Sorting and Transport of Yeast Membrane Proteins

描述(申请人提供)：该项目将探索控制液泡型质子转运ATPase(V-ATPase)在简单模式真核生物-酿酒酵母中的组装、分选和运输的机制。V-ATPase是一种分子机器，它将ATP的水解和质子的转运结合到细胞器的管腔中，在真菌、植物和动物中高度保守。我们在酵母中的遗传分析发现，酵母中有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表征，以表征V-ATPase的组装途径。虽然多年来人们已经知道V-ATPase是高度保守的，但直到最近才清楚的是，我们在酵母中鉴定的V-ATPase组装因子在人类中是保守的。提出了三个具体目标。第一个目的集中在重要的问题上，即每个V-ATPase组装因子在内质网中V-ATPase的6亚单位完整膜域的高度协调组装中如何发挥作用。这一目的还集中在研究V-ATPase的ER退出机制，以及该蛋白复合体的ER退出所需的蛋白质的功能。第二个目标是在酵母中鉴定人的V-ATPase组装因子。我们与Dirk Lefeber教授的合作为我们提供了四种人类V-ATPase组装因子(hVma12、hVma21、hVma22和AC45)的cDNA，以及这些导致人类疾病的基因突变等位基因的未发表信息。这些蛋白质将在简单得多的酵母模型系统中表征V-ATPase的组装功能。最后，第三个目标涉及酵母V-ATPase高尔基内体形式的分选和保留，它包含亚基“a”的第二个亚型，Stv1蛋白。我们将利用最近发现的Stv1分选/保留信号的知识来研究和表征参与Stv1-V-ATPase复合体的分选和保留的蛋白质。从这些研究中获得的知识将为这个复杂的蛋白质机器的组装、运输和分类提供新的见解，并将为越来越多的与V-ATPase相关的人类疾病的机制基础提供见解。

DESCRIPTION (provided by applicant): This project will explore the mechanisms that govern the assembly, sorting and transport of the vacuolar- type proton-translocating ATPase (V-ATPase) in the simple model eukaryote, the yeast Saccharomyces cerevisiae. The V-ATPase is a molecular machine that couples the hydrolysis of ATP with the translocation of protons into the lumen of organelles, and the V-ATPase is highly conserved across fungi, plants and animals. Our genetic analysis in yeast has identified a group of genes in yeast encoding proteins that function 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 characterize the assembly pathway for the V-ATPase. Whereas it has been known for years that the V-ATPase is highly conserved, it has only very recently become clear that the V-ATPase assembly factors that we have identified in yeast are conserved in humans. Three specific aims are proposed. The first aim focuses on the important question of how each of the V- ATPase assembly factors functions in the highly orchestrated assembly of the 6-subunit integral membrane domain of the V-ATPase in the ER. This aim is also focused on investigating the mechanism of ER exit of the V-ATPase, and the function of the proteins identified as required for ER exit of this protein complex. The second aim centers on characterizing the human V-ATPase assembly factors in yeast. Our collaboration with Professor Dirk Lefeber has provided us with the cDNAs of four human V-ATPase assembly factors (hVma12, hVma21, hVma22 and Ac45), as well as unpublished information on mutant alleles of these genes that cause human disease. These proteins will be characterized for V-ATPase assembly function in the much simpler yeast model system. Finally, the third aim addresses the sorting and retention of the Golgi-endosomal form of the yeast V-ATPase that contains the second isoform of subunit "a", the Stv1 protein. We will use the knowledge of the Stv1 sorting/retention signal that we have recently identified to investigate and characterize the proteins involved in the sorting and retention of the Stv1-containing V-ATPase complex. The knowledge gained from these studies will provide new insights into the assembly, transport and sorting of this complex protein machine, and will provide insights into the mechanistic basis for the growing number of V-ATPase associated human diseases.