Characterization of a GDP dissociation inhibitory region of ADP-ribosylation factor domain protein ARD1.
ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity and later recognized as critical components in intracellular vesicular transport and phospholipase D activation. ARF domain protein 1 (ARD1) is a member of the ARF family that differs from other ARFs by the presence of a 46-kDa amino-terminal extension. We previously reported that this extension acts as a GTPase-activating protein for the ARF domain of ARD1 (Vitale, N., Moss, J., and Vaughan, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1941-1944). Both GTP binding and GTP hydrolysis are necessary for physiological function of guanine nucleotide-binding proteins, and the rates of GDP/GTP exchange and GTPase activity are critical in the activation/deactivation cycle. Dissociation of GDP from the ARF domain of ARD1 was faster than from ARD1 itself (both proteins synthesized in Escherichia coli). Using deletion mutations, it was demonstrated that the 15 amino acids directly preceding the ARF domain were responsible for decreasing the rate of GDP dissociation but not guanosine 5-[gamma-thio]triphosphate dissociation. By site-specific mutagenesis it was shown that hydrophobic amino acids in this region were particularly important in stabilizing the GDP-bound form of ARD1. It is suggested that, like the amino-terminal segment of ARF, the equivalent region in ARD1, located between the GTPase-activating protein and ARF domains, may act as a GDP dissociation inhibitor.
Vitale
N
N
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA. vitalen@fido.nhlbi.nih.gov
Moss
J
J
Vaughan
M
M
D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013873:Thionucleotides / Q000378:metabolism; D013917:Thrombin
D017931:DNA Primers; D011994:Recombinant Proteins; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D013917:Thrombin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors