Molecular Analysis of Multivesicular Body Formation

描述（由申请人提供）：核内体将注定要在溶酶体中降解的内吞大分子从循环回到细胞表面或传递到其他细胞内细胞器的分子中分离出来。多泡体（MVB）是一种晚期的核内体，含有由核内体膜向室室管腔内陷形成的小泡。许多生长因子受体和免疫系统的刺激成分被分类成MVB囊泡。此外，人类免疫缺陷病毒-1和其他包膜RNA病毒篡夺MVB囊泡形成所需的成分，以产生病毒粒子，从而使病毒感染传播。本应用程序的目的是确定MVB囊泡介导运输的分子基础。该项目的前期工作已经确定了酿酒酵母中Bro1蛋白参与MVB货物分选途径的机制。提出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。在缺乏运输所需的功能性内体分选复合物（escrt）的细胞中，由于内体形成“E类区室”，这一过程受到损害。初步研究表明，E类室的形成是由内体泛素化蛋白的积累驱动的，并依赖于内体系链复合物。特异性目标1将确定调节内体蛋白去泛素化的机制，特异性目标2将探索泛素化蛋白和系带成分如何合作创建E类室，特异性目标3将研究调节Snf7聚合的机制，Snf7被认为是MVB囊泡出芽的驱动力。所有这三个具体目标将采用遗传、生化和显微研究相结合的方法来研究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.