PROTEIN SORTING IN THE TRANS GOLGI NETWORK OF YEAST

蛋白质如何在反式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 细胞可以建立和维持许多 复杂的特殊功能，如极化、吞噬和 调节分泌。识别控制蛋白质的机制 这些途径以及在功能上决定了这个机器如何 如何运作以及如何监管将是理解 这个一般的过程。 解决这些问题的一个模式系统是研究如何 蛋白质运输到酿酒酵母的溶酶体(液泡)。 之前的研究已经发现了一种新的蛋白质运输途径 从跨高尔基网络运输空泡蛋白的一个子集。 这一途径绕过了通过内体/前泡的运输。 车厢。碱性磷酸酶等蛋白质与液泡 Synaxin Vam3p不进入与血浆结合的分泌囊泡 膜或TGN来源的囊泡结合到内体前腺泡 车厢。相反，这些蛋白质似乎进入了一个新的类别 可以直接与液泡融合的小泡。此分类事件 是由这些蛋白质胞浆尾部的决定因素所介导的。 到目前为止，动力蛋白样蛋白Vps1p和非笼状蛋白相关 接头复合体AP3已被发现参与了这一过程。 重要的是，Vps1p和AP3在哺乳动物细胞中都有密切的同源物 这表明这条新的运输途径在所有 真核细胞。 这项提案的目标将是确定和在功能上 描述控制选择性转运的蛋白质的特征 碱性磷酸酶在液泡发生中的作用 使用一种结合了遗传学和生物化学的方法。 将进行基因筛查以确定所需的成分 用于TGN中碱性磷酸酶的具体分选。语料库的功能分析 然后，将进行相应的蛋白质来描绘 蛋白质：选择性地将碱性磷酸酶包装成ITS的蛋白质相互作用 同族运输中间体。这些研究还将确定和 从功能上鉴定碱性磷酸酶融合所需的基因 含有带有液泡的囊泡。最后，条件等位基因 含碱性磷酸酶小泡与细胞融合所需的基因 将利用液泡来分离这些膜运输 中间体并分析它们的成分。

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.