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Fig. 6. Intragenic complementation between the pkc1-834 and stt1-1 mutations. (A) Diploid strains of ZDS1/ZDS1 (W303-1D), zds1 ${Delta}$ PKC1/zds1 ${Delta}$ PKC1 (YMM125), zds1 ${Delta}$ pkc1-834/zds1 ${Delta}$ pkc1-834 (YMM219), zds1 ${Delta}$ stt1-1/zds1 ${Delta}$ stt1-1 (YMM218), and zds1 ${Delta}$ pkc1-834/zds1 ${Delta}$ stt1-1 (YMM220) were spotted onto YPD plates with the indicated concentration of CaCl₂ and incubated at the indicated temperatures. (B) Alkaline phosphatase colony assay of the various strains. Cells were spotted on YPD plates and cultured at 25°C for 2 days. The plates were further incubated overnight at 25°C or 35°C and alkaline phosphatase released from the cells as a result of cell lysis was detected using BCIP. The blue color (seen here as darker disks) indicates defective cell walls. The mpk1 ${Delta}$ (TNP46) strain was used as a positive control. (C) Mpk1 activation by heat-shock in various strains. Wild-type (DHT22-1b), pkc1-834 (YMM28) and stt1-1 (YMM114) cells were shifted from 25°C to 37°C, and incubated for 2 hours. The Mpk1p phosphorylation was monitored by western blotting. Immunoblot analysis was carried out using anti-phospho-p44/42 MAPK antibody (top panel) or anti-Mpk1p antibody to detect Mpk1p (bottom panel). (D) The temperature sensitivity of stt1-1 is partially suppressed by overexpression of MPK1. scz6 (YMM28) or stt1-1 (YMM114) cells with high-copy plasmids: control (YEp24), PKC1 (YEp24-PKC1) or MPK1 (YEp24-MPK1), were spotted on YPD plates and grown at the indicated temperatures for 2 days.

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