PMC

Swe1 mediates the G₂ arrest caused by Hog1 activation. (A) Deletion of HOG1 or SWE1 abolishes cell cycle arrest at G₂ caused by Sln1 inactivation. sln1^ts4, sln1^ts4 hog1Δ, and sln1^ts4 swe1Δ strains were synchronized with α-factor for 3 h, released into fresh media at 25°C and shifted to 37°C after 50 min in YPD (time 0). Total DNA content was analysed as in . (B) Wild-type, YPC89 (hog1Δ) and YPC166 (swe1Δ) cells containing an empty vector or P_GAL1-Pbs2^DD were grown for 7 h in SD-Ura plus galactose and visualized by differential interference contrast.

Localization of Hsl7 is affected by activation of the HOG pathway. (A) Localization of Hsl7 but not Cdc11 or Hsl1 is affected by osmostress. Wild-type, hog1Δ or hsl1Δ cells containing chromosomal GFP-tagged Cdc11, Hsl1 or Hsl7 were grown in YPD and subjected (NaCl) or not (control) to a brief osmostress (10 min at 0.4 M NaCl). GFP proteins were visualized by direct fluorescence. Numbers represent percentage of cells with GFP fluorescence at the bud neck and is the result of the measurement in triplicate of 200 cells each. (B) Time course of Hsl7GFP localization upon osmostress. Wild-type cells containing Hsl7GFP were subjected to 0.4 M NaCl at the indicated times. Hog1 phosphorylation was followed using specific antibodies against phosphorylated p38 in whole-cell extracts. Numbers represent percentage of cells with GFP fluorescence at the bud neck and is the result of the measurement in triplicate of 200 cells each. (C) Localization of Hsl7 is affected by Hog1 activation. Wild-type cells expressing P_GAL1-Pbs2^DD or a control vector and GFP-tagged Cdc11, Hsl1 or Hsl7 were grown in minimal media containing galactose. Numbers represent percentage of cells with GFP fluorescence at the bud neck and is the result of the measurement in triplicate of 200 cells each.

Activation of Hog1 causes a decrease on Clb2 levels and Clb2–Cdc28 activity. (A) The levels of Clb2 protein decreases upon Hog1 activation. Wild-type and YPC89 (hog1Δ) cells containing P_GAL1-Pbs2^DD were transformed with a plasmid expressing HA-tagged Clb2 under its own promoter. Cells were grown in raffinose and then galactose was added (time 0). Cells were collected at the indicated times and Clb2 was visualized by immunoblotting with monoclonal antibody 12CA5 to HA. Wild-type cells without the Clb2-HA plasmid are shown as negative control (−). (B) Hog1 activation leads to a reduction on the relative levels of Clb2–Cdc28 activity. Cells as in (A); wild type (•) and hog1Δ (▾) were grown in the presence of galactose and collected at the indicated time points. Protein extracts were prepared and Clb2–Cdc28 kinase activity was assessed by an in vitro kinase assay of immunoprecipitated Clb2–Cdc28 using Histone H1 (HH1) as a substrate. Kinase activity was normalized to that of wild type, time 0. Data±s.d. from three independent experiments are shown.

Activation of the HOG pathway results in G₁ and G₂ cell cycle arrest. (A) Cells arrest at G₁ in response to Sln1 inactivation. The sln1^ts4 or wild-type strains were synchronized with α-factor for 2 h, shifted to 37°C for 1 h and then released into YPD medium at 37°C. Total DNA content was assessed by FACS analysis and presented as cell counts (y-axis) versus 1C and 2C DNA content (x-axis). The percentage of budding cells is depicted on the right-hand side of each graph. (B) Cells arrest at G₂ in response to Sln1 inactivation. The sln1^ts4 or wild-type strains were synchronized with α-factor for 3 h, released into fresh media at 25°C and shifted to 37°C after 50 min in YPD (time 0). Total DNA content was analysed as in (A). (C) Cells arrest at G₁ in the presence of osmostress. Wild-type cells were synchronized in G₁ phase with α-factor and released into YPD medium containing 0.4 M NaCl. Total DNA content was assessed as in (A). (D) Cells arrest at G₂ in the presence of osmostress. Wild-type cells were synchronized with α-factor and released into YPD medium. After 50 min, NaCl was added (0.4 M) (time 0). Total DNA content was assessed as in (A).

Hog1 phosphorylates Ser1220 within the Hsl7-binding site in Hsl1. (A) Hog1 interacts with Hsl1 in vivo. Yeast extracts containing GST or GST-Hog1 and untagged or tagged HA-Hsl1 (from its chromosomal locus) were precipitated using glutathione beads and precipitated Hsl1 was probed using anti-HA antibodies. GST proteins were detected using anti-GST antibodies. (B) Hsl1 is phosphorylated upon osmostress in a HOG1-dependent manner. hsl1 cells or hog1 cells were transformed with a plasmid expressing a catalytically inactive Hsl1, contains the K110A mutation (HSL1) or in addition the S1220A mutation (hsl1^SA) under the GAL1 promoter. Cells were synchronized with pheromone treatment in the presence of galactose and 50 min after release, cells were subjected (+) to a brief osmotic shock (0.4 M NaCl, 10 min). Extracts were treated (+) with alkaline phosphatase (AP). The effect of AP was prevented by the addition of phosphatase inhibitors (not shown). The presence of Hsl1 and Cdc28 was probed. (C) Hog1 directly phosphorylates the C-terminal region of Hsl1 in vitro. Hog1 and the constitutively activated Pbs2 allele (Pbs2^EE) purified from E. coli were incubated in the presence of kinase buffer and ATP. Then, catalytically inactive Hsl1 kinase domain (1–600), an Hsl1 fragment (from aa 600 to 900) or the C-terminal domain of Hsl1 that contains aa 717–1517, purified from E. coli, were added in the presence of radioactive ATP. Phosphorylated proteins were detected by autoradiography (P32) or Coomassie staining. Position of Hsl1 fragments is indicated on the left. (D) Hog1 directly phosphorylates the Ser1220 of Hsl1 in vitro. The wild-type Hsl1 and the Hsl1 S1220A mutant were expressed in E. coli and assayed as in (C). Phosphorylated proteins were detected by autoradiography (P32) or Coomassie staining. Position of Hsl1 proteins is indicated on the left.

Swe1 stabilization is critical for cell survival upon stress. (A) Swe1 phosphorylation is affected by osmostress. Cells expressing epitope-tagged Swe1 from its chromosomal locus were synchronized by pheromone treatment and 50 min after release, cells were subjected (NaCl) or not (control) to 0.4 M NaCl. Swe1 and Cdc28 (loading control) were detected from cell extracts by immunoblotting using specific antibodies. Control strain (without tagged Swe1) was wild type W303 (−). (B) Swe1 accumulates in response to osmostress. Tagged Swe1 was expressed from its chromosomal locus in wild-type cells (YPC130). Cells were synchronized as in (A) and subjected (NaCl) or not (control) to 0.4 M NaCl. Swe1 was detected from yeast extracts using monoclonal anti-myc-specific antibodies. Cdc28 is presented as loading control. Control strain was wild type W303 (−). The percentage of acrylamide on (A) was lower than in (B) to increase the separation between the phosphorylated forms of Swe1. (C) YPC130 strain (which expresses Swe1-myc) containing either control vector (vector) or P_GAL1-Pbs2^DD plasmid were grown in SD-Ura plus raffinose and after addition of galactose cells were collected at the indicated times. The presence of myc-tagged Swe1 was detected as in (C). The asterisk indicates an unspecific protein detected by the myc antibodies that is kept constant along the culture. It is presented as a loading control. (D) Deletion of SWE1 or SIC1 results in osmosensitive cells. The wild-type (W303) strain and its derivatives hog1Δ, sic1Δ, swe1Δ and sic1Δ swe1Δ mutants were spotted on YPD plates or YPD plates containing 0.4 M, 0.8 M or 1 M NaCl. Growth was scored after 3 days at 30°C.

Phosphorylation of Hsl1 Ser1220 by Hog1 determines Hsl7 localization and is essential for G₂ arrest and survival upon stress. (A) Mutation of Hsl1 Ser1220 affects Hsl7 localization. The hsl1Δ HSL7GFP strain transformed with plasmids containing wild-type Hsl1 or the mutated alleles Hsl1 Ser1220A (Hsl1^SA) or Hsl1 Ser1220E (Hsl1^SE) and P_GAL1-Pbs2^DD or a control vector were grown as in . Hsl7GFP was visualized by direct fluorescence. Numbers represent percentage of cells with GFP fluorescence at the bud neck and is the result of the measurement in triplicate of 200 cells each. (B) Cells as in (A) were grown in minimal media and subjected (NaCl) or not (control) to a brief osmostress (10 min, 0.4 M NaCl). Hsl7GFP was visualized as in (A). Numbers represent percentage of cells with GFP fluorescence at the bud neck and is the result of the measurement in triplicate of 200 cells each. (C) The interaction between Hsl7 and Hsl1 decreases upon osmostress depending on Hog1 phosphorylation. Two-hybrid analyses were performed in liquid assays with cells carrying wild-type Hsl1 and mutant Hsl1 (Hsl1^SA or Hsl1^SE) fused to the GAL4 activator domain together with Hsl7 fused to the LexA BD. β-Galactosidase activity was assayed in cells grown to mid-log phase that were subjected (NaCl, filled bars) or not (control, open bars) to hyperosmotic stress (0.4 M NaCl for 60 min). β-Galactosidase activity is given in nmol/min/mg and is the result of the measurement in duplicate of three independent transformants. Error bars indicate standard error of the mean. (D) Accumulation of Swe1 by osmostress depends on phosphorylation of Hsl1 S1220. hsl1 cells carrying HA-tagged wild-type (Hsl1) or the Hsl1S1220A mutant (Hsl1^SA) were grown as in and collected after the indicated time points under osmostress (filled bars). Swe1 was detected by using anti-HA antibodies. Cdc28 was detected by specific antibodies on the same membranes and used as a loading control. Data were quantified by phosphoimager and is the mean of two independent experiments. (E) Cells with the Hsl1^SA mutant fail to arrest at G₂ in response to Sln1 inactivation. Wild-type cells (SLN1 HSL1) or an sln1^ts hsl1 strain containing wild-type HSL1 or Hsl1^SA in a plasmid were synchronized with α-factor for 3 h, released into fresh media at 25°C and shifted to 37°C after 75 min in YPD (time 0). Total DNA content was assessed by FACS analysis and presented as cell counts (y-axis) versus 1C and 2C DNA content (x-axis). The percentage of budding cells is depicted on the right-hand side of each graph. (F) Cells containing an unphosphorylatable allele of Hsl1 become osmosensitive. hsl1Δ cells as in (E) were spotted on YPD plates containing NaCl or Sorbitol. Growth was scored after 3 days.