Pdr3-11 elevates drug resistance and Pdr5 expression in the absence of Med15. (A) Isogenic strains containing or lacking MED15 were transformed with the empty vector plasmid pRS315 (Vector) or the same plasmid containing the hyperactive PDR3-11 allele (PDR3-11). Transformants were grown to midlog phase and then 1000 cells/spot were placed on a YPD plate containing a gradient of cycloheximide (Cyh; increasing concentration indicated by the bar of increasing width). Except where noted, cycloheximide was present at 0.2 μg/ml. (B) Whole cell protein extracts were prepared from the transformants above. Equal amounts of protein were resolved on SDS-PAGE and analyzed by Western blotting using an antibody directed against Pdr5 or the vacuolar ATPase subunit Vph1. (C) A strain lacking both Pdr1 and Pdr3 (pdr1Δ pdr3Δ) and expressing a Med15-TAP fusion protein was transformed with a low-copy-number plasmid (Vector) or the same plasmid expressing wild-type Pdr3 (Ace-PDR3) or the hyperactive form of Pdr3 (Ace-PDR3-11) under control of the copper responsive Ace1 transcription factor. Transformants were grown to early log phase and split into subcultures. Subcultures were untreated (−) or induced with copper (+) to control expression of the different forms of Pdr3. After this treatment, whole cell protein extracts were prepared under native conditions. Aliquots of this total protein extract were reserved as controls for input protein with the remainder subjected to immunoprecipitation using an anti-TAP antibody. Immunoprecipitates were washed and then denatured in Laemmli sample buffer. Both immunoprecipitated and input samples were electrophoresed on SDS-PAGE and analyzed by Western blotting for Pdr3 (anti-HA antibody) and Med15 (anti-TAP antibody).

Mitochondrial signals still induce PDR5 in med15Δ cells. (A) Isogenic wild-type and med15Δ cells containing (ρ⁺) or lacking (ρ⁰) their mitochondrial genomes were tested for cycloheximide resistance by spotting 10-fold serial dilution of midlog phase cells on rich medium (YPD) or YPD medium containing cycloheximide (Cyh). (B) Strains from above were transformed with low-copy-number plasmids containing gene fusions between PDR5- or SNQ2- and Escherichia coli lacZ. Transformants were assayed for β-galactosidase activity as described (Guarente, 1983).

Mediator components influence resistance phenotypes in nonidentical ways. (A) The various subdomains of the Mediator complex are illustrated at the top of the figure and discussed in the text. This model is adapted from an earlier publication (Casamassimi and Napoli, 2007). (B) Isogenic ρ⁺ and ρ⁰ cells containing the disruption mutations indicated at the top of the figure were grown to midlog phase, and 10-fold serial dilutions were placed on media indicated. Strains containing all Mediator components are denoted as wild-type (wt) in both ρ⁺ and ρ⁰ backgrounds. The two media lacking all drugs are YPD and YPGE. Cycloheximide (Cyh) plates are based on YPD medium. (C) Isogenic ρ⁺ and ρ⁰ cells containing the indicated Mediator disruption mutations listed at the right of the figure were transformed with plasmids containing the PDR5- or SNQ2-lacZ reporter genes and grown to midlog phase. The levels of β-galactosidase activity were measured as described above.

Nonidentical roles of Cdk module subunits of Mediator in PDR5 activation. (A) Isogenic ρ⁺ and ρ⁰ cells containing the indicated disruption mutations in the four subunits of the Cdk module were grown to midlog phase, serially diluted as above and tested for their ability to grow on rich medium (YPD), YPD containing cycloheximide (Cyh), or rich medium with glycerol/ethanol as the carbon source (YPGE). (B) The strains described above were transformed with the indicated reporter plasmids and β-galactosidase activities determined.

Differential roles of Med12 and Med15 in autoregulation of PDR3 expression. (A) Isogenic ρ⁺ and ρ⁰ cells with the indicated relevant genotypes were transformed with a low-copy-number plasmid containing an epitope-tagged allele of PDR3 or the empty vector plasmid (Vector). Transformants were grown to midlog phase, and protein extracts prepared and analyzed by Western blotting using an anti-HA antibody. The position of epitope-tagged Pdr3 is indicated at the left, and the asterisk (*) denotes the position of a nonspecific signal that cross reacts with the anti-HA antibody. Levels of phosphoglycerate kinase were assessed as a loading control. (B) Quantitative reverse transcription-PCR analysis of PDR3 and PDR5 mRNA levels in ρ⁺ and ρ⁰ cells containing or lacking MED12 was carried out as described (Shahi et al., 2007). Total RNA was extracted from these strains, and specific primers were used to detect PDR3 and PDR5 mRNA. Transcript levels are relative to the level seen in ρ⁺ cells, and ACT1 mRNA was analyzed in every sample as a control transcript.

Med12 is not required for Psd1 signaling to PDR5 in ρ⁺ cells. (A) Wild-type cells or isogenic med15Δ and med12Δ derivatives were transformed with high-copy-number plasmids containing (2 μm PSD1) or lacking (Vector) the PSD1 gene. Transformants were grown to midlog phase and spotted as 10-fold serial dilutions on YPD plates with or without cycloheximide (Cyh). (B) Cells expressing a Med15-TAP fusion protein and containing the wild-type gene dosage of PSD1 (wt) or the high-copy-number plasmid bearing PSD1 (2 μm PSD1) were processed for ChIP as described (Gulshan et al., 2005). Aliquots of total chromatin (Input) or chromatin that was immunoprecipitated with anti-TAP antibody (Med15-associated chromatin) were analyzed by qPCR by using primers specific for the PDR5 promoter (left) or the PDR3 promoter (right). (C) Relative transcript level analysis by reverse transcriptase qPCR analysis of DLD1 and ACO1 mRNA levels in ρ⁺ and ρ⁰ cells containing or lacking MED12 was carried out as described above. Quantitation of qPCR was carried out as described above.

Comparison of Med12 and Med15 roles in control of PDR5 expression. (A) Isogenic ρ⁺ and ρ⁰ strains containing disruption mutations of the either MED15 and/or MED12 were grown to midlog phase, and serial dilutions were tested for their ability to grow on YPD or YPD-containing cycloheximide (Cyh) media. (B) The strains described above were transformed with the indicated reporter genes and assayed for the levels of β-galactosidase produced in appropriate transformants.

Med12 recruitment to the PDR5 promoter and its association with Pdr3 is highly inducible in ρ⁰ cells. (A) Isogenic ρ⁺ and ρ⁰ cells expressing the indicated Mediator subunit-TAP fusion protein were processed for ChIP as described above. Immunoprecipitated (IP) DNA was analyzed by qPCR using primers designed to amplify the PDR5 promoter. Antibody independent DNA recovery was estimated by performing identical immunoprecipitations but without the addition of any primary antibody (No IgG) The ratio of IP to input signals was calculated for the signal from the PDR5 promoter primer pairs divided by the corresponding value for the control (No IgG). The mean and SEs of the resulting normalized IP/Input ratios from replicate cultures, calculated with three PCR measurements, are plotted. (B) Strains with the indicated relevant genotypes (top of panel) expressing both Med12-TAP and epitope-tagged Pdr3 (under copper control: Ace1-Pdr3) were processed for coimmunoprecipitation using anti-TAP antibody as described in Figure 1. Immunoprecipitated samples were analyzed by Western blotting with anti-TAP (detects Med12-TAP) and anti-HA (detects epitope-tagged Pdr3). (C) Isogenic ρ⁺ and ρ⁰ pdr1Δ pdr3Δ strains expressing the indicated Mediator subunit-TAP fusion protein were transformed with a low-copy-plasmid expressing Myc-Pdr1 and processed for coimmunoprecipitation using anti-TAP antibody. Input and anti-TAP immunoprecipitated (IP) samples were electrophoresed on SDS-PAGE and subjected to Western blotting using anti-TAP (detects mediator subunit) and anti-Myc (detects epitope-tagged Pdr1) antibodies.