Figure 4.

Effect of cytohesin-1 on release of bound GTP[γS] from ARF3. Samples of native ARF3 (1.5 μg, 75 pmol) were incubated with 4 μM [³⁵S]GTP[γS] (2 × 10⁶ cpm), as described (12), in 100 μl containing 20 mM Tris (pH 8.0), 50 mM NaCl, 20 μg of phosphatidylserine, 50 μg of BSA, 12 mM DTT, 0.65 mM MgCl₂, and 1 mM EDTA for 40 min at 36.5°C. Tubes were placed in ice and 20-μl samples (15 pmol of ARF3) were transferred to tubes containing 50 μM unlabeled GTP[γS] (▪), GDP (•), GDP[βS] (▴), or no nucleotide (▾), with (solid line) or without (dotted line) cytohesin-1 (0.5 μg) and other additions, as indicated in Fig. 2, in a final volume of 100 μl. After incubation for the indicated time, samples were filtered for radioassay of protein-bound [³⁵S]GTP[γS]. One hundred percent binding (3.1–3.2 pmol) was the mean of values from samples of ARF3 ± cytohesin-1 and unlabeled nucleotide kept at 0°C for 60 min. Without ARF3, 0.03–0.05 pmol of [³⁵S]GTP[γS] was bound; these control values have been subtracted from the data presented.