Figure 9.

Influence of Pmp1p activity on the cell cycle-dependent activation of Pmk1p. (A) Pmk1p activates periodically during the cell cycle. Top, cells from strains MI600 (cdc25-22, pmk1-HA6H) and MI602 (cdc25-22, pmp1Δ, pmk1-HA6H) were grown to an A₆₀₀ of 0.3 at 25°C, shifted to 37°C for 3.5 h, and then released from the growth arrest by transfer back to 25°C. Aliquots were taken at different time intervals, and Pmk1-HA6H was purified by affinity chromatography. Activated or total Pmk1p were detected by immunoblotting with anti-phospho-p42/44 or anti-HA antibodies, respectively, and anti-Cdc2 antibody was used as loading control. Bottom, septation index of strains MI600 (filled circles), MI601 (cdc25-22, pmk1Δ; open circles), and MI602 (filled triangles). (B) Quantitative analysis of Pmk1p activity during cell cycle in strains MI600 (filled bars) and MI602 (empty bars) obtained from data in A. (C) Defective cell separation in pmk1Δ and pmp1Δ cells. Cells from strains MI600 (Control), MI601, and MI602 were taken at the times shown during cell cycle experiments, stained with Calcofluor white, and observed by fluorescence microscopy. Arrows indicate cells with multiseptate phenotype and defective cell separation.