Ali Rashid, Brett Christopher L, Mukherjee Sanchita, Rao Rajini
Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Biol Chem. 2004 Feb 6;279(6):4498-506. doi: 10.1074/jbc.M307446200. Epub 2003 Nov 10.
Endosomal Na+/H+ exchangers are important for salt and osmotolerance, vacuolar pH regulation, and endosomal trafficking. We show that the C terminus of yeast Nhx1 interacts with Gyp6, a GTPase-activating protein for the Ypt/Rab family of GTPases, and that Gyp6 colocalizes with Nhx1 in the endosomal/prevacuolar compartment (PVC). The gyp6 null mutant exhibits novel phenotypes consistent with loss of negative regulation of Nhx1, including increased tolerance to hygromycin, increased vacuolar pH, and decreased plasma membrane potential. In contrast, overexpression of Gyp6 increases sensitivity to hygromycin, decreases vacuolar pH, and results in a slight missorting of vacuolar carboxypeptidase Y to the cell surface. We conclude that Gyp6 is a negative regulator of Nhx1-dependent trafficking out of the PVC. Taken together with its GTPase-activating protein-dependent role as a negative regulator of Ypt6-mediated retrograde traffic to the Golgi, we propose that Gyp6 coordinates upstream and downstream events in the PVC to Golgi pathway. Our findings provide a possible molecular link between intraendosomal pH and regulation of vesicular trafficking.
内体Na+/H+交换蛋白对于盐耐受性、渗透压耐受性、液泡pH调节以及内体运输至关重要。我们发现酵母Nhx1的C末端与Gyp6相互作用,Gyp6是Ypt/Rab家族GTP酶的GTP酶激活蛋白,并且Gyp6与Nhx1在内体/前液泡区室(PVC)中共定位。gyp6缺失突变体表现出与Nhx1负调控丧失一致的新表型,包括对潮霉素的耐受性增加、液泡pH升高以及质膜电位降低。相反,Gyp6的过表达增加了对潮霉素的敏感性,降低了液泡pH,并导致液泡羧肽酶Y轻微错误分选至细胞表面。我们得出结论,Gyp6是Nhx1依赖的从PVC输出运输的负调控因子。结合其作为Ypt6介导的向高尔基体逆行运输的负调控因子的GTP酶激活蛋白依赖性作用来看,我们提出Gyp6协调PVC到高尔基体途径中的上游和下游事件。我们的发现为内体pH与囊泡运输调节之间提供了一种可能的分子联系。