Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A.
Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A.
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Rabin3 的相互作用克隆，这是一种与 Ras 样 GTP 酶 Rab3A 相关的新型蛋白质。
DOI：
10.1128/mcb.15.3.1137
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发表时间：
1995
影响因子：
5.3
通讯作者：
Macara,IG
中科院分区：
文献类型：
--
作者：
Brondyk,WH;McKiernan,CJ;Fortner,KA;Stabila,P;Holz,RW;Macara,IG
Rab 3A是一种小的Ras样GTP酶，在神经内分泌细胞中表达，与分泌囊泡膜相关并调节胞吐作用。使用酵母双杂交系统，我们已经确定了一个大鼠脑cDNA编码一种新的50 kDa的蛋白质，我们命名为Rabin 3，与Rab 3A和Rab 3D相互作用，但不与其他小GTP酶（Rab 3C，Rab 2，Ran或Ras）。Rab 3A的效应域中的几个独立的点突变（F51 L、V55 E和G56 D）不改变GT3的核苷酸结合，从而消除了与Rabin 3的相互作用，而另一个突变（V52 A）似乎增加了相互作用。这些结果表明，相互作用是高度特异性的。然而，谷胱甘肽S-转移酶-Rabin 3融合蛋白在体外仅与重组Rab 3A弱相关，并且不具有可检测的GTP酶激活蛋白或核苷酸交换活性，并且Rabin 3在肾上腺嗜铬细胞中过表达对分泌没有可观察到的影响。该蛋白具有卷曲螺旋结构域的特征序列和与Saccharomycesgae蛋白Sec 2 p序列相似的第二个小区域，Sec 2 p是酵母细胞组成性分泌所必需的，并与Rab 3A GT3的近亲Sec 4p相互作用。Rabin 3 mRNA和蛋白广泛表达，但在睾丸中特别丰富。
Rab3A is a small, Ras-like GTPase expressed in neuroendocrine cells, in which it is associated with secretory vesicle membranes and regulates exocytosis. Using the yeast two-hybrid system, we have identified a rat brain cDNA encoding a novel 50-kDa protein, which we have named Rabin3, that interacts with Rab3A and Rab3D but not with other small GTPases (Rab3C, Rab2, Ran, or Ras). Several independent point mutations in the effector domain of Rab3A (F51L, V55E, and G56D) which do not alter nucleotide binding by the GTPase abolish the interaction with Rabin3, while another mutation (V52A) appears to increase the interaction. These results demonstrate that the interaction is highly specific. However, a glutathione S-transferase-Rabin3 fusion protein associates only weakly in vitro with recombinant Rab3A and possesses no detectable GTPase-activating protein or nucleotide exchange activity, and Rabin3 overexpressed in adrenal chromaffin cells has no observable effect on secretion. The protein possess a sequence characteristic of coiled-coil domains and a second small region with sequence similarity to aSaccharomyces cerevisiaeprotein, Sec2p, Sec2p is essential for constitutive secretion in yeast cells and interacts with Sec4p, a close relative of the Rab3A GTPase. Rabin3 mRNA and protein are widely expressed but are particularly abundant in testes.