Specific functional interaction of human cytohesin-1 and ADP-ribosylation factor domain protein (ARD1)

Activation of ADP-ribosylation factors (ARFs) is mediated by guanine nucleo tide-exchange proteins, which accelerate conversion of inactive ARF-GDP to active ARF-GTP. ARF domain protein (ARD1), a 64-kDa GTPase with a C-termina l ADP-ribosylation factor domain, is localized to lysosomes and the Golgi a pparatus. When ARD1 was used as bait to screen a human liver cDNA library u sing the yeast two-hybrid system, a cDNA for cytohesin-1, a similar to 50-k Da protein with ARF guanine nucleotide-exchange protein activity, was isola ted. In this system, ARD1-GDP interacted well with cytohesin-1 but very poo rly with cytohesin-2. In agreement, cytohesin-1, but not cytohesin-2, marke dly accelerated [S-35]guanosine 5'-3-O-(thio)triphosphate binding to ARD1. The effector region of the ARF domain of ARD1 appeared to be critical for t he specific interaction with cytohesin-1. Replacement of single amino acids in the Sec7 domains of cytohesin-1 and -2 showed that residue 30 is critic al for specificity. In transfected COS-7 cells, overexpressed ARD1 and cyto hesin-1 were partially colocalized, as determined by confocal fluorescence microscopy. It was concluded that cytohesin-1 is likely to be involved in A RD1 activation, consistent with a role for ARD1 in the regulation of vesicu lar trafficking.