Figure 1. Rho1 GTPase is a positive regulator of the cell integrity MAPK pathway.

(A) Strains MI200 (WT) and GB3 (pck2Δ) were separately transformed with pREP3X-rho2⁺ plasmid and grown for 18 h in the presence (+B1) or absence (−B1) of thiamine. Activated Pmk1 was detected by immunoblotting with anti-phospho-p42/44 and total Pmk1 with anti-HA antibodies. (B) Strains MI200 (Control), MI700 (rho2Δ), and GB3 (pck2Δ) were separately transformed with pREP3X-rho1+(G15V) plasmid, grown for 18 h with or without thiamine, and both total and activated Pmk1 were detected as above. (C) Strains MI200 (WT), GB3 (pck2Δ), and LS203 (rho1-596 pck2Δ) were grown in YES medium to mid log-phase. Aliquots were harvested and Pmk1-HA6H was purified by affinity chromatography. Pmk1-HA6H was purified and the activated and total Pmk1 was detected as already indicated. *,P<0.05 in mutant strains as compared to the wild type. **,P<0.04 in rho1-596 rho2Δ cells as compared to the rho2Δ mutant. (D) Strains MI200 (WT), LS201 (rho1-596), MI700 (rho2Δ), GB3 (pck2) and LS202 (rho1-596 rho2Δ) were grown in YES medium to mid log-phase. And both activated and total Pmk1 was detected as above. (E) VIC assays for strains MI200 (WT), MI700 (rho2Δ), GB3 (pck2Δ), LS201 (rho1-596), GB29 (rho2Δ pck2Δ), LS202 (rho1-596 rho2Δ), LS203 (rho1-596 pck2Δ), and LS207 (rho1-596 rho2Δ pck2Δ). After growth in YES medium, 10⁴, 10³ or 10² cells were spotted onto YES plates supplemented with 0.5 µg/ml FK506 plus 0.1, 0.2, 0.3, 0.35 or 0.4 M MgCl₂, and incubated for 4 days at 28°C before being photographed.