Wilson A L, Maltese W A
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
J Biol Chem. 1993 Jul 15;268(20):14561-4.
Low molecular mass GTP-binding proteins encoded by the Rab gene family are posttranslationally modified by a specific geranylgeranyltransferase (GGTase II), which catalyzes the thioether linkage of geranylgeranyl isoprenoids to cysteines within one of the following carboxyl-terminal sequence motifs: GGCC, CXC, CCSN. Short peptides containing these sequences are poor substrates for isoprenylation in vitro, suggesting that structural domains remote from the carboxyl terminus are required for interactions between Rab proteins and GGTase II. To begin to define these domains, deletions and point mutations were created within the Rab1B gene, and the ability of the mutant translation products to undergo isoprenylation was evaluated in reticulocyte lysates. Deletion of amino acids 2-9 diminished but did not eliminate isoprenylation of Rab1B, suggesting that the extreme amino-terminal region is not absolutely required for interaction with GGTase II. Longer deletions in the amino-terminal region, which probably disrupt the overall conformation of Rab1B, completely prevented isoprenylation. Site-directed mutations predicted to lie in the amino-terminal variable region (Y5N), the beta 3 strand (Q60E), and Loop 7 (A110D) of the Rab1B structure did not reduce isoprenylation. However, two mutations (I41N, D44N) in the effector domain, which appears to mediate interactions with proteins that stimulate GTP hydrolysis or GDP dissociation, essentially abolished the ability of Rab1B to undergo isoprenylation. These findings imply that the effector domain plays a key role in the isoprenylation of Rab proteins, either by serving as a prenyltransferase binding site or by facilitating interactions with accessory proteins that allow Rab1B to assume a specific guanine nucleotide-dependent conformation that is recognized by GGTase II.
Rab基因家族编码的低分子量GTP结合蛋白在翻译后被一种特异性的香叶基香叶基转移酶（GGTase II）修饰，该酶催化香叶基香叶基类异戊二烯与以下羧基末端序列基序之一中的半胱氨酸形成硫醚键：GGCC、CXC、CCSN。含有这些序列的短肽在体外是异戊二烯化的不良底物，这表明Rab蛋白与GGTase II之间的相互作用需要远离羧基末端的结构域。为了开始确定这些结构域，在Rab1B基因内创建了缺失和点突变，并在网织红细胞裂解物中评估了突变翻译产物进行异戊二烯化的能力。删除氨基酸2 - 9会减少但不会消除Rab1B的异戊二烯化，这表明与GGTase II相互作用并非绝对需要极端的氨基末端区域。氨基末端区域更长的缺失可能会破坏Rab1B的整体构象，从而完全阻止异戊二烯化。预测位于Rab1B结构的氨基末端可变区（Y5N）、β3链（Q60E）和环7（A110D）的定点突变并没有降低异戊二烯化。然而，效应结构域中的两个突变（I41N、D44N），该结构域似乎介导与刺激GTP水解或GDP解离的蛋白质的相互作用，基本上消除了Rab1B进行异戊二烯化的能力。这些发现表明，效应结构域在Rab蛋白的异戊二烯化中起关键作用，要么作为异戊二烯基转移酶结合位点，要么通过促进与辅助蛋白的相互作用，使Rab1B呈现出被GGTase II识别的特定鸟嘌呤核苷酸依赖性构象。
