Figure 1.
Hog1 is activated in response to AA through a lateral intracellular pathway. (A) HOG pathway from the plasma membrane to Hog1. (B) Bypass of the upper components of the HOG pathway for Hog1 activation by AA. WT cells (DL3187), a sho1Δ ssk1Δ mutant (DL4306) transformed with a plasmid-expressing Ste11ΔN (p1230), and an ssk2Δ ssk22Δ ste11Δ mutant (DL2344) transformed with a plasmid expressing Pbs2-DD (p3370) were treated with AA (100 mM; pH 4.5). Molecular mass markers (in kDa) are on the right. (C) Basal phosphorylation of Hog1 by Pbs2 is required for Hog1 activation by AA. A sho1Δ ssk1Δ mutant (DL4306) transformed with a plasmid-expressing Pbs2-DD (p3675) or empty vector (pRS316) was treated as above. (D) A constitutive form of Pbs2 (PBS2-DD) is as effective as WT for Hog1 activation by AA treatment. A pbs2Δ mutant (DL4278) transformed with a plasmid expressing Pbs2 (p3671) or Pbs2-DD (p3675) was treated as above. Extracts were subjected to SDS-PAGE and immunoblot analysis for detection of total Hog1 and phosphorylated Hog1 (P-Hog1). (E) AA treatment does not induce phosphorylation of Pbs2. WT cells transformed with a plasmid expressing Pbs2-HA (p3671) were preadapted to growth at pH 4.5 and treated with AA (100 mM, 10 min), NaCl (200 mM, 3 min), or untreated (Con). Extracts were subjected to SDS-PAGE and immunoblot analysis for detection of total Hog1, phosphorylated Hog1 (P-Hog1), total Pbs2 (Pbs2-HA), and phosphorylated Pbs2 (P-T518-Pbs2). Molecular mass markers (in kDa) are on the right. (F) Hog1 activation by AA is retained in the absence of protein phosphatases that negatively regulate Hog1. WT cells (DL3187) or a ptp2Δ ptp3Δ ptc1Δ mutant (DL4309) were treated with AA and processed as above. Source data are available for this figure: SourceData F1. Refer to the image caption for details.

Hog1 is activated in response to AA through a lateral intracellular pathway. (A) HOG pathway from the plasma membrane to Hog1. (B) Bypass of the upper components of the HOG pathway for Hog1 activation by AA. WT cells (DL3187), a sho1Δ ssk1Δ mutant (DL4306) transformed with a plasmid-expressing Ste11ΔN (p1230), and an ssk2Δ ssk22Δ ste11Δ mutant (DL2344) transformed with a plasmid expressing Pbs2-DD (p3370) were treated with AA (100 mM; pH 4.5). Molecular mass markers (in kDa) are on the right. (C) Basal phosphorylation of Hog1 by Pbs2 is required for Hog1 activation by AA. A sho1Δ ssk1Δ mutant (DL4306) transformed with a plasmid-expressing Pbs2-DD (p3675) or empty vector (pRS316) was treated as above. (D) A constitutive form of Pbs2 (PBS2-DD) is as effective as WT for Hog1 activation by AA treatment. A pbs2Δ mutant (DL4278) transformed with a plasmid expressing Pbs2 (p3671) or Pbs2-DD (p3675) was treated as above. Extracts were subjected to SDS-PAGE and immunoblot analysis for detection of total Hog1 and phosphorylated Hog1 (P-Hog1). (E) AA treatment does not induce phosphorylation of Pbs2. WT cells transformed with a plasmid expressing Pbs2-HA (p3671) were preadapted to growth at pH 4.5 and treated with AA (100 mM, 10 min), NaCl (200 mM, 3 min), or untreated (Con). Extracts were subjected to SDS-PAGE and immunoblot analysis for detection of total Hog1, phosphorylated Hog1 (P-Hog1), total Pbs2 (Pbs2-HA), and phosphorylated Pbs2 (P-T518-Pbs2). Molecular mass markers (in kDa) are on the right. (F) Hog1 activation by AA is retained in the absence of protein phosphatases that negatively regulate Hog1. WT cells (DL3187) or a ptp2Δ ptp3Δ ptc1Δ mutant (DL4309) were treated with AA and processed as above. Source data are available for this figure: SourceData F1.

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