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

1997; National Academy of Sciences; Volume: 94; Issue: 5 Linguagem: Inglês

10.1073/pnas.94.5.1745

1091-6490

Elisabetta Meacci, Su-Chen Tsai, Ronald Adamik, Joel Moss, Martha Vaughan,

Lipid Membrane Structure and Behavior

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 αLβ2 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 35 S-labeled guanosine 5′-[γ-thio]triphosphate [ 35 S]GTP[γS] or [ 3 H]GDP to ARF purified from bovine brain (i.e., it appeared to function as an ARF-GEP). Addition of cytohesin-1 to ARF3 with [ 35 S]GTP[γS] bound, accelerated [ 35 S]GTP[γS] release to a similar degree in the presence of unlabeled GDP or GTP[γS] and to a lesser degree with GDP[βS]; 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.