Down-regulation of Hsp90 promotes the development of mating projections.

(A) Morphologies of the control and tetON-HSP90/hsp90 mutant. Control, GH1013 + pACT1-WOR1; tetON-HSP90/hsp90, a tetON-promoter–controlled conditional expression strain of HSP90 with ectopically expressed WOR1 (+ pACT1-WOR1). 1 × 105 cells were spotted on YPD-K and YP-K media with or without Dox as indicated and cultured at 25°C for three, five, or seven days. Percentages of projected cells are indicated in the corresponding images. Scale bar for colonies, 2 mm (inset); scale bar for cells, 10 μm. (B) Relative expression of mating-related genes. 1 × 105 cells of each strain were cultured on YPD-K medium (for seven days) or on YP-K medium (for three days) with 40 μg/mL Dox at 25°C. Relative expression levels were not tested on YP-K medium without Dox since cell viability of the tetON-HSP90/hsp90 mutant was severely impaired. Error bars represent standard errors of technical duplicates. *p t test. Experiment was repeated in a biological replicate, and a representative image is shown. The numerical data are presented in S3 Data. Dox, doxycycline; FIG1, Factor-Induced Gene 1; FUS1, cell FUSion 1; Hsp90, Heat shock protein 90; MFA1, Mating type A1; MFα, Mating α factor precursor; NA, not analyzed; p, mating projection; pACT1, plasmid pACT1; tetON, tetracycline-induced; WOR1, White–Opaque Regulator 1; YPD-K, yeast extract-peptone-glucose-K2HPO4; YP-K, yeast extract-peptone-K2HPO4.