PMC

The distances were calculated using PAML . The bars to the left indicate the distances (ratio) between S288c and YJM789 while the bars to the right measure the distances (ratio) between S288c and S. paradoxus.

A. The strains carrying the GFP-tagged version of Pdr5p were exponentially grown in YPD media and visualized by tagged-GFP signal. Fluorescence (left) and DIC (right) images were background-subtracted and scaled identically. The results clearly show that Pdr5p localizes at plasma membrane in YJM789 strain. B. Western blot analysis of Pdr5p (GFP-tagged) in YJM789 strain by using anti-GFP antibody. Lane 1: YJM789 WT, lane 2: YJM789 expressing GFP-tagged Pdr5p. The result indicates that intact Pdr5p could express normally in YJM789 strain.

BY4741, YJM789,their PDR5 null strains and YJM789Δpdr5::BPDR5(GY02) were grown in YPD overnight and reinoculated to OD₆₀₀ = 0.1. 90 µL of the above media with strains were treated with 10 µL of water or different concentrations of fluconazole, and then grown for 24 h at 30°C. OD₆₀₀ values at 24 h are shown in the figure. Measurements were made in triplicate with standard deviations shown in the figures.

A. All strains were grown in YPD overnight at 30°C and reinoculated to OD₆₀₀ = 0.1. 90 µL of media with strains were treated with 10 µL of water or different concentrations of AmB, and then grown for 20 h at 30°C. The values are the averages from three experiments. B. YJM789Δpdr5 (GY03) and YJM789Δpdr5:: BPDR5 (GY02) mutants grew in YPD medium overnight at 30°C and reinoculated to OD₆₀₀ = 0.1. 90 µL of media with strains were treated with 10 µL of water or different concentrations of AmB, and then grown for 20 h at 37°C. Measurements were made in triplicate with standard deviations shown in the figures. C. BY4741, YJM789, BY4741Δpdr5 and YJM789Δpdr5 were grown overnight and reinoculated to OD₆₀₀ = 0.2, then 4 µL of ten-fold serial dilutions were spotted onto YPD agar containing AmB (5 µg/mL, 10 µg/mL), and the plates were incubated at 30°C for 2 days.

The strains were grown in YPD overnight at 30°C and reinoculated to OD₆₀₀ = 0.1. 90 µL media of strains were treated with 10 µL of water or a pharmacological compound (A: itraconazole, B: ketoconazole, C: fluconazole), respectively, and then grown for 24 h. Only OD₆₀₀ values at 24 h are shown in the figure. Measurements were made in triplicate with standard deviations shown in the figures. D. Strains were grown overnight and reinoculated to OD₆₀₀ = 0.2, then 4 µL of ten-fold serial dilutions were spotted onto YPD agar containing one of the drugs (itraconazole: 2 µg/mL, ketoconazole: 1 µg/mL, fluconazole 5 µg/mL), and the plates were incubated at 30°C for 2 days.

A: Schematics of PDR5 gene regions; B: Amino acid difference for whole PDR5 sequence; C: Amino acid difference for transmembrane domain regions of PDR5 sequence. The topology information for the WT Pdr5p was downloaded from UniProtKB/Swiss-Prot database (http://www.uniprot.org/uniprot/P33302). PDR5 DNA sequences of eight strains of S. cerevisiae (DBVPG1788, DBVPG1853, K11, NCYC361, S288c, YJM789, YJM981 and YPS606) were downloaded from a recent study . Only the DNA sequences without any frame-shift mutations were used in this study. DNA sequences of PDR5 gene in S. paradoxus and S. bayanus were downloaded from NCBI database. The phylogenetic tree of these species was adapted from Fitzpatrick et al. . The data were analyzed by Matlab and the different color schemes represent levels of amino acid similarity.

A. Cell growth on Fluconazole. 4 µl five-fold serial dilutions of BY4741 WT cells, pdr5 null mutant cells, and cells expressing PDR5-1, which was reconstructed with YPDR5 TMDR1 and BPDR5 TMDR2 and PDR5-2, which was reconstructed with BPDR5 TMDR1 and YPDR5 TMDR2, were spotted on SC-uracil drug agar plates. Before spotting, all strains were grown to exponential phase, diluted to 0.2 OD₆₀₀. Plates were incubated for 3 days at 30°C. The results indicate that the TMDR1 in YJM789 PDR5 is functional (row #1 vs. row #3), but the TMDR2 in YJM789 PDR5 cannot conduct its original function (row #1 vs. row #4). B. Expression of hybrid constructs. Electrophoresis result for RT- PCR products was depicted. Total RNA (lane 1) and cDNA (lane 2) of pdr5Δ null mutant cells harboring YEplac195-PDR5-1, total RNA (lane 3) and cDNA (lane 4) of pdr5Δ null mutant cells harboring YEplac195-PDR5-2 were amplified by specific primer pairs. The result indicates that both constructs can be expressed successfully in the BY4741Δpdr5 background.