Guanine Nucleotide Exchange on ADP-ribosylation Factors Catalyzed by Cytohesin-1 and Its Sec7 Domain*
- From the Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892
Abstract
ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins that require specific guanine nucleotide-exchange proteins (GEPs) to accelerate the conversion of inactive ARF-GDP to active ARF-GTP. Cytohesin-1, a 46-kDa ARF GEP, contains a central Sec7 domain of 188 amino acids similar in sequence to a region of the yeast Sec7 protein. Cytohesin-1 and its 22-kDa Sec7 domain (C-1 Sec7), synthesized in Escherichia coli, were assayed with recombinant non-myristoylated ARFs and related proteins to compare their GEP activities. Both were effective with native mammalian ARFs 1 and 3. Cytohesin-1 accelerated GTPγS (guanosine 5′-3-O-(thio)triphosphate) binding to recombinant human ARF1 (rARF1), yeast ARF3, and ARD1 (a 64-kDa guanine nucleotide-binding protein containing a C-terminal ARF domain). In contrast, C-1 Sec7 enhanced GTPγS binding to recombinant human ARFs 1, 5, and 6; yeast ARFs 1, 2, and 3; ARD1; two ARD1 mutants that contain the ARF domain; and Δ13ARF1, which lacks the N-terminal α-helix. Neither C-1 Sec7 nor cytohesin-1 increased GTPγS binding to human ARF-like ARL proteins 1, 2, and 3. Thus, ARLs, initially differentiated from ARFs because of their inability to activate cholera toxin, differ also in their failure to interact functionally with C-1 Sec7 or cytohesin-1. As C-1 Sec7 was much less substrate-specific than cytohesin-1, it appears that structure outside of the Sec7 domain is important for ARF specificity. Data obtained with mutant ARF constructs are all consistent with the conclusion that the ARF N terminus is an important determinant of cytohesin-1 specificity.
Footnotes
-
↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵‡ To whom correspondence should be addressed: Rm. 5N-307, Bldg. 10, 10 Center Dr., MSC 1434, National Institutes of Health, Bethesda, MD 20892-1434. Tel.: 301-402-1454; Fax: 301-402-1610; E-mail:PachecoG{at}gwgate.nhlbi.nih.gov.
-
↵§ Present address: Dept. of Biochemical Sciences, University of Florence, 50134 Firenze, Italy.
-
↵¶ Present address: Biologie de la Communication Cellulaire, INSERM U-388, Centre de Neurochimie, 5 rue Blaise Pascal, 67084 Strasbourg, France.
- Abbreviations:
- ARF
-
ADP-ribosylation factor
- BFA
-
brefeldin A
- GEP
-
guanine nucleotide-exchange protein
- C-1 Sec7
-
cytohesin-1 Sec7 domain
- GTPγS
-
guanosine 5′-3-O-(thio)triphosphate
- PS
-
l-α-phosphatidyl-l-serine
- PIP2
-
phosphatidylinositol 4,5-bisphosphate
- PH
-
pleckstrin homology
- ARL
-
ARF-like protein.
-
- Received June 19, 1998.
- Revision received July 27, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
