ADP-ribosylation factors (ARFs), a family of 20-kDa guanine nucleotide- binding proteins, were discovered as activators of cholera toxin A subunit (CTA)-catalyzed ADP-ribosylation of the stimulatory GTP-binding protein of the adenylyl cyclase system (Gs alpha) and participate in intracellular vesicular membrane trafficking. ARFs are activated when bound GDP is replaced by GTP and inactivated by hydrolysis of bound GTP to yield ARF-GDP. Usually, ARFs are isolated in an inactive GDP-bound state and require the addition of GTP along with detergent or phospholipid for activity. Purified mutant recombinant ARF1 lacking the first 13 amino acids (rdelta13ARF1-P) stimulated cholera toxin activity essentially equally, with or without added GTP (and phospholipid or detergent). In this study, rdelta13ARF1-P was shown to contain bound nucleotides, which later were identified as GTP and GDP. Nucleotide- free rdelta13ARF1 (rdelta13ARF1-F), prepared by dialysis against 7M urea, was active without added GTP in the absence of SDS, but inactive without added GTP in its presence. Renaturation of rdelta13ARF1-F in the presence of GTP, ITP, GDP or IDP yielded, respectively, rdelta13ARF1-GTP, and rdelta13ARF1-ITP, which were active, and rdelta13ARF1-GDP and rdeltaARF1-IDP, which were inactive. These studies are consistent with the hypothesis that the amino terminus affects nucleotide binding and that an amino-terminally truncated ARF can assume an active conformation in the absence of GTP.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL000656-05
Application #
5203497
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
1995
Total Cost
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Vichi, Alessandro; Payne, D Michael; Pacheco-Rodriguez, Gustavo et al. (2005) E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1). Proc Natl Acad Sci U S A 102:1945-50
Vitale, N; Pacheco-Rodriguez, G; Ferrans, V J et al. (2000) Specific functional interaction of human cytohesin-1 and ADP-ribosylation factor domain protein (ARD1). J Biol Chem 275:21331-9
Vitale, N; Patton, W A; Moss, J et al. (2000) GIT proteins, A novel family of phosphatidylinositol 3,4, 5-trisphosphate-stimulated GTPase-activating proteins for ARF6. J Biol Chem 275:13901-6
Sata, M; Moss, J; Vaughan, M (1999) Structural basis for the inhibitory effect of brefeldin A on guanine nucleotide-exchange proteins for ADP-ribosylation factors. Proc Natl Acad Sci U S A 96:2752-7
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Stevens, L A; Moss, J; Vaughan, M et al. (1999) Effects of site-directed mutagenesis of Escherichia coli heat-labile enterotoxin on ADP-ribosyltransferase activity and interaction with ADP-ribosylation factors. Infect Immun 67:259-65
Morinaga, N; Adamik, R; Moss, J et al. (1999) Brefeldin A inhibited activity of the sec7 domain of p200, a mammalian guanine nucleotide-exchange protein for ADP-ribosylation factors. J Biol Chem 274:17417-23
Rudolph, A E; Stuckey, J A; Zhao, Y et al. (1999) Expression, characterization, and mutagenesis of the Yersinia pestis murine toxin, a phospholipase D superfamily member. J Biol Chem 274:11824-31
Moss, J; Vaughan, M (1999) Activation of toxin ADP-ribosyltransferases by eukaryotic ADP-ribosylation factors. Mol Cell Biochem 193:153-7
Togawa, A; Morinaga, N; Ogasawara, M et al. (1999) Purification and cloning of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factors. J Biol Chem 274:12308-15

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