Molecular Analysis of Multivesicular Body Formation

描述（由申请方提供）：内体是异质的膜结合区室，其将预定在溶酶体中降解的内吞大分子与再循环回细胞表面或导向其他细胞内目的地的分子分离。多泡体（MVB）是一种晚期内吞隔室，含有内体膜向内向隔室腔内陷形成的囊泡。许多控制真核生物生理学的蛋白质被分选到MVB囊泡中，包括活化的生长因子受体和免疫系统的刺激组分。MVB转运途径在多种异常病理条件下受到干扰。因此，对MVB生物发生的机制理解可能最终导致用于治疗人类疾病的新的治疗或诊断靶点。本申请的长期目标是了解MVB分选的分子机制。一种遗传策略已被用来隔离突变，阻止MVB途径的芽殖酵母酿酒酵母。这种方法已经发现了BR01基因，该基因编码与晚期内体相关的保守的可溶性细胞质蛋白（Bro1p）。在BR01基因缺失的酵母突变体中不存在MVB囊泡。该提案的具体目的是1）定义Brolp在MVB途径中发挥作用的阶段，2）确定Brolp与晚期内体的关联机制，以及3）鉴定在MVB途径中与Brolp功能性合作的辅因子。将在Brolp氨基酸序列中构建突变，并将使用功能测定、定位研究和电子显微镜的组合来确定这些突变的结果。生物化学和遗传学研究将用于表征参与Brolp向晚期内体募集或在MVB途径中与Bro1p功能性合作的其他因子。

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.