Figure 1.
Identification of residues important for Pdr5p function and distribution of mutations in YJM789 Pdr5p.
(A) Segmental replacement of BY4741 PDR5 and drug resistance profiles of pdr5Δ S. cerevisiae cells expressing wild type and chimeric PDR5s by spot assay. Various regions of BY4741 PDR5 are segmentally replaced by corresponding segments from YPDR5. Segments from BY4741 and YJM789 PDR5 are shown as white and grey rectangles, respectively. The unique Bsp I, Mlu I, Bsp1407 I restriction sites, which were used to construct PDR5 chimeras are marked. Five-fold serial dilutions of pdr5Δ mutant carrying different PDR5 constructs were spotted on SD/-Ura media in the absence and the presence of 3 µg/mL fluconazole or 75 ng/mL cycloheximide. Plates were incubated for 72 hours at 30°C. (B) The topological model is based on the molecular modeling study of Pdr5p [11]. Two-dimensional schematic cartoon of Pdr5p shows the 12 predicted membrane spanning helices (white spheres) and two NBDs (blue spheres). The green boxed areas indicate the TMDs. Divergent residues between BY4741 and YJM789 are shown as red, purple and dark blue spheres, respectively. Ala 1352 is indicated by black arrow.
Figure 2.
Drug resistance profiles of cells expressing PDR5 variants.
(A) Nine mutated sites distributed throughout blocks S2A and S2B were separated by site-directed mutagenesis and fluconazole sensitivities of mutant variants were determined by spot assay. Cells were grown overnight and reinoculated to OD600 = 0.1, then 4 µL of five-fold serial dilutions were spotted onto drug free or drug containing (3 µg/mL fluconazole or 75 ng/mL cycloheximide) SD/-Ura plates. The plates were incubated at 30°C for 72 hours. (B) Resistance of BPDR5 (⧫), pdr5Δ (▴) and A1352 (▪) mutants to increasing concentrations of fluconazole. Cells from overnight cultures were inoculated to OD600 = 0.1. Growth was estimated on the basis of OD600 and plotted as percentage growth, where the growth in drug free media was taken to be 100%. The mean values of three independent experiments are plotted and the error bars represent the S.D. (C) Growth of BPDR5 (⧫), pdr5Δ (▴) and A1352 (▪) mutants was compared as described in B, except that the experiment was performed in the presence of increasing concentrations of cycloheximide.
Table 1.
Strains and plasmids.
Figure 3.
Pdr5p-mediated efflux of rhodamine 6G in BPDR5 and mutant cells.
(A) Rhodamine 6 G resistance of BPDR5 (□), pdr5Δ (▴) and A1352 (▪) mutants were compared in liquid culture as described in materials and methods. Growth in the absence of R6G was served as control. Growth at each concentration of R6G is shown as (% control). The mean values of three independent experiments are plotted and the error bars represent the S.D. (B) The transport capabilities of BPDR5, pdr5Δ and A1352M were compared with flow cytometry. The histogram derived from the CellQuest software depicts fluorescence intensities for BPDR5 (black), pdr5Δ (red) and A1352M (blue).
Figure 4.
Drug resistance profiles of pdr5Δ S. cerevisiae cells expressing wild type and A1352 mutant variants of PDR5 on agar plates and in liquid culture.
(A) Drug sensitivity of A1352 mutant variants determined by spot assay. Serial dilutions of BPDR5 cells, pdr5Δ and A1352 mutant variants were spotted on SD/-Ura agar plates in the absence (left panel) and the presence (right panel) of 75 ng/mL cycloheximide, respectively. The plates were incubated at 30°C for 72 hours. (B) Quantitative analysis of drug resistance mediated by Pdr5p mutants. Cells carrying empty plasmid or overexpressing BPDR5, A1352 mutant variants as indicated, were subjected to analysis in liquid culture as described in materials and methods. OD600 values at 12 h were analyzed. Growth in drug free cultures of each strain was served as control. Normalized growth rates are shown as the means ± standard deviations (error bars) for three independent experiments at each concentration of cycloheximide.
Figure 5.
Membrane localization of Pdr5p and its mutant variants.
Isolated plasma membranes (15 µg/lane) were electrophoresed on an SDS–8% polyacrylamide gel and visualized with Coomassie blue (top panel). Pdr5p (middle panel) and Pma1p (bottom panel) were immunodetected with specific antibodies as described in the materials and methods section. Filled arrowheads indicate Pdr5p and open arrowheads indicate Pma1p. Coomassie blue stained SDS-PAGE gel (upper panel) and western blot (lower panel) of the same strains are shown.
Figure 6.
The effect of A1352M mutation on the ATPase activity of Pdr5p.
Plasma membrane preparations (0.25 µg/well) from BPDR5 (dark), pdr5Δ (gray) and A1352M (white) were incubated in 25 µL reaction buffer containing 300 mM Mes-Tris (pH 7.4), 4 mM ATP, 5 mM MgCl2, 0.2 mMammonium molybdate, 50 mM KNO3, and 10 mM NaN3 at 30°C for 20 min. The oligomycin-sensitive activity was determined as the difference in the ATPase activity in the presence or absence of 20 µg/mL oligomycin. The figure shows the means of three independent experiments, and the error bars represent standard error.
Table 2.
Primers used in construction of PDR5 chimeras.