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）：蛋白质分泌是一个从根本上保守的过程，它为细胞提供了一种机制，可以创建稳定的细胞内室，例如ER，Golgi，溶酶体和质膜，并与其他细胞和组织进行通信。酿酒酵母中此过程的分子机械分析已发现所有真核生物之间共享的基因和机制。 该提案扩展了我在以前的竞争更新中启动的一系列新调查，涉及从高尔基体综合体的囊泡交通机理，尤其是从反式高尔基人到细胞表面的交通途径。有证据表明，从反式高尔基体提出了囊泡形成的多个途径，其中一种涉及在前腔室中或晚期内体中对分泌和液泡蛋白的一部分进行分类。另一种涉及对早期内体驻留蛋白和细胞表面膜蛋白的生物合成形式进行排序。提出了遗传和细胞分馏研究，以发现在早期和晚期内体中控制细胞表面前体的基因。描述了一种遗传方法来识别从早期内体到新生分裂隔膜的膜蛋白上的分类信号。 已经开发了一种生化方法，以探索与酵母中反式高尔基体/早期内体的囊泡发芽相关的基因产物的确切功能。转运囊泡的形成已从酵母细胞的裂解物中重构。使用差分离心步骤测定囊泡的形成，该步骤将高尔基膜和分泌囊泡分开。该测定法将用于净化和重建萌芽事件中所需的蛋白质。 来自酵母高尔基膜的囊泡流量的途径推断到人类细胞中的同一事件。由于这种保护，酵母细胞已被用作方便的载体，用于表达和分泌商用量的治疗性人类蛋白质。对酵母囊泡流量细节的机械欣赏也将阐明造成分泌途径的人类疾病的病理。