REGULATION OF GTP BINDING PROTEINS

ARF功能需要在gtp结合的活性形式和gdp结合的非活性形式之间进行调节。GTP的结合由鸟嘌呤核苷酸交换蛋白（GEPs）催化，GTP酶激活蛋白（gap）灭活GTP。目前已经发现了两种常见的GEPs，一种是被BFA（一种干扰蛋白质分泌并导致高尔基池可逆解体的药物）抑制的~200 kda的GEPs家族，另一种是BFA耐药的~55 kda的GEPs家族。所有GEP都有大约200个氨基酸组成的所谓Sec7结构域，这些氨基酸负责GEP的活性及其BFA敏感性。我们先前纯化了两个bfa抑制的GEPs，并克隆了一个cDNA。今年，完成了重组蛋白的详细表征。在Sec7结构域上去除序列c端加上n端596个残基对活性影响不大，但进一步删除36个n端氨基酸会使活性降低约200倍，达到Sec7结构域本身的水平。正在进行的工作将确定导致Sec7结构域催化活性显著增强的结构元件。第二种抑制BFA的GEPs，被称为BIG2，已被克隆、鉴定，并于今年报道。推导出的牛和人蛋白的氨基酸序列与先前鉴定的BIG1相同99.5%，约74%。通过比较它们的功能、表达和亚细胞分布，我们将获得这两种明显相似的bfa敏感gep的生物学作用的线索。-鸟嘌呤核苷酸交换蛋白Sec7，鸟嘌呤苷A

ARF function requires the regulated alternation between GTP-bound active and GDP-bound inactive forms. GTP binding is catalyzed by guanine nucleotide-exchange proteins (GEPs) and inactivation by GTPase- activating proteins (GAPs). Two general types of GEPs have been recognized, a family of ~200-kDa proteins that are inhibited by BFA (a drug that interferes with protein secretion and causes reversible disintegration of Golgi cisternae) and smaller ~55-kDa GEPs that are BFA-resistant. All GEPs have so-called Sec7 domains of ~200 amino acids that are responsible for the GEP activity, as well as its BFA sensitivity. We had earlier purified two BFA-inhibited GEPs and cloned one cDNA. This year, detailed characterization of the recombinant protein was completed. Removal of sequence C-terminal to the Sec7 domain plus up to 596 residues from the N-terminus had little effect on activity, but further deletion of 36 N-terminal amino acids decreased activity ~200-fold to the level of the Sec7 domain itself. Ongoing work will identify the structural elements responsible for the dramatic enhancement of Sec7 domain catalytic activity. The second of the BFA- inhibited GEPs, termed BIG2, was cloned, characterized, and reported this year. Deduced amino acid sequences of the bovine and human proteins are 99.5% identical and ~74% identical to that of the previously characterized BIG1. By comparison of their functions, expression and subcellular distributions, we should obtain clues to the biological roles of these two apparently very similar BFA-sensitive GEPs. - ARF, brefeldin A, guanine nucleotide-exchange protein, Sec7, arfaptin