MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF GTP-BINDING PROTEIN

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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.