Sorting and Transport of Yeast Membrane Proteins

描述（由申请人提供）：本研究的总体目标是在简单的真核生物酵母酵母（Saccharomyces cerevisiae）中对液泡型质子易位atp酶（v - atp酶）的组装、分选和运输进行分子机制的理解。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。酵母的遗传分析已经确定了一组编码蛋白质的基因，这些蛋白质在内质网（ER）内的v - atp酶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化方法进行表征，以研究组装因子与内质网中v - atp酶膜部门亚基的相互作用。我们还发现了可能的内质网定位的货物受体，用于将v - atp酶装载到从内质网出芽的COPII囊泡中。货物受体将被研究与V- atp酶亚基和组装因子的直接相互作用，以及它们在这一过程中的确切作用。了解一个复杂的、多亚基的整体膜蛋白的组装及其装载到离开内质网的囊泡中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。我们已经确定了Stv1p n端结构域的突变，导致stv1相关的v - atp酶错误定位到液泡。我们将对这些突变进行表征，以确定它们是否影响高尔基复合体的保留或从核内体中恢复Stv1p。我们还确定了一大批参与高尔基/内体网络中Stv1相关v - atp酶的分选和保留的基因，我们将对它们编码的蛋白质进行表征，以评估它们是否与Stv1p分选/保留信号结合，或者通常影响更大组高尔基膜蛋白的保留/恢复。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和细胞器生物发生非常有用，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。这些关于酵母细胞膜运输和细胞器酸化的基础研究为我们理解许多与细胞器酸化缺陷和蛋白质错位有关的人类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液泡型atp酶（v - atp酶）在简单模型真核生物酵母酿酒酵母细胞器酸化中的作用。我们将研究由14种不同的蛋白质“亚基”组成的v - atp酶如何在酵母细胞的一个室中组装，然后以极高的保真度运输到不同的细胞室。酵母的膜运输研究已被证明对更广泛地理解所有真核细胞的细胞器酸化非常有用，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。了解v - atp酶的功能将为肾脏疾病（肾小管酸中毒）、骨病（骨质疏松）和肿瘤转移提供重要的见解，我们的研究将产生新的药物靶点。

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.