Characterization of a GTPase-activating Protein That Stimulates GTP Hydrolysis by Both ADP-ribosylation Factor (ARF) and ARF-like Proteins

Min Ding, Nicolas Vitale, Su-Chen Tsai, Ronald Adamik, Joel Moss, Martha Vaughan
1996 Journal of Biological Chemistry  
ADP-ribosylation factors (ARFs) are ϳ20-kDa guanine nucleotide-binding proteins that participate in vesicular transport in the Golgi and other intracellular compartments and stimulate cholera toxin ADP-ribosyltransferase activity. Both GTP binding and hydrolysis are necessary for its physiological functions, although purified mammalian ARF lacks detectable GTPase activity. An ARF GTPase-activating protein (GAP) was purified >15,000-fold from rat spleen cytosol using (NH 4 ) 2 SO 4 precipitation
more » ... and chromatography on Ultrogel AcA 34, DEAE-Sephacel, heparin-Sepharose, hydroxylapatite, and Ultrogel AcA 44. In fractions (ϳ100-kDa proteins) from Ultrogel AcA 44, a major protein band of ϳ50 kDa on SDS-polyacrylamide gel electrophoresis correlated with GAP activity, consistent with it being a homodimer, thus differing from an ARF GAP purified from rat liver (Makler, V., Cukierman, E., Rotman, M., Admon, A., and Cassel, D. (1995) J. Biol. Chem. 270, 5232-5237). Purified spleen GAP accelerated hydrolysis of GTP bound to recombinant ARF1, ARF3, ARF5, and ARF6; no effect of NH 2 -terminal myristoylation was observed. ARF GAP also activated GTP hydrolysis by ARL1, which is 56% identical in amino acid sequence to ARF1, but lacks ARF activity. ARD1 is a 64-kDa guanine nucleotide-binding protein that contains an 18-kDa ARF domain at its carboxyl terminus; the ARF domain lacks the amino-terminal ␣-helix found in native ARF and hence is similar to the amino-terminal truncated mutant ⌬13ARF1. Both the ARF domain of ARD1 and ⌬13ARF1 were poor substrates for ARF GAP. The non-ARF1 domain of ARD1 enhanced the GTPase activity of the ARF domain, but not that of the ARF proteins and ⌬13ARF1, i.e. it lacks the relatively broad substrate specificity exhibited by ARF GAP. ADP-ribosylation factors (ARFs) 1 are 20 kDa guanine nucleotide-binding proteins, which were originally identified and
doi:10.1074/jbc.271.39.24005 pmid:8798635 fatcat:j3nv36qi2rfjhkani5uxynf2mm