MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF GTP-BINDING PROTEIN

adp -核糖基化因子结构域蛋白1 （ARD1）最初是在实验室中克隆的，是adp -核糖基化因子（ARF）鸟嘌呤核苷酸结合蛋白家族的成员，与其他ARF不同的是，它存在一个46 kda的氨基末端延伸（p5），其ARF结构域（p3）的作用是gtpase激活蛋白（GAP）。与ARF的GAP类似，ARD1的GAP结构域含有锌指基序和精氨酸残基，这些残基对活性至关重要。它与其他ARF gap的不同之处在于它与gtp结合结构域的共价结合以及它对ARD1的ARF结构域的特异性。据推测，arf在细胞内运输囊泡的形成及其从一个隔室到另一个隔室的运动中起关键作用。过表达和内源性ARD1都与高尔基体和溶酶体膜有关，与溶酶体的形成或功能以及高尔基体和溶酶体之间的蛋白质运输的作用一致。该小组已经确定了单独合成的p3和p5中的许多突变对GTPase活性和蛋白质相互作用的影响，并在今年描述了p5和p3中的序列基序，它们分别作为细胞中溶酶体和高尔基定位的信号。在酵母双杂交筛选中发现了ARD1与细胞分裂素-1的相互作用。细胞素-2在这个系统中不能相互作用，对ARD1的GEP活性远低于细胞素-1，尽管它们对ARF1的活性相同。在体外也显示出优先的物理相互作用，并且鉴定了负责细胞分裂素-1/ARD1相互作用特异性的残基。

ADP-ribosylation factor domain protein 1 (ARD1) initially cloned in the laboratory, is a member of the ADP-ribosylation factor (ARF) family of guanine nucleotide-binding proteins that differs from other ARFs by the presence of a 46-kDa amino-terminal extension (p5), which acts as a GTPase-activating protein (GAP) for its ARF domain (p3). Similar to ARF GAPs, the GAP domain of ARD1 contains a zinc finger motif and arginine residues that are critical for activity. It differs from other ARF GAPs in its covalent association with the GTP-binding domain and its specificity for the ARF domain of ARD1. ARFs are presumed to play a key role in the formation of intracellular transport vesicles and in their movement from one compartment to another. Both overexpressed and endogenous ARD1 were associated with Golgi and lysosomal membranes, consistent with a role in the formation or function of lysosomes and in protein trafficking between Golgi and lysosomes. The group had defined effects of numerous mutations in separately synthesized p3 and p5 on GTPase activity and protein-protein interactions, and described this year sequence motifs in p5 and p3 that serve, respectively, as signals for lysosomal and Golgi localization in cells. Interaction of ARD1 and cytohesin-1 was found in a yeast two-hybrid screen. Cytohesin-2 failed to interact in this system and had much less GEP activity toward ARD1 than did cytohesin-1, although they were equally active with ARF1. Preferential physical interaction was also shown in vitro and residues responsible for specificity of the cytohesin-1/ARD1 interaction were identified.