Molecular Studies of Eukaryotic Cell Surface Growth

描述(申请人提供)：蛋白质分泌是一个基本保守的过程，为细胞提供了一种机制，以创建稳定的细胞内隔间，如内质网、高尔基体、溶酶体和质膜，并与其他细胞和组织进行交流。对酿酒酵母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。 这一建议扩展了我在之前的竞争性更新中启动的关于高尔基复合体囊泡运输机制的新研究路线，特别是从跨高尔基复合体到细胞表面的运输途径。已有证据表明，从跨高尔基体形成囊泡的途径有多种，其中之一涉及在液泡前隔室或晚内体中分离分泌和空泡蛋白的子集。另一个涉及对早期内体驻留蛋白和生物合成形式的细胞表膜蛋白的分类。遗传学和细胞分级研究被建议用来发现控制早期和晚期内小体中细胞表面前体分类的基因。描述了一种遗传学方法来识别膜蛋白上的分选信号，该蛋白从早期的内吞体内定向到新生的分裂隔膜。 建立了一种生化方法来探索酵母中反式高尔基体/早期内体与囊泡萌发相关的基因产物的确切功能。酵母细胞裂解产物重组了运输泡的形成。用分离高尔基膜和分泌囊泡的差速离心法测定囊泡的形成。这项检测将被用于提纯和重组萌芽事件中所需的蛋白质。 从酵母高尔基膜的囊泡运输途径可以推断出在人类细胞中也发生了同样的事件。由于这种保守，酵母细胞已被用作一种方便的载体，用于表达和分泌商业数量的治疗性人类蛋白。对酵母中囊泡运输细节的机械性评价也将阐明影响分泌途径的人类疾病的病理。

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.