The HOG components function upstream of Crz1 and suppress Crz1’s nuclear localization.

<p>(<b>A</b>) Cells of the wild-type KN99<b>a</b>, <i>crz1</i>Δ (<b>a</b>), <i>ssk2</i>Δ (<b>a</b>), and <i>pbs2</i>Δ (<b>a</b>) were cultured on YP+GlcN (0.5%) medium for 7 days. (<b>B</b>) Cells of the wild-type H99, the <i>ssk2</i>Δ<i>crz1</i>Δ double mutant, and the <i>pbs2</i>Δ<i>crz1</i>Δ double mutant were cultured on YP+GlcN medium for 7 days. (<b>C</b>) The control strain XW252 (GFP-Nop1, P_<i>CRZ1</i>-Crz1-mCherry) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006982#pgen.1006982.ref079" target="_blank">79</a>] and the corresponding <i>ssk1</i>Δ, <i>ssk2</i>Δ, and <i>pbs2</i>Δ mutants in the XW252 background were cultured in YPD or YP+GlcN medium overnight at 22°C. GFP-Nop1 labels the nucleolus within the nucleus [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006982#pgen.1006982.ref079" target="_blank">79</a>]. (<b>D</b>) The strain JL408 (<i>MAT</i>α, <i>hog</i>1Δ; P_<i>GPD1</i>-mCherry-<i>CRZ1</i>) was generated from a cross between the <i>hog1</i>Δ mutant in the mating type <b>a</b> background and the strain JL410 (<i>MAT</i>α, P_<i>GPD1</i>-mCherry-<i>CRZ1</i>). JL408 was cultured in YP or YP+GlcN medium overnight at 22°C. (<b>E</b>) The wild-type strain H99 was grown to mid-logarithmic phase and then exposed to 1 M NaCl or 2% glucosamine (YPGlcN) for the indicated time points. Hog1 phosphorylation levels were monitored using anti-P-p38 antibody. The blot was stripped and then used for detection of Hog1 protein level with a polyclonal anti-Hog1 antibody as a loading control.