Sorting and Transport of Yeast Membrane Proteins

描述（由申请人提供）：本项目将探索在简单模型真核生物酵母酿酒酵母中控制液泡型质子转运ATP酶（V-ATP酶）的组装、分选和运输的机制。V-ATP酶是一种分子机器，它将ATP的水解与质子转移到细胞器的内腔中耦合，并且V-ATP酶在真菌，植物和动物中高度保守。我们在酵母中的遗传分析已经鉴定了酵母中编码在内质网（ER）中在V-ATP酶的膜部分的组装中起作用的蛋白质的一组基因。这些V-ATP酶组装因子将通过遗传和生物化学方法表征，以表征V-ATP酶的组装途径。尽管多年来人们已经知道V-ATP酶是高度保守的，但直到最近才清楚我们在酵母中鉴定的V-ATP酶组装因子在人类中是保守的。 提出了三个具体目标。第一个目标集中在每个V-ATP酶组装因子如何在ER中V-ATP酶的6-亚基整合膜结构域的高度协调组装中起作用的重要问题上。这个目的也集中在调查的V-ATP酶的ER退出的机制，和确定为该蛋白质复合物的ER退出所需的蛋白质的功能。第二个目标集中在表征酵母中的人V-ATP酶组装因子。我们与Dirk Lefeber教授的合作为我们提供了四种人类V-ATP酶组装因子（hVma 12，hVma 21，hVma 22和Ac 45）的cDNA，以及有关导致人类疾病的这些基因突变等位基因的未发表信息。这些蛋白质将在更简单的酵母模型系统中表征V-ATP酶组装功能。最后，第三个目标解决了高尔基体-内体形式的酵母V-ATP酶的分选和保留，其包含亚基“a”的第二种同种型，Stv 1蛋白。我们将使用我们最近发现的Stv 1分选/保留信号的知识来研究和表征包含Stv 1的V-ATP酶复合物的分选和保留所涉及的蛋白质。从这些研究中获得的知识将为这种复杂蛋白质机器的组装，运输和分选提供新的见解，并将为越来越多的V-ATP酶相关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has provided us with the cDNAs of four human V-ATPase assembly factors (hVma12, hVma21, hVma22 and Ac45), as well as unpublished information on mutant alleles of these genes that cause human disease.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.