Fig 4. Calcineurin promotes sexual reproduction via Pbp1.

(A) Pbp1-FLAG was immunoprecipitated from WT (HP181), unexposed or 1 μg/ml FK506 exposed cells grown at 24°C or shifted from 24°C to 37°C for 1 hr, and treated with λ phosphatase and/or phosphatase inhibitor in vitro. (B-D) Pbp1-mCherry co-localized with the PBs component Dcp1 (HP114) (B), SGs component Pub1 (HP311) (C), or Cna1 (HP268) (D) during a temperature shift to 37°C. Each strain was grown at 24°C or shifted from 24°C to 37°C for 1 hr and visualized with a DeltaVision Elite Deconvolution microscope. Arrows indicate the co-localization of Pbp1-mCherry with Dcp1 (B), Pub1 (C), or Cna1 (D). (E) For sexual reproduction assays, the WT (H99, KN99a), and the cna1Δ (HP242, HP243), pbp1Δ (HP6, HP246) mutant strains were co-cultured with the opposite mating type strain on V8 media and incubated at room temperature in the dark for 7 days. Cells and the periphery of colonies were visualized by microscopy (upper panel). The bottom panel shows higher magnification views of the edges of colonies (Scale bars = 50 μm). (F) Pheromone gene expression (MFα1) was analyzed in WT, cna1Δ, and pbp1Δ mutants. The WT (H99, KN99a), cna1Δ (HP242, HP243), and pbp1Δ (HP6, HP246) mutant strains were co-cultured with the opposite mating type cells on V8 media and incubated at room temperature in the dark for 24 hours. Cells were harvested, RNA was isolated, and expression of MFα1 was assessed by real-time PCR and plotted as the mean of three independent determinations with standard deviation as error bars. Results shown in Fig 4A–4E are representative of two independent experiments.