FIGURE 1.

Regulation of enzymatic GDP/GTP exchange activity of cytohesin-2 with myr-Arf6 and myr-Arf1 by V-ATPase-derived a2N(1–17) peptide on a membrane interface. A, preparation of recombinant myr-Arf1 and myr-Arf6 GTP-binding proteins. Human Arf1 was co-expressed with yeast N-myristoyltransferase in Escherichia coli BL21/DE3 strain. Recombinant myr-Arf1 was purified from the E. coli lysate (lane A1) using an AKTA purifier equipped with HiTrapQ, Superdex 200 columns (lane A2), and phenyl-Sepharose HP column (lane A3). Human Arf6 was also co-expressed with yeast N-myristoyltransferase in E. coli BL21/DE3 strain. Recombinant myr-Arf6 was extracted from the insoluble fraction using Triton X-100 (lane B1). The extract was precipitated by 35% ammonium sulfate and myr-Arf6 purified with HiTrapQ column (lane B2). B, time course of nucleotide exchange on myr-Arf1 and myr-Arf6 catalyzed by cytohesin-2. Exchange was followed by measuring the binding of [³⁵S]GTPγS in an assay mixture that included PIP₂-containing liposomes. C, dose-dependent inhibition of enzymatic GEF-activity of cytohesin-2 with myr-Arf6 by V-ATPase-derived a2N(1–17) peptide. Although FITC-a2N(1–17)-TAT peptide potently inhibits (IC₅₀ = 0.9 μm), the GEF activity of cytohesin-2, the control FITC-TAT peptide has no effect on its activity. D, dose-dependent inhibition of enzymatic GEF activity of cytohesin-2 with myr-Arf1 by V-ATPase-derived a2N(1–17) peptide. Although FITC-a2N(1–17)-TAT peptide potently inhibits (IC₅₀ = 0.9 μm), the GEF-activity of cytohesin-2, the control FITC-TAT peptide has no effect on its activity. E, dose-dependent inhibition by SecinH3 (IC₅₀ >150 μm) of enzymatic GEF activity of cytohesin-2 with myr-Arf6. F, dose-dependent inhibition by SecinH3 (IC₅₀ >150 μm) of enzymatic GEF-activity of cytohesin-2 with myr-Arf1.