Figure 4.

Hog1 is slightly activated by zymolyase and the presence of an active form of this MAPK is necessary for the induction of CRH1. (A) Effect of zymolyase in Hog1 activation. Exponentially growing WT cells were collected before and after different intervals of zymolyase treatment, and Hog1 activation was examined by immunoblotting total extracts with an anti-phospho-p38 antibody. The levels of phosphorylation of Hog1 by 0.4 M NaCl are also shown. The phosphorylation of Hog1 by zymolyase was dependent on the presence of the MAPKK Pbs2. (B) Hog1 is activated by zymolyase in a slt2Δ strain. Exponentially growing WT and slt2Δ cells were collected before and after different intervals of zymolyase treatment and Hog1 activation was examined. (C) Hog1 is not translocated to the nucleus after zymolyase-induced stress. A hog1Δ strain was transformed with a GFP-tagged Hog1 (pRS-HOG1-GFP). Cells were grown exponentially, exposed (Zym +) or not (Zym −) to 0.4 U/ml zymolyase 100T at the indicated times and visualized by fluorescence microscopy. (D) Congo Red does not activate the MAPK Hog1 in a WT strain. Exponentially growing WT cells were collected before and after different intervals of Congo Red treatment at 30 μg/ml, and Hog1 activation was examined. (E) CRH1 mRNA levels (represented as the ratio of treated vs. untreated cells) were analyzed by Q-RT-PCR in hog1Δ cells transformed with pRS416 (empty vector), the WT HOG1 allele (pRS416-HOG1), or inactive mutant alleles of hog1 (hog1 TA-174/YA-176 and hog1 KM-78) after 2 h of zymolyase treatment.