用是真核细胞从其环境中内化物质并将其转移到适当的细胞内区室的过程。 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，并从环境中移除物质。 该过程涉及围绕待内化的材料的细胞质膜的一部分的内卷，随后夹断该膜以形成内体。 然后，内体从细胞的外围移动到内部，在那里它可以与其他膜封闭的隔室（例如溶酶体）融合以递送其货物。 对内体形态的详细分析和追踪分子沿胞外和内吞途径沿着运动的大量研究已经引起了对细胞器和途径的复杂性的极大赞赏，其中包括效应蛋白如G蛋白的功能。特别是，小G-蛋白，rab 7，已形成了在这个实验室正在进行的研究的焦点，因为它的独特的本地化的晚期内体和其在调节晚期内吞膜转运的重要性。在上一个CAREER奖项期间，提出了与当前项目相关的几个关键观察结果。首先，rab 7被证明是唯一需要从早期到晚期内体的运输，并在随后的运输从晚期内体到溶酶体没有功能。因此，rab 7是进一步解剖这一重要转运步骤的理想“分子手柄”。作为原理的证明，几个rab 7相互作用的合作伙伴，可能是运输机制的进一步组成部分，已经确定了遗传和生化手段。最值得注意的是，rab 7被证明与磷脂酰肌醇3-激酶，其活性是从早期到晚期内体运输的关键相互作用。还观察到在从晚期内体到溶酶体的运输中对磷脂酰肌醇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物的特异性激活剂的想法一致。其次，阐明了高尔基体和内体之间的相互连接途径。新合成的溶酶体蛋白质和内化分子流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相关。第三，发现rab 7阳性内体（用绿色荧光蛋白-rab 7嵌合体显示）沿沿着微管易位，表明微管马达是转运机制的组成部分。通过CAREER奖资助获得的试剂，技术和综合经验使实验室处于进行当前项目的独特位置。提出了三个具体的目标，将进一步澄清调节后期内吞膜转运的蛋白质组分。 这些是：1，检测vps 34磷脂酰肌醇3-激酶在胞吞转运中的作用，观察到vps 34磷脂酰肌醇3-激酶在内吞转运中以rab 7构象依赖性方式与rab 7相互作用; 2，使用基于VSV G蛋白的顺序切割的已建立的体外转运测定，分析先前在酵母双杂交测定中鉴定的两种新的rab 7相互作用蛋白的功能;和3，使用绿色荧光蛋白-rab 7嵌合体在真实的时间内监测内体动力学。 该项目的完成将推进分子如何通过内体流动以及是什么调节这些过程的知识。这些信息作为所有膜转运过程的分子机制的范例，包括参与病毒进入、抗原加工和呈递以及溶酶体功能的分子机制。

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.