Characterization of a GTPase-activating Protein That Stimulates GTP Hydrolysis by Both ADP-ribosylation Factor (ARF) and ARF-like Proteins
| Autor: | Nicolas Vitale, Min Ding, Joel Moss, Ronald Adamik, Martha Vaughan, Su-Chen Tsai |
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| Rok vydání: | 1996 |
| Předmět: | |
| Zdroj: | Journal of Biological Chemistry. 271:24005-24009 |
| ISSN: | 0021-9258 5232-5237 |
| Popis: | ADP-ribosylation factors (ARFs) are ∼20-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 (NH4)2SO4 precipitation and chromatography on Ultrogel AcA 34, DEAE-Sephacel, heparin-Sepharose, hydroxylapatite, and Ultrogel AcA 44. In fractions (∼100-kDa proteins) from Ultrogel AcA 44, a major protein band of ∼50 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 NH2-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 α-helix found in native ARF and hence is similar to the amino-terminal truncated mutant Δ13ARF1. Both the ARF domain of ARD1 and Δ13ARF1 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 Δ13ARF1, i.e. it lacks the relatively broad substrate specificity exhibited by ARF GAP. |
| Databáze: | OpenAIRE |
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