Sorting and Transport of Yeast Membrane Proteins

描述（由申请人提供）：本研究的总体目标是发展对高尔基膜蛋白保留的机制理解，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。酵母已被证明是一个很好的模型系统，无论是鉴定真核细胞中调节膜运输的蛋白质，还是研究这些蛋白质功能的分子机制。遗传分析表明，大量酵母基因不仅编码酵母液泡质子易位atp酶（v - atp酶）的亚基，而且还编码4个不是该酶亚基的蛋白质，这些蛋白质位于内质网（ER），是v - atp酶组装和运输所必需的。使用遗传和生化方法，v - atp酶组装因子将被表征，以确定哪些因子在组装过程的特定步骤中起作用。这些组装因子还将在v - atp酶装载到er衍生囊泡中，并将v - atp酶护送到高尔基复合体中发挥作用。有两种不同形式的酵母v - atp酶；高尔基和内体形式的复合物与100 kDa亚基的Stv1p异构体组装，液泡膜上的复合物与100 kDa亚基的Vph1p异构体组装。Stv1p异构体中的高尔基/内体定位信号将通过突变分析鉴定，识别和分类stv1相关v - atp酶的蛋白质将通过遗传学鉴定和表征。pH敏感形式的绿色荧光蛋白（GFP）将针对各种酵母胞内细胞器来确定高尔基-内体网络的pH值。酵母中膜运输的研究已被证明对更广泛地理解所有真核细胞中的膜运输非常有用，因为从酵母到人类调节这些过程的机制和蛋白质非常相似。这些酵母的基础研究为我们理解许多与细胞器酸化缺陷有关的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.