MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF GTP-BINDING PROTEIN

ADP-核糖基化因子结构域蛋白1（ARD 1）最初在实验室中克隆，是鸟嘌呤核苷酸结合蛋白的ADP-核糖基化因子（ARF）家族的成员，其与其他ARF的不同之处在于存在46-kDa的氨基末端延伸（p5），其作为其ARF结构域（p3）的GTP酶激活蛋白（GAP）。 与ARF GAP类似，ARD 1差距域包含对活性至关重要的锌指基序和精氨酸残基。 它与其他ARF GAP的不同之处在于其与GTP结合结构域的共价结合及其对ARD 1的ARF结构域的特异性。 ARF被认为在细胞内运输囊泡的形成和它们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ARD 1均与高尔基体和溶酶体膜相关，这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中的作用一致。 该小组已经确定了单独合成的p3和p5中的许多突变对GTdR活性和蛋白质-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，分别作为细胞中溶酶体和高尔基体定位的信号。 在酵母双杂交筛选中发现了ARD 1和cytohesin-1的相互作用。细胞粘连蛋白-2未能在该系统中相互作用，并且对ARD 1的GEP活性比细胞粘连蛋白-1低得多，尽管它们与ARF 1的活性相同。 在体外也显示出优先的物理相互作用，并鉴定了负责cytohesin-1/ARD 1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.