Figure 4.—

Single-round turnover assays of GAP activity on Sec4p and Ypt1p. One micromolar Ypt GTPases His-Sec4p and His-Ypt1p were prebound with [γ-³²P]GTP for 30–60 min and then diluted 10-fold into reactions containing a 200-fold excess of cold GTP and His-Gyp1p, His-Gyp5p, GST, GST-Gyl1p, GST-His-Rvs167p-His-Rvs161p, or combinations of these that had been allowed to incubate together on ice for 20 min. Samples were taken in duplicate at 0, 1, 2, 5, 10, 30, and 60 min and GTP hydrolysis was assayed using the charcoal-binding method. Values shown represent average P_i released in a 100-μl reaction after zero time values have been subtracted. (A) Assay of GAP activity of various concentrations of Gyp5p on the GTPase Sec4. (B) Assay of GAP activity of various concentrations of Gyp5p on the GTPase Ypt1. (C) The initial rates of P_i release are plotted against concentration of Gyp5p for B. (D) Effect of adding Gyl1p and Gyp5p to Ypt1p. (E) Effect of adding Rvs167p-Rvs161p dimer on Gyp5p GAP activity toward Ypt1p.