Analysis of subcellular localization and function of the yeast Rab6 homologue, Ypt6p, using a novel amino-terminal tagging strategy.
Analysis of subcellular localization and function of the yeast Rab6 homologue, Ypt6p, using a novel amino-terminal tagging strategy.
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DOI：
10.1016/j.bbrc.2014.06.002
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发表时间：
2014-07
影响因子：
3.1
通讯作者：
Sonoko Kawamura;M. Nagano;J. Toshima;J. Toshima
中科院分区：
文献类型：
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作者：
Sonoko Kawamura;M. Nagano;J. Toshima;J. Toshima
Ypt6p是哺乳动物Rab6的酵母同源物，参与液泡成熟和膜蛋白循环等膜运输调控的多个过程。尽管一些证据表明Ypt6p可能定位于多个膜室，但内源性Ypt6p的精确定位仍有待阐明。本研究提出了一种基于同源重组的内源蛋白n端标记新方法，并对Ypt6p的亚细胞定位和功能进行了研究。Ypt6p及其gtp结合形式主要定位于细胞壁-内侧-高尔基区室，而gtp结合形式的Ypt6p则定位于细胞质。Ric1p是Ypt6p特异性GEF复合物的一个组成部分，在早期高尔基体中与Ypt6p大量共定位，并且Ypt6p的定位改变为胞浆inric1Δ细胞。另一方面，Ypt6p的推定GAP Gyp6p定位于反式高尔基区，而gyp6的缺失增加了Ypt6p在反式高尔基区的定位，这表明Gyp6p在到达反式高尔基区时促进了Ypt6p与高尔基体的分离。此外，我们证明了Ypt6p的gdp结合形式的过表达导致液泡形成缺陷和Snc1p再循环到质膜。这些结果表明，Ypt6p的gtp结合活性对于早期高尔基腔的高尔基内运输和蛋白质循环是必要的。
Ypt6p, the yeast homologue of mammalian Rab6, is involved in the multiple processes regulated by membrane trafficking such as vacuole maturation and membrane protein recycling. Although several lines of evidence suggest that Ypt6p is possibly localized to multiple membrane compartments, the precise localization of endogenous Ypt6p remains to be elucidated. In this study, we developed a novel method for N-terminal tagging of endogenous protein based on homologous recombination and investigated the subcellular localization and function of Ypt6p. Ypt6p and its GTP-bound form were predominantly localized to thecis- tomedial-Golgi compartments whereas the GDP-bound form of Ypt6p was localized to the cytosol. Ric1p, a component of the specific GEF complex for Ypt6p, largely colocalized with Ypt6p in the early Golgi, and localization of Ypt6p changed to the cytosol inric1Δ cells. On the other hand, Gyp6p, a putative GAP for Ypt6p, was localized to thetrans-Golgi compartment and deletion ofGYP6increased the localization of Ypt6p at thetrans-Golgi, suggesting that Gyp6p promotes the dissociation of Ypt6p from the Golgi when arriving at thetrans-Golgi compartment. Additionally, we demonstrated that overexpression of the GDP-bound form of Ypt6p caused defective vacuole formation and recycling of Snc1p to the plasma membrane. These results suggest that the GTP-binding activity of Ypt6p is necessary forintra-Golgi trafficking and protein recycling in the early Golgi compartment.