Abstract
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.
Highlights
ADP-ribosylation factors (ARFs) domain protein 1 (ARD1) is a 64-kDa protein that contains a 18-kDa carboxylterminal ADP-ribosylation factor (ARF) domain (p3) and a 46-kDa amino-terminal domain (p5) [2]
It was shown that the amino-terminal p5 domain of ARD1 stimulates hydrolysis of GTP bound to the ARF domain p3 and appears to be the GTPase-activating proteins (GAPs) component of this bifunctional protein [18]
We demonstrated that the GAP domain of ARD1 interacts with the effector region of the ARF domain of ARD1 and thereby stimulates GTP hydrolysis [17]
Summary
Construction and Expression of Mutated Forms of ARD1—Fragments of ARD1 with deletions at the 5Ј end (EMBL Data Bank 1993, accession number L04510) from pGEX-5G/LIC [2] were amplified by PCR using the forward primers 5Ј-GATTCAGGTGTCCCGCGGATGAAA-3Ј, 5Ј-TGTCAAACTAGCCCGCGGATGTCG-3Ј, 5Ј-GAAACTCTGTGTCGTCAACCGCGGATGGCT-3Ј, and 5Ј-AATCAGTTGGATGCCCCGCGGATGGTCACTTTTACAAAG-3Ј (differences from the original clones are underlined) and the reverse primer 5Ј-GAATTCCCGGGGATCCAACTGCG-3Ј (italicized sequence is a BamHI restriction site). Assay of CTA-catalyzed ADP-ribosylagmatine Formation—Purified recombinant ARD proteins were incubated for 30 min in 50 l of 20 mM Tris (pH 8.0), 10 mM dithiothreitol, 2.5 mM EDTA with 0.3 mg/ml bovine serum albumin and 1 mg/ml cardiolipin before the addition of 20 l of solution to yield final concentrations of 100 M GTP␥S and 10 mM MgCl2. The data are presented as the means Ϯ S.E. of values from quadruplicate determinations
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