Molecular Studies of Eukaryotic Cell Surface Growth

真核细胞表面生长的分子研究

• 批准号: 6547543
• 负责人: RANDY W. SCHEKMAN
• 金额: $ 17.93万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6547543
• 关键词: Golgi apparatus; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; beta fructofuranosidase; cell cycle; cell membrane; chitin; clathrin; density gradient ultracentrifugation; developmental genetics; electron microscopy; enzyme activity; eukaryote; gene expression; genetic regulation; immunoelectron microscopy; immunoprecipitation; laboratory rabbit; membrane proteins; molecular cloning; protein purification; protein transport; secretory protein; vesicle /vacuole; western blottings

DESCRIPTION (provided by applicant): Protein secretion is a fundamentally conserved process that provides a mechanism for cells to create stable Intracellular compartments such as the ER, Golgi, lysosome and plasma membrane, and to communicate with other cells and tissues. A molecular mechanistic analysis of this process in Saccharomyces cerevisiae has uncovered genes and mechanisms that are shared among all eukaryotes. This proposal extends a new line of investigation that I initiated in my previous competitive renewal concerning the mechanism of vesicle traffic from the Golgi complex and particularly the pathway of traffic from the trans Golgi to the cell surface. Evidence has emerged to suggest multiple paths of vesicle formation from the trans Golgi, one of which involves sorting a subset of secretory and vacuolar proteins in the pre-vacuolar compartment, or late endosome. Another involves sorting of early endosomal resident proteins and biosynthetic forms of cell surface membrane proteins. Genetic and cell fractionation studies are proposed to discover the genes that govern sorting of cell surface precursors in the early and late endosomes. A genetic approach is described to identify the sorting signal on a membrane protein that is directed from the early endosome to the nascent division septum. A biochemical approach has been developed to explore the exact function of gene products associated with vesicle budding from the trans Golgi/early endosome in yeast. Transport vesicle formation has been reconstituted from lysates of yeast cells. Vesicle formation is assayed using a differential centrifugation step that separates Golgi membranes and secretory vesicles. This assay will be used to purify and reconstitute proteins required in the budding event. The pathways of vesicle traffic from the yeast Golgi membrane extrapolate to the same event in human cells. As a result of this conservation, yeast cells have been used as a convenient vehicle for the expression and secretion of commercial quantities of therapeutic human proteins. A mechanistic appreciation of the details of vesicle traffic in yeast will also illuminate the pathology of human diseases that impinge on the secretory pathway.

描述（由申请人提供）：蛋白质分泌是一种基本上保守的过程，为细胞提供了一种机制，以产生稳定的细胞内区室，如ER、高尔基体、溶酶体和质膜，并与其他细胞和组织通讯。对酿酒酵母中这一过程的分子机制分析揭示了所有真核生物共有的基因和机制。 这个建议扩展了一个新的调查线，我在我以前的竞争性更新有关的机制，囊泡交通从高尔基复合体，特别是交通的途径，从transGolgi到细胞表面。有证据表明，从反式高尔基体形成囊泡的多种途径，其中之一涉及在前液泡区室或晚期内体中分选分泌蛋白和液泡蛋白的子集。另一个涉及早期内体驻留蛋白和细胞表面膜蛋白的生物合成形式的分选。遗传和细胞分级分离的研究提出了发现的基因，管理的细胞表面前体细胞在早期和晚期内体的排序。描述了一种遗传学方法来识别从早期内体到新生分裂隔膜的膜蛋白上的分选信号。 一种生物化学方法已经被开发来探索酵母中与来自transGolgi/早期内体的囊泡出芽相关的基因产物的确切功能。运输囊泡的形成已经从酵母细胞的裂解物重建。使用差速离心步骤分离高尔基体膜和分泌囊泡来测定囊泡形成。该测定将用于纯化和重构出芽事件中所需的蛋白质。 从酵母高尔基体膜的囊泡交通的途径外推到人类细胞中的相同事件。由于这种保守性，酵母细胞已被用作表达和分泌商业量的治疗性人蛋白质的方便载体。酵母中囊泡交通的细节的机械欣赏也将照亮人类疾病的病理学，对分泌途径的冲击。