Absence of Nmd5 does not compromise osmoresistance or signaling fidelity. (A) Tenfold serial dilutions of HOG1⁺ NMD5⁺, hog1Δ NMD5⁺, and HOG1⁺ nmd5Δ cells were spotted onto plates containing YPD medium (Left) or YPD containing 1 M sorbitol (Right) and incubated for 48 h at 25°C. (B) HOG1⁺ NMD5⁺, hog1Δ NMD5⁺, and HOG1⁺ nmd5Δ cells carrying an integrated copy of a FUS1^prom-lacZ reporter were grown to midexponential phase, collected, resuspended in either YPD (−) or YPD plus 1 M sorbitol (+) and, after 5 h, assayed for β-galactosidase activity.

Hog1-GFP-CCAAX^Ras2 is plasma membrane-associated and catalytically functional. (A) Schematic depiction of Hog1-GFP-CCAAX^Ras2 and Ste2 (1–296)-Hog1-GFP. S-palmitoyl and S-farnesyl substituents tethering the Hog1-GFP-CCAAX^Ras2 chimera to the plasma membrane are shown as bold zig-zag lines. (B) Cells expressing Hog1-GFP, Hog1-GFP-CCAAX^Ras2, or Ste2 (1–296)-Hog1-GFP were grown to midexponential phase in YPD and harvested at the indicated times after treatment with 1 M sorbitol. Extracts were immunoblotted with anti-phospho-p38 antibodies to detect the dually phosphorylated form of these Hog1 chimeras and with anti-GFP antibodies to detect the total amount of each chimera. (C) HOG1-GFP or HOG1-GFP-CCAAX^Ras2 cells expressing a histone H2B isoform (Htb2) tagged with mCherry (39) to mark the nucleus were grown to midexponential phase in YPD, treated with 1 M sorbitol for 10 min, and examined by deconvolution microscopy. (D) Samples of the cells shown in C were ruptured, clarified, fractionated, and immunoblotted with anti-GFP antibodies. (E) HOG1⁺, hog1Δ, or HOG1-GFP-CCAAX^Ras2 cells carrying an integrated copy of RCK2-(HA)₃ were grown to midexponential phase in YPD and harvested 10 min after treatment with 1 M sorbitol. Extracts were immunoblotted with an anti-HA epitope mAb or with anti-Pgk1 antibodies as a control for equivalent loading. Row 1, unmodified Rck2; row 2, and row 3, hyperphosphorylated Rck2; asterisk, background band present in all lanes.

Membrane-tethered Hog1 confers osmoresistance and maintains signaling fidelity. (A) Ability of strains of the indicated genotypes to grow on YPD, YPD + 1 M sorbitol, or YPD + 1 M sorbitol containing 12 μM 1-NM-PP1 was assessed as in Fig. 1A. (B) HOG1⁺, hog1Δ, or hog1-as-GFP-CCAAX^Ras2 cells carrying an integrated copy of a FUS1^prom-lacZ reporter were grown to midexponential phase, collected, and resuspended in either YPD (black bars), YPD + 1 M sorbitol (gray bars), or YPD + 1 M sorbitol containing 12 μM 1-NM-PP1 (open bars). After 5 h, samples were assayed for β-galactosidase activity.

Membrane tethering of Hog1 prevents gene induction by hyperosmotic stress. (A) HOG1⁺, hog1Δ, and Hog1-GFP-CCAAX^Ras2-expressing cells were grown to midexponential phase in YPD and harvested at the indicated times after exposure to 1 M sorbitol. mRNA was isolated and resolved by electrophoresis in a denaturing agarose gel, transferred to nylon membranes, and hybridized to biotinylated probes specific for the ALD3, CTT1, GPD1, and STL1 transcripts, which were detected by incubation with infrared dye-tagged streptavidin. A probe specific for the IPP1 transcript served as a control for mRNA recovery and equivalent loading and for normalization to quantify the relative intensities of the ALD3, CTT1, GPD1, and STL1 signals, plotted in B. Diamonds, HOG1⁺; triangles, hog1Δ; squares, HOG1-GFP-CCAAX^Ras2.

Transcriptional induction of GPD2 does not occur in cells expressing membrane-tethered Hog1. Ability of HOG1⁺ gpd1Δ and HOG1-GFP-CCAAX^Ras2 gpd1Δ cells to grow at 30°C on YPD, or YPD plus 1 M sorbitol, was assessed as in Fig. 1A.