Rab7 and Accessory Protein Function in Late Endocytosis

本项目是由美国国家科学基金会职业奖MCB9507206资助的研究的延续。总的目标是研究晚期内吞膜运输的分子机制。内吞作用是真核细胞从其环境中吸收物质并将其转移到适当的细胞内隔室的过程。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，并从环境中去除物质。这一过程包括将被内化物质周围的细胞质膜的一部分内化，然后将该膜挤压形成内核体。然后核内体从细胞外围移动到细胞内部，在那里它可能与其他膜封闭的隔室（如溶酶体）融合，以运送货物。对核内体形态的详细分析以及对分子沿外吞和内吞途径运动的大量研究，使人们对细胞器和途径的复杂性有了极大的认识，其中包括效应蛋白（如g蛋白）的功能。特别是小g蛋白rab7，由于其独特的定位于内核体晚期，并且在调节晚期内吞膜运输中具有重要作用，因此已成为该实验室正在进行的研究的焦点。在之前的CAREER奖期间，提出了与当前项目相关的几个关键观察结果。首先，rab7被证明是从早期核内体到晚期核内体的运输所必需的，并且在随后从晚期核内体到溶酶体的运输中没有功能。因此，rab7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rab7相互作用的伙伴，可能是运输机制的进一步组成部分，已经通过遗传和生化手段确定。最值得注意的是，rab7被证明与磷脂酰肌醇3-激酶相互作用，其活性对于从早期到晚期内体的运输至关重要。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也被观察到，这与兔蛋白作为更一般的下游效应物控制膜融合的特定激活剂的观点一致。其次，阐明了连接高尔基体和核内体的途径。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的信息与内吞动力学的研究有关。第三，rab7阳性核内体（用绿色荧光蛋白-rab7嵌合体显示）被发现沿微管转运，这意味着微管马达是运输机械的组成部分。通过CAREER奖资助获得的试剂、技术和综合经验使实验室处于开展当前项目的独特地位。提出了三个具体目标，将进一步阐明调节晚期内吞膜运输的蛋白质成分。这些是：1，检验vps34磷脂酰肌醇3-激酶的作用，观察到它以rab7构象依赖的方式与rab7相互作用，在内吞运输中；2、利用基于VSV G蛋白序列切割的体外转运实验，分析先前在酵母双杂交实验中鉴定出的两个新的rab7相互作用蛋白的功能；3、利用绿色荧光蛋白-rab7嵌合体实时监测核内体动力学。这个项目的完成将促进对分子如何通过核内体流动以及是什么调节这些过程的认识。这些信息可作为所有膜转运过程的分子机制范例，包括那些涉及病毒进入、抗原加工和呈递以及溶酶体功能的过程。

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.