Fig. 2. Mck1 functions downstream of the Mpk1 pathway to induce G₂ delay through the down-regulation of Hsl1. (A) The effect of overexpression of Cmp2ΔC (constitutively active form of the Cmp2 calcineurin catalytic subunit) or Mpk1 in the Δzds1 mck1-1 strain was examined. The Δzds1 mck1-1 (YMM2144) strain was transformed with the GAL-regulated plasmids pGAL-Cmp2ΔC (pYES2::Cmp2ΔC), pGAL-Mpk1 (pNV7::Mpk1) or vector alone (pYES2). The transformants were grown in liquid synthetic complete medium (SD minus Ura) at 28°C until early log growth phase. Cells were collected by centrifugation, washed, and suspended in SG (galactose medium, GAL promoter on) and the cells were then incubated at 28°C for 10 h. Cell morphology (DIC), FACS and the DNA content of PI-stained cells were analyzed by flow cytometry analysis. (B) The effect of overexpression of Mpk1 or Mck1 in Δzds1 (YAT1), Δzds1Δmpk1 (YMM3) or Δhsl1 (YMM52) strains was determined. These strains were transformed with pGAL-Mpk1 (pNV7::Mpk1), pGAL-Mck1 (pYES2::Mck1) or vector alone (pYES2). Experimental conditions were as described in (A). (C) Presumed pathway for the regulation of the Hsl1 kinase by Mck1 as suggested by the genetic analyses.