Sorting and Transport of Yeast Membrane Proteins

描述(由申请人提供)：这项研究的总体目标是发展对高尔基膜蛋白保留、离子梯度在膜运输中的作用以及VATPase在简单真核生物酿酒酵母中的组装、运输和靶向的机制的理解。酵母已被证明是一个很好的模型系统，无论是在真核细胞中识别调节细胞膜运输的蛋白质，还是研究这些蛋白质发挥作用的分子机制。遗传分析表明，大量的酵母基因不仅编码酵母液泡质子转运酶(V-ATPase)的亚基，而且还编码四(4)个编码蛋白质的基因，这些基因不是该酶的亚基，而是定位于内质网(ER)，是组装和运输V-ATPase所必需的。使用遗传和生化方法，将确定V-ATPase组装因子的特征，以确定哪些因子在组装过程的特定步骤中起作用。这些组装因子也将被描述为在V-ATPase装载到内质网衍生的小泡中，以及在护送V-ATPase到高尔基复合体中的作用。酵母V-ATPase有两种不同的形式：高尔基体和内体形式的复合体与100 kDa亚基的Stv1p亚基组装，而液泡膜上的复合体与100 kDa亚基的Vph1p亚基组装。Stv1p亚型中的高尔基体/内体定位信号将通过突变分析来鉴定，识别和分类Stv1相关V-ATPase的蛋白质将通过遗传学来鉴定和表征。PH敏感形式的绿色荧光蛋白(GFP)将针对各种酵母细胞内细胞器，以确定高尔基体-内体网络的pH。酵母膜运输的研究已被证明对更广泛地理解所有真核细胞的膜运输是非常有用的，因为调节这些过程的机制和蛋白质在从酵母到人类的过程中有着惊人的相似性。这些在酵母中的基础研究为我们理解与细胞器酸化缺陷有关的许多人类疾病提供了重要的见解。

DESCRIPTION (provided by applicant): The overall goal of this research is to develop a mechanistic understanding of Golgi membrane protein retention, the role of ion gradients in membrane traffic, and the assembly, transport and targeting of the VATPase in the simple eukaryote Saccharomyces cerevisiae. Yeast has proved to be an excellent model system, both for identifying the proteins regulating membrane traffic in eukaryotic cells and for investigating the molecular mechanisms by which these proteins function. Genetic analysis has revealed a large collection of yeast genes encoding not only subunits of the yeast vacuolar proton-translocating ATPase (V-ATPase), but also four (4) genes encoding proteins that are not subunits of the enzyme but instead are localized to the endoplasmic reticulum (ER) and required for assembly and transport of the V-ATPase. Using both genetic and biochemical approaches, the V-ATPase assembly factors will be characterized to determine which factors function in specific steps of the assembly process. These assembly factors will also be characterized for a role in the loading of the V-ATPase into ER-derived vesicles, and in escorting the V-ATPase to the Golgi complex. There are two different forms of the yeast V-ATPase; the Golgi and endosomal form of the complex assembles with the Stv1p isoform of the 100 kDa subunit, and the complex on the vacuole membrane assembles with the Vph1p isoform of the 100 kDa subunit. The Golgi/endosomal localization signals in the Stv1p isoform will be identified by mutational analysis, and the proteins that recognize and sort the Stv1-associated V-ATPase will be identified by genetics and characterized. pH-sensitive forms of the Green Fluorescent Protein (GFP) will be targeted to the various yeast intracellular organelles to determine the pH of the Golgi-endosomal network. Studies of membrane traffic in yeast have proven tremendously useful to a broader understanding of membrane transport in all eukaryotic cells because of the remarkable similarity in mechanisms and proteins that regulate these processes from yeast to humans. These basic studies in yeast are providing important insights into our understanding of many diseases in humans related to defects in organelle acidification.