Figure 5

Pmk1 allows full adaptation to respiratory metabolism in fission yeast by reinforcing the SAPK pathway. A. Strains MI200 (Pmk1-Ha6H, Control), MI204 (sty1Δ, Pmk1-Ha6H), MI102 (pmk1Δ), and LS116 (pmk1Δ, Pmk1(K52E):GFP), were grown in YES medium plus 7% glucose to early-log phase, and 10⁵, 10⁴, 10³, 10², or 10 cells were spotted on YES plates supplemented with either 7% glucose or 2% glycerol plus 3% ethanol, in the presence or absence of 30 mM NAC. Plates were incubated for either 3 (glucose plates) or 5 (glycerol plates) days at 28 °C before being photographed. B. Strains MI200 (Pmk1-Ha6H, Control), and MI102 (pmk1Δ), were grown in YES medium plus 7% glucose to early-log phase and transferred to the same medium with 2% glycerol plus 3% ethanol. Total RNA was extracted, and both fbp1+ and pyp2+ mRNA levels were detected by Northern blot analysis after hybridization with ³²P-labelled probes for fbp1⁺, pyp2⁺, and leu1⁺ (loading control) genes. C. Strains MI702 (Pyp2-13myc, Control) and LS134 (pmk1Δ, Pyp2-13myc), were grown in YES medium plus 7% glucose to early-log phase and transferred to the same medium with 2% glycerol plus 3% ethanol. Pyp2 protein levels were detected with anti-c-myc antibody. Anti-Cdc2 antibody was used as loading control. D. Strains JM1521 (Sty1-Ha6H, Control) and MI100 (pmk1Δ, Sty1-Ha6H), were grown in YES medium plus 7% glucose to early-log phase and transferred to the same medium with 2% glycerol plus 3% ethanol. Either activated or total Sty1 were detected with anti-phospho-p38 or anti-HA antibodies, respectively. E. Strains JM1821 (Atf1-Ha6H, Control) and AF390 (pmk1Δ, Atf1-Ha6H), were grown in YES medium plus 7% glucose to early-log phase and transferred to the same medium with 2% glycerol plus 3% ethanol. Atf1 was purified by affinity chromatography and detected with anti-HA antibody. Anti-Cdc2 antibody was used as loading control.