Yeast Rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p
Yeast Rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p
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DOI：
10.1091/mbc.12.5.1215
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发表时间：
2001-05-01
影响因子：
3.3
通讯作者：
Novick, P
中科院分区：
文献类型：
--
作者：
Du, LL;Novick, P
酿酒酵母中的一个相关蛋白家族在体外对Rab GTP酶具有GTP酶激活蛋白活性。然而，它们在体内的功能仍然不清楚。其中Gyp1p在体外作用于Sec4p、Ypt1p、Ypt7p和Ypt51p。在这里，我们提供的数据揭示了它在体内的底物和它在Rab GTP酶功能中所起的作用。红色荧光蛋白标记的Gyp1p集中在细胞质的点状结构上，这些结构主要与顺式高尔基体标记共存。酵母裂解物的亚细胞分离证实Gyp1p与膜外围相关，并与高尔基标记协同分离。这种定位表明Gyp1p可能只作用于高尔基体上的Rab GTP酶。一株Gyp1 Delta菌株在37℃的合成培养基上表现出生长缺陷。过表达Ypt1p，而不是其他Rab GTP酶，强烈抑制Gyp1 Delta细胞的生长。相反，YPT1的部分功能缺失等位基因ypt1-2可以抑制Gyp1 Delta细胞的生长缺陷。此外，GYP1的缺失可以部分抑制与运输蛋白颗粒复合体亚单位中突变相关的生长缺陷，运输蛋白颗粒复合体是一种催化Ypt1p上核苷酸交换的复合体。这些结果表明，Gyp1p在高尔基体上作为Ypt1p的负调控因子发挥作用。
A family of related proteins in yeast Saccharomyces cerevisiae is known to have in vitro GTPase-activating protein activity on the Rab GTPases. However, their in vivo function remains obscure. One of them, Gyp1p, acts on Sec4p, Ypt1p, Ypt7p, and Ypt51p in vitro. Here, we present data to reveal its in vivo substrate and the role that it plays in the function of the Rab GTPase. Red fluorescent protein-tagged Gyp1p is concentrated on cytoplasmic punctate structures that largely colocalize with a cis-Golgi marker. Subcellular fractionation of a yeast lysate confirmed that Gyp1p is peripherally associated with membranes and that it cofractionates with Golgi markers. This localization suggests that Gyp1p may only act on Rab GTPases on the Golgi. A gyp1 Delta strain displays a growth defect on synthetic medium at 37 degreesC. Overexpression of Ypt1p, but not other Rab GTPases, strongly inhibits the growth of gyp1 Delta cells. Conversely, a partial loss-of-function allele of YPT1, ypt1-2, can suppress the growth defect of gyp1 Delta cells. Furthermore, deletion of GYP1 can partially suppress growth defects associated with mutants in subunits of transport protein particle complex, a complex that catalyzes nucleotide exchange on Ypt1p. These results establish that Gyp1p functions on the Golgi as a negative regulator of Ypt1p.