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简单模型真核生物（酵母酿酒酵母）中液泡型质子转运ATP酶（V-ATP酶）的组装、分选和转运的分子机制进行理解。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。酵母中的遗传分析已经鉴定了一组编码蛋白质的基因，所述蛋白质在内质网（ER）中在V-ATP酶的膜部分的组装中起作用。这些V-ATP酶组装因子将通过遗传和生物化学方法表征，以研究组装因子与ER中V-ATP酶膜扇区亚基的相互作用。我们还确定了可能的ER定位的货物受体装载到COPII囊泡出芽的V-ATP酶从ER。将研究货物受体与V-ATP酶亚基和组装因子的直接相互作用，以及它们在该过程中的确切作用。了解一个复杂的，多亚基整合膜蛋白的组装和它的加载到囊泡退出ER是细胞生物学的一个基本问题。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。我们已经确定了突变的Stv 1 p N-末端结构域，导致错误定位的Stv 1相关的V-ATP酶的液泡。我们将描述这些突变，以确定它们是否影响保留在高尔基复合体或检索Stv 1 p从内体回来。我们还确定了一个大组的基因参与的分选和保留的Stv 1相关的V-ATP酶在高尔基体/核内体网络，我们将其编码的蛋白质的特征，以评估他们是否结合到Stv 1 p分选/保留信号或一般影响保留/检索的一个更大的群体的高尔基体膜蛋白。酵母细胞膜运输的研究已被证明是非常有用的膜运输和细胞器的生物合成在所有真核细胞中的更广泛的理解，因为显着的相似性机制和蛋白质调节这些过程从酵母到人类。酵母细胞膜运输和细胞器酸化的这些基础研究为我们理解与细胞器酸化和蛋白质错误定位缺陷相关的人类许多疾病提供了重要的见解。公共卫生相关性：本研究的总体目标是了解在简单的真核生物模型中，酿酒酵母中的细胞器的酸化的液泡型ATP酶（V-ATP酶）的作用。我们将研究V-ATP酶，这是由14个不同的蛋白质“亚基”，是如何组装在一个区室的酵母细胞，然后以极大的保真度运输到不同的细胞区室。酵母细胞膜运输的研究已被证明是非常有用的细胞器酸化在所有真核细胞中的更广泛的理解，因为在机制和蛋白质，调节这些过程从酵母到人类显着的相似性。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。了解V-ATP酶的功能将为肾脏疾病（肾小管酸中毒），骨骼疾病（骨硬化症）和肿瘤转移提供重要的见解，我们的研究应该产生新的药物靶点。

DESCRIPTION (provided by applicant): The overall goal of this research is to develop a molecular mechanistic understanding of the assembly, sorting and transport of the vacuolar-type proton-translocating ATPase (V-ATPase) in the simple model eukaryote, the yeast 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 in yeast has identified a group of genes encoding proteins that function in the in the endoplasmic reticulum (ER) in assembly of the membrane sector of the V-ATPase. These V-ATPase assembly factors will be characterized by genetic and biochemical approaches, to investigate the interactions of the assembly factors with the V-ATPase membrane sector subunits in the ER. We have also identified possible ER-localized cargo receptors for loading the V-ATPase into COPII vesicles budding from the ER. The cargo receptors will be investigated for direct interactions with the V- ATPase subunits and assembly factors, and for their precise role in the process. Understanding the assembly of a complex, multisubunit integral membrane protein and its loading into vesicles exiting the ER is a fundamental issue in cell biology. 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. We have identified mutations in the Stv1p N-terminal domain that lead to mislocalization of the Stv1-associated V-ATPase to the vacuole. We will characterize these mutations to determine whether they affect retention in the Golgi complex or retrieval of Stv1p back from the endosome. We have also identified a large group of genes involved in the sorting and retention of the Stv1- associated V-ATPase in the Golgi/endosome network, and we will characterize their encoded proteins to assess whether they bind to the Stv1p sorting/retention signals or generally affect the retention/retrieval of a larger group of Golgi membrane proteins. Studies of membrane traffic in yeast have proven tremendously useful to a broader understanding of membrane transport and organelle biogenesis 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 membrane trafficking and organelle acidification in yeast are providing important insights into our understanding of many diseases i humans related to defects in organelle acidification and protein mislocalization. PUBLIC HEALTH RELEVANCE: The overall goal of this research is to understand the role of the vacuolar-type ATPase (V-ATPase) in the acidification of cellular organelles in the simple model eukaryote, the yeast Saccharomyces cerevisiae. We will investigate how the V-ATPase, which is composed of 14 different protein "subunits", is assembled in one compartment of the yeast cell and then transported with great fidelity to different cellular compartments. Studies of membrane traffic in yeast have proven tremendously useful to a broader understanding of organelle acidification 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. Understanding V-ATPase function will provide important insights into diseases of the kidney (renal tubular acidosis), bone diseases (osteopetrosis), and in tumor metastasis, and new drug targets should arise from our studies.