Stress resistance and signal fidelity independent of nuclear MAPK function

Abstract

Elevated external solute stimulates a conserved MAPK cascade that elicits responses that maintain osmotic balance. The yeast high-osmolarity glycerol (HOG) pathway activates Hog1 MAPK (mammalian ortholog p38alpha/SAPKalpha), which enters the nucleus and induces expression of >50 genes, implying that transcriptional up-regulation is necessary to cope with hyperosmotic stress. Contrary to this expectation, we show here that cells lacking the karyopherin required for Hog1 nuclear import or in which Hog1 is anchored at the plasma membrane (or both) can withstand long-term hyperosmotic challenge by ionic and nonionic solutes without exhibiting the normal change in transcriptional program (comparable with hog1Delta cells), as judged by mRNA hybridization and microarray analysis. For such cells to survive hyperosmotic stress, systematic genetic analysis ruled out the need for any Hog1-dependent transcription factor, the Hog1-activated MAPKAP kinases, or ion, glycerol, and water channels. By contrast, enzymes needed for glycerol production were essential for viability. Thus, control of intracellular glycerol formation by Hog1 is critical for maintenance of osmotic balance but not transcriptional induction of any gene.

Fig. 1.

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.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.

Fig. 5.

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.