Molecular Analysis of Multivesicular Body Formation

描述(申请人提供)：内体将溶酶体中注定要被降解的内吞大分子与被循环回细胞表面或路由到其他细胞内细胞器的分子分开。多囊泡体(MVB)是一种晚期的内小体，它含有内体膜向腔室内陷而形成的小泡。免疫系统的许多生长因子受体和刺激成分被分类为MVB囊泡。此外，人类免疫缺陷病毒-1和其他包膜RNA病毒侵占MVB囊泡形成所需的成分，以产生病毒粒子，从而使病毒感染得以传播。本应用的目的是确定MVB囊泡介导转运的分子基础。该项目之前的工作已经确定了酿酒酵母中Bro1蛋白参与MVB货物分拣途径的机制。这项拟议的工作旨在扩大这一分析的范围，以了解调节MVB囊泡形成的分子机制。这一过程在缺乏运输所需的功能性内小体分选复合体(ESCRT)的细胞中受到损害，这是内小体形成“E类隔室”的结果。初步研究表明，E类隔室的形成是由内体泛素化蛋白的积累驱动的，并依赖于内体系留复合体。特异靶1将确定调节内体蛋白质去泛素化的机制，特异靶2将探索泛素化的蛋白质和拴系组分如何合作创建E类隔室，特异靶3将研究调控Snf7聚合的机制，Snf7被认为是MVB囊泡萌发的驱动力。所有三个特定的目标都将通过结合遗传、生化和显微研究来实现，以检查各种突变的酵母菌株。

DESCRIPTION (provided by applicant): Endosomes segregate endocytosed macromolecules destined to be degraded in the lysosome from molecules that are either recycled back to the cell surface or routed to other intracellular organelles. The multivesicular body (MVB) is a late endosome that contains vesicles formed by invagination of the endosomal membrane toward the compartmental lumen. Many growth factor receptors and stimulatory components of the immune system are sorted into MVB vesicles. Furthermore, human immunodeficiency virus-1 and other enveloped RNA viruses usurp components required for MVB vesicle formation in order to produce virions, thereby enabling the spread of viral infection. The objective of this application is to determine the molecular basis of MVB vesicle-mediated transport. Previous work on this project has identified mechanisms by which the Bro1 protein in Saccharomyces cerevisiae participates in the MVB cargo sorting pathway. The proposed work seeks to expand the scope of this analysis to understand the molecular mechanisms that regulate MVB vesicle formation. This process is impaired in cells lacking functional endosomal sorting complexes required for transport (ESCRTs) as a result of endosomes forming `class E compartments.' Preliminary studies suggest that class E compartment formation is driven by the accumulation of ubiquitinated proteins at endosomes and depends on endosomal tethering complexes. Specific aim 1 will determine the mechanism that regulates protein deubiquitination at endosomes, specific aim 2 will explore how ubiquitinated proteins and tethering components cooperate to create class E compartments, and specific aim 3 will investigate the mechanism that regulates polymerization of Snf7, which has been implicated as the driving force of MVB vesicle budding. All three specific aims will be pursued using a combination of genetic, biochemical, and microscopic studies to examine a variety of mutant yeast strains.