Specific functional interaction of human cytohesin-1 and ADP-ribosylation factor domain protein (ARD1).
Activation of ADP-ribosylation factors (ARFs) is mediated by guanine nucleotide-exchange proteins, which accelerate conversion of inactive ARF-GDP to active ARF-GTP. ARF domain protein (ARD1), a 64-kDa GTPase with a C-terminal ADP-ribosylation factor domain, is localized to lysosomes and the Golgi apparatus. When ARD1 was used as bait to screen a ... human liver cDNA library using the yeast two-hybrid system, a cDNA for cytohesin-1, a approximately 50-kDa protein with ARF guanine nucleotide-exchange protein activity, was isolated. In this system, ARD1-GDP interacted well with cytohesin-1 but very poorly with cytohesin-2. In agreement, cytohesin-1, but not cytohesin-2, markedly accelerated [(35)S]guanosine 5'-3-O-(thio)triphosphate binding to ARD1. The effector region of the ARF domain of ARD1 appeared to be critical for the specific interaction with cytohesin-1. Replacement of single amino acids in the Sec7 domains of cytohesin-1 and -2 showed that residue 30 is critical for specificity. In transfected COS-7 cells, overexpressed ARD1 and cytohesin-1 were partially colocalized, as determined by confocal fluorescence microscopy. It was concluded that cytohesin-1 is likely to be involved in ARD1 activation, consistent with a role for ARD1 in the regulation of vesicular trafficking.
Mesh Terms:
Amino Acid Sequence, Amino Acid Substitution, Cell Adhesion Molecules, Cell Line, Cloning, Molecular, GTP Phosphohydrolases, GTP-Binding Proteins, Gene Library, Guanine Nucleotide Exchange Factors, Guanosine 5'-O-(3-Thiotriphosphate), Guanosine Diphosphate, Humans, Kinetics, Liver, Molecular Sequence Data, Mutagenesis, Site-Directed, Point Mutation, Recombinant Fusion Proteins, Recombinant Proteins, Saccharomyces cerevisiae, Sequence Alignment, Sequence Homology, Amino Acid, Transfection
Amino Acid Sequence, Amino Acid Substitution, Cell Adhesion Molecules, Cell Line, Cloning, Molecular, GTP Phosphohydrolases, GTP-Binding Proteins, Gene Library, Guanine Nucleotide Exchange Factors, Guanosine 5'-O-(3-Thiotriphosphate), Guanosine Diphosphate, Humans, Kinetics, Liver, Molecular Sequence Data, Mutagenesis, Site-Directed, Point Mutation, Recombinant Fusion Proteins, Recombinant Proteins, Saccharomyces cerevisiae, Sequence Alignment, Sequence Homology, Amino Acid, Transfection
J. Biol. Chem.
Date: Jul. 14, 2000
PubMed ID: 10748148
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