Identification of EPI64 as a GTPase-activating protein specific for Rab27A
Identification of EPI64 as a GTPase-activating protein specific for Rab27A
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DOI：
10.1074/jbc.m603808200
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发表时间：
2006-10-20
影响因子：
4.8
通讯作者：
Fukuda, Mitsunori
中科院分区：
文献类型：
--
作者：
Itoh, Takashi;Fukuda, Mitsunori
小GTPase Rab27A在黑素细胞的黑素小体运输和各种分泌细胞的分泌中起关键作用。由于GTP或GTP锁定的Rab27A突变体引起黑素小体的核周聚集，因此Rab27A的适当的GTP- gdp循环对于黑素小体的运输是必不可少的，并且在黑素细胞中必须存在Rab27A的某些鸟嘌呤核苷酸交换因子和gtpase激活蛋白（gap）。然而，Rab27A的此类调节因子尚未被确定。在这项研究中，我们开发了快速筛选40种不同的TBC （Tre2/Bub2/Cdc16）蛋白（假定的Rab27A-GAP）的新方法：(i)寻找在黑素细胞中诱导黑素体聚集的TBC蛋白；（ii）在同时表达Rab27A和TBC蛋白的培养细胞中，用Rab27A效应域（即Slac2-a的SHD）捕获GTP-Rab27A；（iii）测定体外Rab27AGAP活性。这些方法使我们能够将EPI64鉴定为特异性的Rab27A-GAP，该蛋白先前被表征为含有孤儿TBC结构域的ebp50结合蛋白。我们进一步发现，EPI64催化区域的突变导致其诱导黑素小体聚集的能力完全丧失。这是基于功能相互作用筛选Rab27A-GAP的第一篇报道，我们的筛选方法可以应用于其他未表征的TBC蛋白。
Small GTPase Rab27A plays a pivotal role in melanosome transport in melanocytes and in secretion by various secreting cells. Because the GTP- or GDP-locked mutant of Rab27A causes perinuclear aggregation of melanosomes, appropriate GTP-GDP cycling of Rab27A is essential for melanosome transport, and certain guanine nucleotide exchange factors and GTPase-activating proteins (GAPs) of Rab27A must be present in melanocytes. However, no such regulators of Rab27A have ever been identified. In this study we developed novel methods of rapidly screening 40 different TBC (Tre2/Bub2/Cdc16) proteins, putative Rab-GAPs, for Rab27A-GAP by: (i) searching for TBC proteins that induce melanosome aggregation in melanocytes; (ii) trapping GTP-Rab27A with a Rab27A effector domain (i. e. the SHD of Slac2-a) in cultured cells that express both Rab27A and TBC proteins; and (iii) measuring in vitro Rab27AGAP activity. These methods allowed us to identify EPI64, previously characterized as an EBP50-binding protein that contains an orphan TBC domain, as a specific Rab27A-GAP. We further showed that mutations in the catalytic domain of EPI64 caused complete loss of its ability to induce melanosome aggregation. This is the first report of screening for Rab27A-GAP based on functional interactions, and our screening methods can be applied for other uncharacterized TBC proteins.