ARD1, a 64-kDa bifunctional protein containing an 18-kDa GTP-binding ADP-ribosylation factor domain and a 46-kDa GTPase-activating domain.
The alpha subunits of the heterotrimeric guanine nucleotide-binding proteins (G proteins) hydrolyze GTP at a rate significantly higher than do most members of the Ras family of approximatelly 20-kDa GTP-binding proteins, which depend on a GTPase-activating protein (GAP) for acceleration of GTP hydrolysis. It has been demonstrated that an inserted domain in the G-protein alpha subunit, not present in the much smaller Ras-like proteins, is responsible for this difference [Markby, D. W., Onrust, R. & Bourne, H. R. (1993) Science 262, 1895-1900]. We report here that ARD1, a 64-kDa protein with an 18-kDa carboxyl-terminal ADP-ribosylation factor (ARF) domain, exhibited significant GTPase activity, whereas the ARF domain, expressed as a recombinant protein in Escherichia coli, did not. Addition of the 46-kDa amino-terminal extension (similarly synthesized in E. coli) to the GTP-binding ARF-domain of ARD1 enhanced GTPase activity and inhibited GDP dissociation. The kinetic properties of mixtures of the ARF and non-ARF domains were similar to those of an intact recombinant ARD1. Physical association of the two proteins was demonstrated directly by gel filtration and by using the immobilized non-ARF domain. Thus, like the alpha subunits of heterotrimeric G proteins, ARD1 appears to consist of two domains that interact to regulate the biological activity of the protein.
Vitale
N
N
Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Moss
J
J
Vaughan
M
M
D020727:ADP-Ribosylation Factors; D002474:Cell-Free System; D002772:Cholera Toxin / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D019204:GTP-Binding Proteins / Q000737:chemistry*; D020690:GTPase-Activating Proteins; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D020703:ras GTPase-Activating Proteins
D018076:DNA, Complementary; D020690:GTPase-Activating Proteins; D011506:Proteins; D011994:Recombinant Proteins; D020703:ras GTPase-Activating Proteins; D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors