A receiver domain (amino acids 177∼240) near the N-terminus of SSK2 is needed for the activation of SSK2 independent of SSK1.

<p>A. Three mutants <i>ssk2Δ^(1–176)</i>, <i>ssk2Δ^(1–240)</i>, <i>ssk2Δ^(177–239)</i> and wild type Ssk2 were expressed in <i>ste11Δssk1Δssk2Δ</i> mutant, and the Hog1p phosphorylation was determined under osmotic stress. B. Three mutants <i>ssk2Δ^(1–176), ssk2Δ^(1–240), ssk2Δ^(177–239)</i> and wild type Ssk2 were expressed in <i>ste11Δssk2Δssk22Δ</i> mutant, and the Hog1p phosphorylation was determined under osmotic stress. C. The osmosensitivity of the mutants of Ssk2p. D. N- terminal portion of Ssk2p (amino acid 177–240) is required for the activation of Ssk2p by the X factor under osmotic stress.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Comparison of protein sequences of Ssk2p and Ssk22p.

<p>The alignment was carried out by software Vector NTI 10.</p

Strains used in this study.

<p>Strains used in this study.</p

Three MAPKKKs involved in HOG pathway have different properties.

<p>A. Hog1p phosphorylation pattern of <i>ssk2Δssk22Δ</i> mutant. B. Hog1p phosphorylation pattern of <i>ssk22Δste11Δ</i> mutant. C. Hog1p phosphorylation pattern of <i>ssk2Δste11Δ</i> mutant. D. Hog1p phosphorylation pattern of <i>ste11Δssk22Δssk2 Δ^(1–240)</i>. E. Scheme of HOG pathway.</p

Hog1p phosphorylation level and growth phenotypes for the wild type (WT) and mutant yeast cells under various osmotic and salt stress conditions.

<p>A. Hog1p MAPK phosphorylation (P-Hog1p) was detected in the <i>ssk1Δste11Δ</i> mutant under hyperosmotic stress. Cells were exposed to different level of osmotic stress induced by sorbitol (concentration shown) in YPD medium for the time indicated. B. Same experiment as in A but for the wild type strain which shows higher sensitivity and a longer duration of the response. C. Hog1p phosphorylation was not detected in the <i>ste11Δssk2Δssk22Δ</i> mutant. D. Hog1p phosphorylation assay under ionic osmotic stress in the <i>ssk1Δste11Δ</i> double mutant. Cells were exposed to a different levels of salt stress induced by NaCl (concentration shown) in YPD medium for the time indicated. E. Same as in D but for the wild type cells. F. The <i>ssk1Δste11Δ</i> mutant exhibited better growth than <i>hog1Δ</i> mutant under osmotic stress. Serial dilutions (from left to right in each panel) of indicated strains were spotted onto YPD and salt plates and growth was scored after 3 days.</p

Ssk2p can be activated independent of Ssk1p under severe osmotic stress.

<p>A. Hog1p was phosphorylated in the <i>ste11Δssk1Δssk22Δ</i> mutant under severe osmotic stress (higher than 0.5 M sorbitol). B. Hog1p could not be phosphorylated in the <i>ste11Δssk1Δssk2Δ</i> mutant under 0.4 M or 1.0 M sorbitol. C. Actin disassembly did not activate the HOG pathway through Ssk2p. Within Lat B treatment, wild type strain and <i>ste11Δssk1Δ</i> mutant did not display activation of Hog1p. D.The effect of Lat B on actin structures in yeast cells. Rd-phalloidin was used to observe the effects of Lat B addition to yeast cells. Both the wild type cells and <i>ste11Δssk1Δ</i> mutant cells were incubated in the absence of Lat B and for 20 min in the presence of 200 mM Lat B. E. The osmosensitivity phenotype of budding yeast HOG pathway mutants. Serial dilutions (from left to right in each panel) of indicated strains were spotted onto YPD and salt plates and growth was scored after 3 days.</p