PROTEIN SORTING IN THE TRANS GOLGI NETWORK OF YEAST

酵母反式高尔基体网络中的蛋白质分类

• 批准号: 6019503
• 负责人: ROBERT C PIPER
• 金额: $ 17.62万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-05 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019503
• 关键词: Golgi apparatus; Saccharomyces cerevisiae; alkaline phosphatase; flow cytometry; fungal genetics; gene mutation; green fluorescent proteins; immunoprecipitation; intermolecular interaction; membrane fusion; membrane transport proteins; protein structure function; protein transport; vesicle /vacuole; yeasts

The process of how proteins are segregated and sorted in the trans-Golgi Network and endosomal system of eukaryotic cells is one of the fundamental mechanisms by which cells can establish and maintain many complex specialized functions such as polarization, phagocytosis, and regulated secretion. Identifying the protein machinery that controls these pathways as well as functionally determining how this machinery operates and how it might be regulated will be the key to understanding this general process. One model system for addressing these issues has been the study of how proteins traffic to the lysosome (vacuole) of Saccharomyces cerevisiae. Previous studies have uncovered a new protein trafficking pathway that transports a subset of vacuolar proteins from the trans-Golgi Network. This pathway bypasses transit through the endosomal/prevacuolar compartment. Protein such as Alkaline Phosphatase and the vacuolar syntaxin Vam3p do not enter into secretory vesicles bound for the plasma membrane or TGN-derived vesicles bound for the endosomal prevacuolar compartment. Instead, these proteins appear to enter a new class of vesicles that may fuse directly with the vacuole. This sorting event is mediated by determinants within the cytosolic tail of these proteins. So far, the dynamin-like protein Vps1p and the non-clathrin associated adaptor complex AP3 have been found to participate in this process. Importantly, both Vps1p and AP3 have close homologues in mammalian cells indicating that this novel trafficking pathway operates in all eukaryotic cells. The goal of this proposal will be to identify and functionally characterize the proteins that control the selective transport of Alkaline Phosphatase to the vacuole along this new vacuolar biogenesis pathway using an approach that combines genetics and biochemistry. Genetic screens will be conducted to identify the components required for the specific sorting of ALP in the TGN. A functional analysis of the corresponding proteins will then be conducted to delineate the protein:protein interactions that selectively package ALP into its cognate transport intermediate. These studies will also identify and functionally characterize the genes required for the fusion of ALP- containing vesicles with the vacuole. Finally, conditional alleles of the genes required for the fusion of ALP-containing vesicles with the vacuole will be exploited to isolate these membrane trafficking intermediates and analyze their constituents.

蛋白质在反高尔基体中分离和分类的过程 真核细胞的网络和内体系统是其中之一 细胞建立和维持许多功能的基本机制 复杂的专门功能，例如极化、吞噬作用和 调节分泌。 识别控制蛋白质机制 这些途径以及功能上决定了该机器如何 运作以及如何监管将是理解的关键 这个一般过程。 解决这些问题的一种模型系统是研究如何 蛋白质运输至酿酒酵母的溶酶体（液泡）。 先前的研究发现了一种新的蛋白质运输途径 从跨高尔基体网络运输液泡蛋白的子集。 该途径绕过内体/前液泡的转运 隔间。 蛋白质，如碱性磷酸酶和液泡 Syntaxin Vam3p 不进入与血浆结合的分泌囊泡 膜或 TGN 衍生的囊泡与内体前液泡结合 隔间。 相反，这些蛋白质似乎进入了一类新的蛋白质 可能与液泡直接融合的囊泡。 本次排序活动 由这些蛋白质的胞质尾部内的决定簇介导。 到目前为止，动力样蛋白 Vps1p 和非网格蛋白相关 已发现衔接复合体AP3参与了这一过程。 重要的是，Vps1p 和 AP3 在哺乳动物细胞中都有密切的同源物 表明这种新的贩运途径在所有国家都有效 真核细胞。 该提案的目标是确定并在功能上 表征控制选择性转运的蛋白质 碱性磷酸酶沿着这个新的液泡生物发生进入液泡 使用结合遗传学和生物化学的方法的途径。 将进行基因筛选以确定所需的成分 用于 TGN 中 ALP 的特定排序。的功能分析 然后将进行相应的蛋白质来描绘 蛋白质：蛋白质相互作用，选择性地将 ALP 包装到其 同源运输中间体。这些研究还将确定和 功能表征 ALP-融合所需的基因 含有带有液泡的囊泡。 最后，条件等位基因 含有 ALP 的囊泡与 将利用液泡来分离这些膜运输 中间体并分析其成分。