MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF GTP-BINDING PROTEIN

ADP-核糖基化因子（ARF）是约20 kDa的鸟嘌呤 核苷酸结合蛋白被认为是 胞内囊泡转运和磷脂酶D活化。两 鸟嘌呤核苷酸交换蛋白（GEPs）和GTP酶激活 最近已经克隆了ARF的GAP蛋白。一个锌指 基序，存在于ARF 1 GAP的氨基末端附近，是 刺激GTP水解。ARD 1是ARF家族的一员， 与其他ARF的不同之处在于存在一个46 kDa的氨基末端 延伸，其作为ARD 1的ARF结构域的GAP，但不作为ARD 1的ARF结构域的GAP。 ARF蛋白。在E.大肠杆菌，刺激的 与ARD 1的ARF结构域结合的GTP水解。使用ARD 1 截短，看来氨基酸101至190对于GAP是关键的。 活性，而残基190至304参与物理活性， ARD 1的两个结构域之间的相互作用，是GTP所必需的 水解 ARD 1氨基端延伸差距功能 需要两个碱基，一个完整的锌指基序和一组 类似于Rho/Rac GAP中存在的序列的残基。相互作用 在ARD 1的两个结构域之间需要两个带负电荷的残基 （Asp 427和Glu 428）位于ARF结构域的效应区 和两个碱性氨基酸（Arg 249和Lys 250）位于 氨基末端延伸因此，ARD 1差距域类似于ARF GAP，但与其他GAP的不同之处在于其与 GTP结合域。使用ARD 1截断，我们还确定了一个 结构域的15个残基直接前面的ARF结构域，具有 GDI样作用（即，它抑制了GDP与ARF的分离 域）。通过定点突变，我们证明了疏水性 氨基酸对于这种GDI样活性是关键的。这些结果 进一步证明了ARD 1是一个独特的单体成员。 G蛋白家族

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins recognized as critical components in intracellular vesicular transport and phospholipase D activation. Both guanine nucleotide-exchange proteins (GEPs) and GTPase-activating proteins (GAPs) for ARFs have been recently cloned. A zinc- finger motif, present near the amino terminus of the ARF1 GAP, was required for stimulation of GTP hydrolysis. ARD1, is a member of the ARF family that differs from other ARFs by the presence of a 46-kDa amino-terminal extension, which acts as a GAP for the ARF domain of ARD1, but not for ARF proteins. The GAP domain of ARD1, synthesized in E. coli, stimulated hydrolysis of GTP bound to the ARF domain of ARD1. Using ARD1 truncations, it appears that amino acids 101 to 190 are critical for GAP activity, whereas residues 190 to 304 are involved in physical interaction between the two domains of ARD1 and are required for GTP hydrolysis. The GAP function of the amino-terminal extension of ARD1 required two arginines, an intact zinc-finger motif and a group of residues that resembles a sequence present in Rho/Rac GAPs. Interaction between the two domains of ARD1 required two negatively charged residues (Asp427 and Glu428) situated in the effector region of the ARF domain and two basic amino acids (Arg249 and Lys250) located in the amino-terminal extension. The GAP domain of ARD1 thus is similar to ARF GAPs, but differs from the other GAPs by its covalent association with the GTP-binding domain. Using ARD1 truncations, we also identified a domain of 15 residues directly preceding the ARF domain that has a GDI-like action (i.e., it inhibits GDP dissociation from the ARF domain). By site-specific mutagenesis we demonstrated that hydrophobic amino acids are critical for this GDI-like activity. These results provide further evidence that ARD1 is a unique member of the monomeric G protein family.