Guanine nucleotide exchange on ADP-ribosylation factors catalyzed by cytohesin-1 and its Sec7 domain.
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 GTPgammaS (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 GTPgammaS 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 Delta13ARF1, which lacks the N-terminal alpha-helix. Neither C-1 Sec7 nor cytohesin-1 increased GTPgammaS 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.
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA. PacheocoG@gate.nhlbi.nih.gov
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Pacheco-Rodriguez
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Meacci
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Vitale
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Moss
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Vaughan
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D002384:Catalysis; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*
D015815:Cell Adhesion Molecules; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; C066755:Sec7 guanine nucleotide exchange factors; C100723:cytohesin-1; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors