REGULATION OF GTP BINDING PROTEINS

ARF功能需要GTP结合的活性形式和GDP结合的非活性形式之间的调节交替。GTP结合由鸟嘌呤核苷酸交换蛋白(GEP)催化，GTP酶激活蛋白(GAP)失活。目前已认识到两种类型的GEP，一类是被BFA(一种干扰蛋白质分泌并导致高尔基池可逆性解体的药物)抑制的~200 kDa蛋白质家族，另一类是较小的~55 kDa GEP，它们对BFA具有抗性。所有的GEP都有大约200个氨基酸组成的所谓Sec7结构域，负责GEP的活性及其对BFA的敏感性。该小组早些时候从牛脑胞浆中纯化了两个BFA抑制的GEP(BIG1和BIG2)，它们在凝胶过滤中表现为~670 kDa的分子。为了确定它们是相同还是不同大小的蛋白质复合体的一部分，并确定它们在细胞内的定位，制备了特异性抗肽抗体并进行了亲和纯化。免疫荧光显微镜显示两者在整个细胞中呈点状分布，集中在核周区域，部分与高尔基体特异的p58蛋白共存。所有的观察结果，包括BIG1和BIG2特异性抗体从胞浆中连续免疫沉淀蛋白质的结果，都与这两种蛋白的大部分存在于胞浆中相同的大分子复合体中，并且在培养细胞中的定位相似的结论一致。对存在于复合体中或与细胞内GEP相互作用的其他蛋白质的鉴定正在进行中。

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. This group had earlier purified, from bovine brain cytosol, two BFA- inhibited GEPs (BIG1 and BIG2) that behaved on gel filtration as molecules of ~670 kDa. To determine whether they were parts of the same or different protein complexes of similar size and to define their intracellular localization, specific anti-peptide antibodies were prepared and affinity purified. Immunofluorescence microscopy revealed a punctate distribution of both throughout cells with concentration in the perinuclear region, partially colocalized with Golgi-specific p58 protein. All observations, including results of sequential immunoprecipitation of proteins from cytosol with BIG1- and BIG2- specific antibodies were consistent with the conclusion that significant fractions of both proteins exist in the same macromolecular complexes in cytosol and are similarly localized in cultured cells. Identification of other proteins present in the complexes or interacting with the GEPs in cells is ongoing.