Sorting and Transport of Yeast Membrane Proteins

描述（由申请人提供）：本研究的总体目标是对高尔基体膜蛋白保留、离子梯度在膜运输中的作用以及简单真核生物酿酒酵母中VATP酶的组装、运输和靶向的机制进行理解。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的蛋白质调节膜交通和调查的分子机制，这些蛋白质的功能。遗传分析已经揭示了大量酵母基因的集合，这些基因不仅编码酵母液泡质子转运ATP酶（V-ATP酶）的亚基，而且编码四（4）种蛋白质的基因，这些蛋白质不是酶的亚基，而是定位于内质网（ER）并且是V-ATP酶的组装和运输所需的。使用遗传和生物化学方法，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。这些组装因子也将被表征为在将V-ATP酶装载到ER衍生的囊泡中以及在护送V-ATP酶到高尔基体复合体中的作用。有两种不同形式的酵母V-ATP酶;高尔基体和内体形式的复合物与100 kDa亚基的Stv 1 p同种型组装，而液泡膜上的复合物与100 kDa亚基的Vph 1 p同种型组装。Stv 1 p同种型中的高尔基体/内体定位信号将通过突变分析进行鉴定，识别和分选Stv 1相关V-ATP酶的蛋白质将通过遗传学进行鉴定和表征。pH敏感形式的绿色荧光蛋白（GFP）将靶向各种酵母细胞内细胞器，以确定高尔基体-内体网络的pH。酵母细胞膜运输的研究已被证明是非常有用的膜运输在所有真核细胞的更广泛的理解，因为在机制和蛋白质，调节这些过程从酵母到人类的显着相似性。酵母的这些基础研究为我们理解与细胞器酸化缺陷有关的人类许多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.