Cytohesin-1, a cytosolic guanine nucleotide-exchange protein for ADP-ribosylation factor

Cytohesin-1, a protein abundant in cells of the immune system, has been proposed to be a human homolog of the Saccharomyces cerevisiae Sec7 gene product, which is crucial in protein transport, More recently, the same protein has been reported to be a regulatory factor for the alpha L beta 2 integrin in lymphocytes. Overexpression of human or yeast ADP-ribosylation factor (ARF) genes rescues yeast with Sec7 defects, restoring secretory pathway function, ARFs, 20-kDa guanine nucleotide-binding proteins initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity and now recognized as critical components in intracellular vesicular transport, exist in an inactive cytosolic form with GDP bound (ARF-GDP), Interaction with a guanine nucleotide-exchange protein (GEP) accelerates exchange of GDP for GTP, producing the active ARF-GTP, Both soluble and particulate GEPs have been described. To define better the interaction between ARF and Sec7-related proteins, effects of cytohesin-1, synthesized in Escherichia coli, on ARF activity were evaluated, Cytohesin-1 enhanced binding of S-35-labeled guanosine 5'-[gamma-thio]triphosphate [S-35]GTP[gamma S] or [H-3] GDP to ARF purified from bovine brain (i.e., it appeared to function as an ARF-GEP), Addition of cytohesin-1 to ARF3 with [S-35]GTP[gamma S] bound, accelerated [S-35]GTP[gamma S] release to a similar degree in the presence of unlabeled GDP or GTP[gamma S] and to a lesser degree with GDP[beta S]; release was negligible without added nucleotide, Cytohesin-1 also increased ARF1 binding to a Golgi fraction, but its effect was not inhibited by brefeldin A (BFA), a drug that reversibly inhibits Golgi function, In this regard, it differs from a recently reported BFA-sensitive ARF-GEP that contains a Sec7 domain.