Figure 2

Glucose deprivation signaling is channelled to the Pmk1 cascade by a Rho-GTPase independent mechanism which involves Pck2. A. Strains MI200 (Pmk1-Ha6H; Control), MI700 (rho2Δ, Pmk1-Ha6H), GB3 (pck2Δ, Pmk1-Ha6H), GB35 (pck1Δ, Pmk1-Ha6H), GB29 (rho2Δ pck2Δ, Pmk1-Ha6H), and MM539 (rho2Δ pck1Δ, Pmk1-Ha6H), were grown in YES medium plus 7% glucose to early-log phase and transferred to the same medium with 3% glycerol. Aliquots were harvested at timed intervals and Pmk1 was purified by affinity chromatography. Either activated or total Pmk1 were detected by immunoblotting with anti-phospho-p44/42 or anti-HA antibodies, respectively. B. Strain MI200 (Pmk1-Ha6H; Control) was transformed with plasmid pREP41-rho1(T20N), grown in EMM2 medium plus 7% glucose with or without thiamine (B1), and transferred to the same mediums with 3% glycerol. C. Strain MI700 (rho2Δ, Pmk1-Ha6H) was transformed with plasmid pREP41-rho1(T20N). Purification and detection of active or total Pmk1 was performed as described in A. D. Strains MI200 (Pmk1-Ha6H; Control), MI700 (rho2Δ, Pmk1-Ha6H), JFZ1001 (rho5Δ, Pmk1-Ha6H), and JFZ1004 (rho2Δ rho5Δ, Pmk1-Ha6H), were grown in YES medium plus 7% glucose to early-log phase and transferred to the same medium with 3% glycerol. E. Strains MI200 (Pmk1-Ha6H; Control), JFZ1001 (rho5Δ, Pmk1-Ha6H), JFZ1002 (rho5Δ pck2Δ, Pmk1-Ha6H), and JFZ1003 (rho5Δ pck1Δ, Pmk1-Ha6H), were grown in YES medium plus 7% glucose to early-log phase and transferred to the same medium with 3% glycerol. F. Strain JFZ1001 (rho5Δ, Pmk1-Ha6H) was transformed with plasmid pREP41-rho1(T20N), grown in EMM2 medium plus 7% glucose with or without thiamine (B1), and transferred to the same mediums with 3% glycerol. G. Strain MI700 (rho2Δ, Pmk1-Ha6H) was transformed with plasmid pREP41-cdc42(T17N), grown in EMM2 medium plus 7% glucose without thiamine, and transferred to the same medium with 3% glycerol.