Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148.
Genetics. 2013 Sep;195(1):115-25. doi: 10.1534/genetics.113.152538. Epub 2013 Jun 21.
Constitutive transport of cellular materials is essential for cell survival. Although multiple small GTPase Rab proteins are required for the process, few regulators of Rabs are known. Here we report that EAT-17, a novel GTPase-activating protein (GAP), regulates RAB-6.2 function in grinder formation in Caenorhabditis elegans. We identified EAT-17 as a novel RabGAP that interacts with RAB-6.2, a protein that presumably regulates vesicle trafficking between Golgi, the endoplasmic reticulum, and plasma membrane to form a functional grinder. EAT-17 has a canonical GAP domain that is critical for its function. RNA interference against 25 confirmed and/or predicted RABs in C. elegans shows that RNAi against rab-6.2 produces a phenotype identical to eat-17. A directed yeast two-hybrid screen using EAT-17 as bait and each of the 25 RAB proteins as prey identifies RAB-6.2 as the interacting partner of EAT-17, confirming that RAB-6.2 is a specific substrate of EAT-17. Additionally, deletion mutants of rab-6.2 show grinder defects identical to those of eat-17 loss-of-function mutants, and both RAB-6.2 and EAT-17 are expressed in the terminal bulb of the pharynx where the grinder is located. Collectively, these results suggest that EAT-17 is a specific GTPase-activating protein for RAB-6.2. Based on the conserved function of Rab6 in vesicular transport, we propose that EAT-17 regulates the turnover rate of RAB-6.2 activity in cargo trafficking for grinder formation.
细胞物质的组成型运输对于细胞存活至关重要。虽然该过程需要多种小 GTPase Rab 蛋白，但已知的 Rab 蛋白调节剂却很少。在这里，我们报告了一种新型 GTP 酶激活蛋白 (GAP) EAT-17，它调节 Caenorhabditis elegans 中研磨器形成过程中的 RAB-6.2 功能。我们鉴定出 EAT-17 是一种与 RAB-6.2 相互作用的新型 RabGAP，RAB-6.2 蛋白可能调节高尔基体、内质网和质膜之间的囊泡运输，以形成功能正常的研磨器。EAT-17 具有一个关键的保守 GAP 结构域，对于其功能至关重要。针对 C. elegans 中 25 个已确认和/或预测的 Rab 进行 RNA 干扰，结果显示 RNAi 针对 rab-6.2 会产生与 eat-17 相同的表型。以 EAT-17 作为诱饵，用 25 种 Rab 蛋白中的每一种作为猎物进行定向酵母双杂交筛选，鉴定出 RAB-6.2 是 EAT-17 的相互作用伙伴，证实 RAB-6.2 是 EAT-17 的特定底物。此外，rab-6.2 的缺失突变体显示出与 eat-17 功能丧失突变体相同的研磨器缺陷，并且 RAB-6.2 和 EAT-17 都在研磨器所在的咽末端球中表达。综上所述，这些结果表明 EAT-17 是 RAB-6.2 的一种特异性 GTP 酶激活蛋白。基于 Rab6 在囊泡运输中的保守功能，我们提出 EAT-17 调节货物运输中 RAB-6.2 活性的周转率，以促进研磨器的形成。
