Fig 4. Pbs2 is a client of Hsp90 in C. albicans.

(A) Pbs2 and Hsp90 physically interact. Immunoprecipitation of Pbs2-HA with anti-HA agarose co-purified Hsp90, while Hsp90 did not co-purify with anti-HA agarose in control cells lacking tagged Pbs2. There was no difference in Hsp90 levels between input samples. (B) Pbs2-HA is destabilized upon transcriptional repression of HSP90. Cells were grown overnight in ±0.5 μg/mL DOX to repress HSP90 in the tetO-HSP90/hsp90Δ strain and then subcultured into medium ± 5 μg/mL DOX for 4 hours before protein extraction and western blotting. Protein levels were normalized to the tubulin loading control and quantified compared with the no DOX control. (C) Pbs2-HA protein levels decrease upon fluconazole but not caspofungin stress. Cells were incubated with 8 μg/mL fluconazole (Fluc) or 100 nM caspofungin (Caspo) before protein extraction and western blotting. Protein levels were normalized to the tubulin loading control and quantified compared with the no drug control. (D) PBS2 transcript levels are not altered by transcriptional repression of HSP90 or treatment with antifungal drugs. Cells were grown overnight in ±0.5 μg/mL DOX to repress HSP90 in the tetO-HSP90/hsp90Δ strain and then subcultured into medium ± 5 μg/mL DOX for 4 hours before RNA extraction and qRT-PCR. Cells were incubated with 8 μg/mL fluconazole or 100 nM caspofungin before RNA extraction and qRT-PCR. PBS2 transcript levels were normalized to PMA1 and TEF1. Significance was determined by one-way ANOVA. **** indicates P-value < 0.001. Raw data for this figure can be found in S1 Data. Caspo, caspofungin; DOX, doxycycline; Fluc, fluconazole; HA, hemagglutinin; IP, immunoprecipitation; qRT-PCR, quantitative reverse transcription PCR; YPD, yeast extract peptone dextrose.