MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF GTP-BINDING PROTEIN

ADP-核糖化因子(ARF)是一个约20 kDa的鸟嘌呤家族 核苷酸结合蛋白最初鉴定和纯化基于 它们增强霍乱ADP-核糖基转移酶活性的能力 毒素。最近，ARF已被确定为关键参与者 磷脂酶D的囊泡运输途径和激活剂。 含18 kDa羧基末端ARF结构域和46 kDa结构域的蛋白 该基因的氨基末端为ARD1，最初是通过克隆获得的。vbl.使用 重组融合蛋白，我们已经证明了氨基末端结构域 (P5)通过刺激其结构域(P3)的活性来控制其活性。 固有的GTPase活性。我们现在已经确认ARD1是一个成员 鸟嘌呤核苷酸结合蛋白家族并定义了 其异常的GTPase活性的特征。我们发现，无论是哪种情况 ARd1或其ARF结构域(P3)，心磷脂促进GTP-GammaS结合 与其他磷脂相比，CTA的激活更有效 测试过。利用嵌合蛋白和定点突变，我们展示了 ARF1，它不是GTP酶激活结构域的底物 ARD1(P5)并且没有与其进行身体互动，两者都获得了 Gly 40和Thr 45后的特性分别替换为， ASP和Pro.猪瘟病毒ARF区(P3)的氨基酸Asp 25和Pro 30 因此，ARD1被确定为功能性和 与ARD1的GTP酶激活结构域的物理相互作用。终于 我们证明了P5的羧基末端部分稳定了ARF- ARD1的结构域处于GDP绑定状态，即充当GDP解离 抑制剂。这些结果与ARD1是一种 它是单体G蛋白家族中唯一的成员，就像α- 异源三聚体G蛋白亚基具有自身的GTP水解性。 正在激活域。

ADP-ribosylation factors (ARFs) are a family of about 20-kDa guanine nucleotide-binding proteins originally identified and purified based on their ability to enhance the ADP-ribosyltransferase activity of cholera toxin. More recently, ARFs have been identified as critical participants in vesicular trafficking pathways and activators of a phospholipase D. A 64-kDa protein with an 18-kDa carboxy-terminal ARF domain and a 46-kDa amino-terminal ARD1, was initially identified by cDNA cloning. Using recombinant fusion proteins, we have shown that the amino-terminal domain of ARD1 (p5) controls the activity of the domain (p3) by stimulating its intrinsic GTPase activity. We have now confirmed that ARD1 is a member of the guanine nucleotide-binding protein family and defined some characteristics of its unusual GTPase activity. We found that for either ARD1 or its ARF domain (p3), cardiolipin promoted both GTPgammaS binding and CTA activation more effectively than did the other phospholipids tested. Using chimeric proteins and site-directed mutagenesis, we showed that ARF1, which is not substrate for the GTPase-activating domain of ARD1 (p5) and does not physically interact with it, acquired both characteristics after Gly 40 and Thr 45 were replaced with, respectively, Asp and Pro. The amino acids Asp 25 and Pro 30 of the ARF-domain (p3) of ARD1 were, therefore identified as participants in functional and physical interaction with the GTPase-activating domain of ARD1. Finally we demonstrated that the carboxy-terminal part of p5 stabilized the ARF- domain of ARD1 in a GDP-bound state, i.e., acted as a GDP-dissociation inhibitor. These results are consistent with the view that ARD1 is a unique member of the monomeric G protein family as it, like the alpha- subunits of hetero trimeric G proteins possesses its own GTP hydrolysis- activating domain.