Rab7 and Accessory Protein Function in Endocytosis

Rab7 和辅助蛋白在内吞作用中的功能

• 批准号: 0446179
• 负责人: Angela Wandinger-Ness
• 金额: --
• 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: 2005
• 资助国家: 美国
• 起止时间: 2005-04-15 至 2010-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0446179&HistoricalAwards=false
• 关键词: Rab7; Accessory; Protein; Function; Endocytosis

This project addresses three specific aims relevant to understanding late endocytic transport and sorting in mechanistic detail. This work is relevant for understanding membrane transport mechanisms and the regulation of receptor trafficking. Studies in Dr. Wandinger-Ness's laboratory have identified Rab7 and its interacting partners (lipid kinases and phosphatases and a proteasome subunit) as a key regulators of late endocytic transport and sorting. They have also demonstrated that late endosomal motility on microtubules is bidirectional and dependent on functional Rab7. Since individual Rab GTPases have largely distinct rather than shared effectors, a comprehensive understanding of membrane trafficking will require the detailed study of individual transport steps and molecules involved. Having identified several key components in late endocytic transport and sorting with support provided by MCB9982161, her plan is to establish in mechanistic terms how these components function together to orchestrate and regulate late endosomal transport and sorting. Planned studies will examine phophoinositide metabolism, proteasome function and microtubule transport all in the context of how these are coordinately regulated by Rab7 and its interacting partners on late endosomes. Based on evidence that the hVPS34 lipid kinase and its p150 adapter form mutually exclusive complexes with the myotubularin 1 lipid phosphatase or with Rab7, Dr. Wandinger-Ness hypothesizes that these selective interactions serve to coordinately regulate local phosphatidyl inositol 3-phosphate (PI3P) levels. In Aim1, she will use in vitro and in vivo assays to test the postulate and establish the mechanisms governing late endosomal phosphoinositide metabolism. Because phosphatidyl inositides play a central role in transport by serving as membrane platforms for recruiting molecules that facilitate membrane fusion and intraluminal vesicle formation, addressing how phosphoinositides are synthesized and turned over will provide new insights into how this aspect of transport is regulated. XAPC7 is an alpha subunit of the proteasome that has been shown by Dr. Wandinger-Ness to complex with Rab7. Based on her initial published data, the proteasome seems to negatively regulate transport, but the exact function of the proteasome on late endosomes remains unclear. Aim2 is geared toward determining the function of the proteasome in the regulation of transport. It is expected that these studies will interface with the growing literature implicating ubiquitination and proteasome activity in the proper trafficking and downregulation of membrane signalling receptors. Finally, data from Dr. Wandinger-Ness' own work and others demonstrate that functional Rab7 and its interacting partner RILP are important for centrosome-directed late endosomal motility on microtubules. Aim3 will explore how the dynactin associated/RILP complex and the KIF1Bb kinesin motor may cooperate to regulate bidirectional late endosomal motility. Together, these studies promise to offer a more comprehensive understanding of late endosomal transport and sorting that is a fundamental facet of the life of eukaryotic cells. In addition, NSF funding of this project will continue to facilitate resource development as exemplified by past contributions in reagents, educational training and resource development. The studies will continue to expand the repertoire of reagents that Dr. Wandinger-Ness has already freely disseminated to over 100 investigators, resulting in numerous citations. The research serves as an underpinning for educational innovation in an interdisciplinary graduate training program. Prior NSF funding provided seed monies for the establishment of a state-of-the-art microscopy facility at the University of New Mexico that is now heavily utilized by students and fellows in various Federally funded research projects. Finally, the project has with prior support been central to the training of a total of 21 undergraduate and graduate students and postdoctoral fellows, more than half of which are women and underrepresented minorities. It is expected this extensive training track record will be expanded with this renewal support.

这个项目解决了三个具体的目标相关的理解后期内吞运输和分选的机械细节。 这项工作是相关的了解膜运输机制和受体贩运的监管。 Wandinger-Ness博士实验室的研究已经确定Rab 7及其相互作用伙伴（脂质激酶和磷酸酶以及蛋白酶体亚基）是晚期内吞转运和分选的关键调节因子。 他们还证明了微管上的晚期内体运动是双向的，并且依赖于功能性Rab 7。 由于单个Rab GTP酶具有很大程度上不同的而不是共享的效应子，因此对膜运输的全面理解将需要对所涉及的单个运输步骤和分子进行详细研究。 在MCB 9982161的支持下，她确定了晚期内吞转运和分选中的几个关键组分，她的计划是从机制上确定这些组分如何共同发挥作用，以协调和调节晚期内体转运和分选。 计划中的研究将检查磷酸肌醇代谢，蛋白酶体功能和微管转运，所有这些都是如何协调调节Rab 7及其相互作用的合作伙伴在晚期内体的背景下。 基于hVPS 34脂质激酶及其p150接头与肌管蛋白1脂质磷酸酶或Rab 7形成互斥复合物的证据，Wandinger-Ness博士假设这些选择性相互作用用于协调调节局部磷脂酰肌醇3-磷酸（PI 3 P）水平。 在Aim 1中，她将使用体外和体内试验来测试假设并建立控制晚期内体磷酸肌醇代谢的机制。 由于磷脂酰肌醇在运输中发挥着核心作用，作为膜平台招募分子，促进膜融合和腔内囊泡的形成，解决磷酸肌醇是如何合成和翻转将提供新的见解，这方面的运输是如何调节。 XAPC 7是蛋白酶体的α亚基，Wandinger-Ness博士已经证明可以与Rab 7复合。 基于她最初发表的数据，蛋白酶体似乎负调节转运，但蛋白酶体对晚期内体的确切功能仍不清楚。aim 2是为了确定蛋白酶体在运输调节中的功能。 预计这些研究将与越来越多的文献接口，涉及泛素化和蛋白酶体活性的适当贩运和下调膜信号受体。 最后，来自Wandinger-Ness博士自己的工作和其他人的数据表明，功能性Rab 7及其相互作用伙伴RILP对于微管上的中心体定向晚期内体运动是重要的。 目的3将探讨如何dynactin相关/RILP复合物和KIF 1Bb驱动蛋白电机可能合作，以调节双向晚期内体运动。 总之，这些研究有望提供一个更全面的了解晚期内体运输和分选，这是真核细胞生命的一个基本方面。 此外，国家科学基金会对该项目的资助将继续促进资源开发，过去在试剂、教育培训和资源开发方面的贡献就是一个例证。 这些研究将继续扩大Wandinger-Ness博士已经免费分发给100多名研究人员的试剂库，导致大量引用。 该研究为跨学科研究生培养计划的教育创新奠定了基础。 先前的NSF资金为在新墨西哥州大学建立一个最先进的显微镜设施提供了种子资金，该设施现在被学生和研究员大量利用在各种联邦资助的研究项目中。 最后，该项目在之前的支持下对总共21名本科生、研究生和博士后研究员的培训至关重要，其中一半以上是女性和代表性不足的少数族裔。 预计这一广泛的培训记录将随着这一更新支持而扩大。