REGULATION OF GTP BINDING PROTEINS

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To activate inactive ARF-GDP, a guanine nucleotide-exchange protein (GEP) is required to promote dissociation of GDP and binding of GTP, a process that can be inhibited by brefeldin A (BFA). BFA-sensitive and insensitive GEPs have been observed in both soluble and particulate cell fractions. Earlier, this group had purified two soluble BFA-insensitive GEPs. This year, a BFA-inhibited GEP was purified from bovine brain cytosol. Four tryptic peptides from the purified GEP had amino acid sequences that were 47% identical to sequences in Sec7 from Saccharomyces cerevisiae, consistent with the view that the BFA-sensitive 200-kDa protein may be a mammalian counterpart of Sec7, which plays a critical role in yeast vesicular transport, and Sec7 may be a GEP for one or more yeast ARFs. Cytohesin-1, a protein abundant in cells of the immune system, has been proposed to be a human homologue of the S. cerevisiae Sec7 gene product, which is crucial in protein transport. Overexpression of human or yeast ARF genes rescued yeast with Sec7 defects, restoring secretory pathway function. 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 [35S]GTP[yS] or [3H]GDP to ARF (i.e., it acted as an ARF-GEP). Addition of cytohesin-1 to ARF with [35S]GTP[yS]bound, accelerated [35S]GTP[yS] release, which was negligible without it. Cytohesin-1 also increased ARF binding to Golgi membranes, but its effect was not inhibited by BFA, a drug that reversibly inhibits Golgi function. In this regard, it differs from the BFA-sensitive ARF-GEP that contains a Sec7 domain also described by the group this year.