Figure 2.

Residues 201–300 in the noncatalytic domain of Wis1 MAPKK are essential for Spc1 MAPK activation. (A) Overexpression of Wis1ΔN100 and Wis1ΔN200, but not Wis1ΔN300, induces Spc1 activation. A Δwis1 strain carrying chromosomal spc1⁺ tagged with the HA6H sequence (CA795) was transformed with pREP1 (vector), pREP1-wis1:HA6H, pREP1-wis1ΔN100:HA6H, pREP1-wis1ΔN200:HA6H, pREP1-wis1ΔN300:HA6H, or pREP1-wis1DDΔN300:HA6H. Transformants were grown to mid-log phase at 30°C for 18 h in EMM2 medium without thiamine. Spc1HA6H and various Wis1HA6H proteins were purified by Ni²⁺-nitrilotriacetic acid chromatography, followed by immunoblotting with anti-phospho-p38 MAPK to detect phosphorylated Spc1 as well as with anti-HA antibodies. (B) Wis1ΔN200, but not Wis1ΔN300, can mediate stress-induced Spc1 activation. Wild-type (KS1376) cells (wt) transformed with the pREP1 vector or Δwis1 (CA795) cells transformed with the plasmids described in A were cultured to mid-log phase at 30°C for 18 h in EMM2 medium supplemented with appropriate amounts of thiamine (see MATERIALS AND METHODS). The transformants were exposed to 0.6 M KCl and activation of Spc1 was analyzed.