Molecular Analysis of Multivesicular Body Formation

描述(申请人提供)：内小体是异质的膜结合室，它将溶酶体中注定要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目的地的分子分开。多囊泡体(MVB)是一种晚期的内吞小体，它含有由内体膜向腔室内陷形成的小泡。许多控制真核生物生理的蛋白质被分成MVB囊泡，包括激活的生长因子受体和免疫系统的刺激成分。MVB的转运途径在各种异常的病理条件下受到干扰。因此，对MVB生物发生机制的理解可能最终导致人类疾病治疗的新的治疗或诊断靶点。这项应用的长期目标是了解MVB分选的分子机制。一种遗传策略已经被用来在萌芽酵母中分离阻断MVB途径的突变。这种方法已经发现了BR01基因，它编码一种保守的可溶性细胞质蛋白(Bro1p)，与晚期内体相关。在BR01基因缺失的酵母突变株中，MVB囊泡缺失。该提案的具体目的是：1)确定BROLP在MVB途径中的作用阶段；2)确定BROLP与晚期内小体结合的机制；3)确定在MVB途径中与BROLP功能协同的辅助因子。突变将在Brop氨基酸序列中构建，这些突变的后果将使用功能分析、定位研究和电子显微镜相结合的方法来确定。生化和遗传学研究将被用来表征其他参与布罗普招募到晚期内小体或在功能上与MVB途径中的Bro1p合作的因素。

DESCRIPTION (provided by applicant): Endosomes are heterogeneous membrane-bound compartments that segregate endocytosed macromolecules destined to be degraded in the lysosome from molecules that are either recycled back to the cell surface or routed toward other intracellular destinations. The multivesicular body (MVB) is a late endocytic compartment that contains vesicles formed by inward invagination of the endosomal membrane toward the compartmental lumen. Many proteins that control the physiology of eukaryotic organisms are sorted into MVB vesicles, including activated growth factor receptors and stimulatory components of the immune system. The MVB transport pathway is perturbed in a variety of abnormal pathological conditions. Therefore, a mechanistic understanding of MVB biogenesis may ultimately lead to new therapeutic or diagnostic targets for the treatment of human diseases. The long-term objective of this application is to understand the molecular mechanisms of MVB sorting. A genetic strategy has been used to isolate mutations that block the MVB pathway in the budding yeast Saccharomyces cerevisiae. This approach has uncovered the BR01 gene, which encodes a conserved soluble, cytoplasmic protein (Bro1p) that associates with late endosomes. MVB vesicles are absent in yeast mutants in which the BR01 gene has been deleted. The specific aims of this proposal are 1) to define the stage at which Brolp functions in the MVB pathway, 2) to determine the mechanism for association of Brolp with late endosomes, and 3) to identify co-factors that functionally cooperate with Bro1p in the MVB pathway. Mutations will be constructed in the Brolp amino acid sequence, and the consequences of these mutations will be determined using a combination of functional assays, localization studies, and electron microscopy. Biochemical and genetic studies will be used to characterize other factors that participate in the recruitment of Brolp to late endosomes or functionally cooperate with Bro1p in the MVB pathway.