Liu Ya-Wen, Huang Chun-Fang, Huang Kai-Bin, Lee Fang-Jen S
Institute of Molecular Medicine, College of Medicine, and Department of Medical Research, National Taiwan University Hospital, National Taiwan University, Taipei 100, Taiwan.
Mol Biol Cell. 2005 Sep;16(9):4024-33. doi: 10.1091/mbc.e05-01-0023. Epub 2005 Jun 22.
ADP-ribosylation factor (ARF) and ARF-like (ARL) proteins are members of the ARF family, which are critical components of several different vesicular trafficking pathways. ARFs have little or no detectable GTPase activity without the assistance of a GTPase-activating protein (GAP). Here, we demonstrate that yeast Gcs1p exhibits GAP activity toward Arl1p and Arf1p in vitro, and Arl1p can interact with Gcs1p in a GTP-dependent manner. Arl1p was observed both on trans-Golgi and in cytosol and was recruited from cytosol to membranes in a GTP-dependent manner. In gcs1 mutant cells, the fraction of Arl1p in cytosol relative to trans-Golgi was less than it was in wild-type cells. Increasing Gcs1p levels returned the distribution toward that of wild-type cells. Both Arl1p and Gcs1p influenced the distribution of Imh1p, an Arl1p effector. Our data are consistent with the conclusion that Arl1p moves in a dynamic equilibrium between trans-Golgi and cytosol, and the release of Arl1p from membranes in cells requires the hydrolysis of bound GTP, which is accelerated by Gcs1p.
ADP核糖基化因子（ARF）和ARF样（ARL）蛋白是ARF家族的成员，它们是几种不同囊泡运输途径的关键组成部分。在没有GTP酶激活蛋白（GAP）的协助下，ARF几乎没有或没有可检测到的GTP酶活性。在此，我们证明酵母Gcs1p在体外对Arl1p和Arf1p表现出GAP活性，并且Arl1p可以以GTP依赖的方式与Gcs1p相互作用。在反式高尔基体和细胞质中均观察到Arl1p，并且它以GTP依赖的方式从细胞质募集到膜上。在gcs1突变细胞中，相对于反式高尔基体，细胞质中Arl1p的比例低于野生型细胞。增加Gcs1p水平可使分布恢复到野生型细胞的分布。Arl1p和Gcs1p均影响Arl1p效应器Imh1p的分布。我们的数据与以下结论一致：Arl1p在反式高尔基体和细胞质之间以动态平衡移动，并且细胞中Arl1p从膜上的释放需要结合的GTP水解，这由Gcs1p加速。
