FIGURE 3:

Increased permeability and decreased density of the PM in the crf1∆ lem3∆ sfk1 triple mutants. (A) Rhodamine uptake is increased in the crf1∆ lem3∆ sfk1∆ triple mutant. Cells were cultured in YPDA medium at 30°C, preincubated in SD medium in the absence (+ATP) or presence (–ATP) of 1 mM sodium azide for 30 min at 30°C, and incubated with rhodamine 6G for 60 min at 30°C. Rhodamine accumulation was measured as described in Materials and Methods. Values represent the mean ± SD from three independent experiments. Asterisks indicate a significant difference, as determined by the Tukey–Kramer test (p < 0.05). (B) Sucrose density gradient centrifugation analysis of PM proteins, Pdr5-GFP and Pma1, and a TGN/endosome protein, Kex2, in the crf1∆ lem3∆ sfk1-2 triple mutant. Cells were cultured as in Figure 2E. Cell lysates were prepared from the wild type and the crf1∆ lem3∆ sfk1-2 triple mutant expressing Pdr5-GFP and fractionated in 22–60% sucrose step density gradients as described in Materials and Methods. Equivalent volumes from each fraction were subjected to SDS–PAGE, and proteins were detected by immunoblotting.