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Figure S1. Activation of the HOG pathway is not induced by altered membrane composition or by proteolytic degradation. (A) Alternations in membrane sterol composition do not affect regulation of the HOG pathway. Treatment by nystatin could have potential side effects on yeast physiology, such as alternation of membrane sterol composition. In order to find out if the HOG pathway regulation depends on membrane sterols, we analyzed the phosphorylation of Hog1 in mutants with defective ergosterol biosynthesis. The uninduced and induced (by 0.4 M NaCl for 5 min) levels of the Hog1 phosphorylation were determined in mutants with a complete deletion of the ERG6 gene in the wild-type or the ste11 strain background. The ERG6-null mutants are viable, but lack ergosterol. The activation of Hog1 was similar between ERG6 strains and erg6 mutants. These results suggest that HOG pathway stimulation by nystatin is not caused by alternation of membrane sterols. (B and C) Proteases from zymolyase do not induce the HOG pathway. Zymolyase is an enzyme preparation containing enzymes involved in ß-glucans degradation, but may also contain unspecified proteolytic activity (according to manufacturer's data sheet; Seikagaku Co.). We tested if proteolytic activity could have contributed to the Hog1 activation by zymolyase. First, we estimated the overall protease activity of zymolyase preparation by incubation with 1 &microg BSA in a mixture identical to that used for cell wall removal (at a final concentration of 50 mU &microl^-1). After a 30-min incubation, the amount of BSA had not changed, even without protease inhibitors (B), suggesting that the concentration of zymolyase used in our experiments to prepare spheroplast did not have significant proteolytic activity. Next, we asked if activation of the HOG pathway by zymolyase is blocked by protease inhibitors. We determined the Hog1 phosphorylation in wild-type, ste11, and ssk2 ssk22 strain after cell wall removal in the absence or the presence of a mixture of protease inhibitors (complete; Boehringer). The levels of Hog1 phosphorylation were indistinguishable between these samples (C). These data suggest that HOG pathway activation is induced specifically by removal of the cell wall, and not by proteolytic activity of zymolyase. The efficiency of protoplast preparations was similar between samples treated by zymolyase with or without protease inhibitors.