FIGURE 3.

Interaction of nucleotides with WT and S558Y. A, colloidal blue staining and immunoblotting of purified PM vesicles prepared from various strains of yeast. Lanes 1–4 depict a colloidal blue-stained gel and lanes 5–8 an immunoblot with the anti-Pdr5 antibody yC-18. Lanes 1 and 5, Δpdr5; lanes 2 and 6, WT Pdr5, single copy; lanes 3 and 7, WT Pdr5, double copy; lanes 4 and 8, mutant Pdr5, S558Y, double copy. Arrow shows the position of the Pdr5 protein. B, photoaffinity labeling of Pdr5 with 0.5 mm 8-azido-[α-³²P]ATP. Cross-linking was carried out in purified plasma membrane vesicles at 4 °C as described under “Experimental Procedures.” Upper panel, 8-azido-[α-³²P]ATP incorporated into the Pdr5 band; lower panel, quantification of the [³²P] incorporated with a STORM 860 PhosphorImager system. The cross-linking in the absence of ATP is shown as filled bars and the cross-linking the presence of ATP as empty bars. C, 8-azido-[α-³²P]ATP binding as a function of ATP concentration (0–1 mm) was determined in WT Pdr5 (•) and the S558Y mutant (□). The 8-azido-[α-³²P]ATP incorporated in the Pdr5-bands was quantified with a PhosphorImager, and the curves were generated with the curve-fitting Prism GraphPad software (GraphPad Software, La Jolla, CA). The inset depicts the data with an expanded x axis in the range 0–0.125 mm ATP. The x and y axis legends for the inset are the same as those for the main figure. Figure shows representative data from two independent experiments.