Sorting and Transport of Yeast Membrane Proteins

描述(申请人提供)：本研究的总体目标是发展对液泡型质子转运ATPase(V-ATPase)在简单模式真核生物-酿酒酵母中的组装、分类和运输的分子机制的理解。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。在酵母中的遗传分析已经确定了一组编码蛋白的基因，这些蛋白在内质网(ER)中发挥作用，组装V-ATPase的膜部分。这些V-ATPase组装因子将通过遗传和生化方法进行鉴定，以研究组装因子与内质网中V-ATPase膜扇区亚基的相互作用。我们还鉴定了可能的内质网定位的货物受体，用于将V-ATPase装载到从内质网萌发的COPII囊泡中。将研究货物受体与V-ATPase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、多亚单位完整的膜蛋白的组装及其在退出内质网的囊泡中的负载是细胞生物学中的一个基本问题。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。我们已经鉴定了Stv1p N-末端结构域的突变，这些突变导致Stv1相关的V-ATPase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体复合体中的保留或从内体收回Stv1p。我们还鉴定了一大组与高尔基体/内体网络中Stv1相关的V-ATPase的分选和保留有关的基因，并将对它们的编码蛋白进行分析，以评估它们是否与Stv1p分选/保留信号结合，或者通常影响更大一组高尔基体膜蛋白的保留/检索。酵母膜运输的研究已被证明对于更广泛地理解所有真核细胞的膜运输和细胞器生物发生是非常有用的，因为调节从酵母到人类的这些过程的机制和蛋白质具有显着的相似性。这些关于酵母膜运输和细胞器酸化的基础研究为我们理解与细胞器酸化缺陷和蛋白质错误定位有关的许多人类疾病提供了重要的见解。公共卫生相关性：这项研究的总体目标是了解液泡型ATPase(V-ATPase)在简单模式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个不同蛋白质“亚基”组成的V-ATPase是如何在酵母细胞的一个隔室中组装，然后以极高的保真度运输到不同的细胞隔室的。对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中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具有惊人的相似性。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。了解V-ATPase的功能将为肾脏疾病(肾小管性酸中毒)、骨骼疾病(骨化症)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.