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- Fig. 1.
Intracellular localization of CbHog1 under conditions of high temperature. (A) Microscopic images of the Cbhog1Δ strain expressing CbHog1-Venus under conditions of normal and high temperature. (B) Quantification of the number of Venus dots per cell in the Cbhog1Δ strain expressing CbHog1-Venus; the same strain was used for the microscopic imaging analysis shown in A. (C) Intracellular dot structures of three yeast species (P. pastoris, S. cerevisiae, S. pombe) expressing fluorescence-tagged Hog1 fusion proteins under high-salt or high-temperature stress. The Pphog1Δ strain expressing PpHog1-YFP, the Spsty1Δ strain expressing SpSty1-GFP, and the Schog1Δ strain expressing ScHog1-GFP were analyzed by fluorescence microscopy. Cells were grown to early log phase and then subjected to high-salt stress (0.5 M NaCl, 5 min), or high-temperature stress (42°C, 30 min). (D) Microscopic images of the Cbhog1Δ strain expressing CbHog1-Venus after removal of high-temperature stress. Cells grown to early log phase in SD medium at 28°C were subjected to high-temperature stress (39°C, 30 min), and then the intracellular dynamics of CbHog1-Venus was observed by fluorescence microscopy during recovery at 28°C for 60 min. (E) Quantification of the number of Venus dots per cell in the Cbhog1Δ strain expressing CbHog1-Venus; the same strain was used for the microscopic imaging analysis shown in D. Each sample contained a minimum of 50 cells. Error bars show ±s.d. of all cells. Scale bars: 2 μm.
- Fig. 2.
CbHog1 is localized to SGs under high-temperature stress. (A) Microscopic images of the C. boidinii strain expressing two SG marker proteins, CbPbp1-mCherry and CbPab1-Venus. Cells were grown to early log phase, and then subjected to high-temperature stress at 42°C for 30 min. Merged images were generated by combining the mCherry and Venus fluorescence images. Arrowheads indicate representative dots in which CbPbp1-mCherry colocalized with CbPab1-Venus. (B) Microscopic images of the C. boidinii strain expressing CbHog1-mCherry and CbPab1-Venus. Cells were grown to early log phase, and then subjected to indicated high-temperature stress for 30 min. Merged images were generated by combining the mCherry (red) and Venus (green) fluorescence images. Arrowheads indicate representative dots in which CbHog1-mCherry colocalized with CbPab1-Venus. (C) Intracellular localization of CbHog1-mCherry and CbPab1-Venus derived from quantification of 50 cells after high temperature stress for 30 min. Errors bars represent +s.d. of the triplicate measurements. (D) Microscopic images of the Cbhog1Δstrain expressing CbPab1-Venus. Cells were grown to early log phase, and subjected to indicated high-temperature stress for 30 min. (E) Microscopic images of the Cbhog1Δ strain expressing CbHog1(K50R)-Venus. Cells were grown to early log phase, and then subjected to high-temperature stress at 42°C for 30 min. (F) Quantification of the number of Venus dots per cell in the Cbhog1Δ strain expressing CbHog1(K50R)-Venus; the same strain was used for the microscopic imaging analysis shown in E. Each sample contained a minimum of 50 cells. Error bars show +s.d. of all cells. (G) Microscopic images of the C. boidinii strain expressing CbHog1(K50R)-mCherry and CbPab1-Venus. Cells were grown to early log phase, and subjected to high-temperature stress at 42°C for 30 min. Merged images were generated by combining the mCherry and Venus fluorescence images. Arrowheads indicate representative dots in which CbHog1(K50R)-mCherry colocalized with CbPab1-Venus. (H) Intracellular localization of CbHog1(K50R)-mCherry and CbPab1-Venus derived from quantification of 50 cells after high temperature stress for 30 min. Error bars represent +s.d. of the triplicate measurements. Scale bars: 2 μm.
- Fig. 3.
The N-terminal region of CbHog1 is necessary and sufficient for dot formation. (A) Schematic diagram of the CbHog1. PPD, putative phosphorylation domain; KD, kinetic domain; CD, catalytic domain; PBD-2, Pbs2-binding domain 2; K50, conserved lysine residue; T172, conserved threonine residue; Y174, conserved tyrosine residue. (B) Microscopic images and numbers of Venus dots per cell in Cbhog1Δ strains expressing CbHog1Δ(1-10)-Venus, CbHog1Δ(1-17)-Venus, CbHog1Δ(1-20)-Venus, CbHog1Δ(1-30)-Venus, CbHog1Δ(1-41)-Venus, CbHog1Δ(348-398)-Venus, CbHog1Δ(318-398)-Venus, or CbHog1Δ(300-398)-Venus. Cells were grown to early log phase in SD medium, and then subjected to high-temperature stress (42°C, 30 min). (C) Microscopic images and the numbers of Venus dots per cell in C. boidinii hog1Δ strains expressing CbHog1(1-20)-Venus, CbHog1Δ(1-30)-Venus, CbHog1Δ(1-40)-Venus, CbHog1(1-50)-Venus, or CbHog1(346-398)-Venus. Cells were grown to early log phase in SD medium, and then subjected to high-temperature stress (42°C, 30 min). N.D., not determined. Error bars show +s.d.
- Fig. 4.
A β-sheet structure in the N-terminal region of CbHog1 contributes to dot formation. (A) Left panel, predicted structure of CbHog1 based on homology modeling (SWISS-MODEL: http://swissmodel.expasy.org/). The β-sheet structure in the N-terminal region (∼50 aa residues) is encircled by a red line. Right panel, each underlined aa residue is predicted to constitute one of the β-sheet strands. (B) Microscopic images of Cbhog1Δ strains expressing CbHog1Δ(25-28)-Venus, CbHog1Δ(35-40)-Venus, CbHog1Δ(38-45)-Venus, CbHog1Δ(39-45)-Venus, CbHog1Δ(40-45)-Venus, or CbHog1Δ(43-45)-Venus. Cells were grown to early log phase in SD medium, and then subjected to high-temperature stress (42°C, 30 min). (C) Quantification of the number of Venus dots per cell in the Cbhog1Δ strain expressing CbHog1-Venus; the same strain was used for the microscopic imaging analysis shown in B. Each sample contained a minimum of 50 cells. Error bars show +s.d. of all cells. Scale bar: 2 μm.
- Fig. 5.
Intracellular localization of ScHog1 in C. boidinii and S. cerevisiae under high-temperature stress. (A) Microscopic images of the Cbhog1Δ strain expressing ScHog1-Venus under the control of the CbACT1 promoter. Cells were grown to early log phase in SD medium, and then subjected to high-temperature stress (42°C, 30 min). (B) Microscopic images of the Cbhog1Δ strain or the Schog1Δ strain expressing ScHog1(1-350). Cells were grown to early log phase in SD medium, and then subjected to high-temperature stress (42°C, 30 min). Scale bars: 2 μm.
- Fig. 6.
Dot formation by CbHog1 under high-temperature stress is important for cell survival. Growth assay of cells grown under conditions of high-salt or high-temperature stress. Cells were grown to early log phase, adjusted to OD610=1, and 3 μl of tenfold serial dilutions were dropped onto YPD plates with or without 0.5 M NaCl. Subsequently, one plate containing 0.5 M NaCl and one without additional NaCl were incubated at 28°C, and one plate without additional NaCl was treated subjected to temperature stress (37°C, 24 h) before incubation at 28°C. Cell growth was scored after 2 days. (A) Growth of the Cbhog1Δ strain expressing CbHog1Δ(38-45)-Venus was compared to that of the Cbhog1Δ strain expressing CbHog1-Venus and Cbhog1Δ strain. (B) Growth of the Cbhog1Δ strain expressing CbHog1Δ(25-28)-Venus or CbHog1Δ(35-40)-Venus was compared to that of the Cbhog1Δ strain expressing CbHog1-Venus and Cbhog1Δ strain. (C) Growth of the Cbhog1Δ strain expressing CbHog1Δ(1-10)-Venus or CbHog1Δ(1-17)-Venus was compared to that of the Cbhog1Δ strain expressing CbHog1-Venus and the Cbhog1Δ strain alone.
- Fig. 7.
Intracellular sequestration of Hog1 suppresses cytotoxicity of ScHog1 under high-temperature stress in C. boidinii. (A) Microscopic images of the Cbhog1Δ strains expressing CbHog1-Venus, ScHog1-Venus, ScHog1(K52R)-Venus, ScHog1(T174A/Y176F)-Venus, and ScHog1-Venus-CAAXRas2. Cells were grown to early log phase in SD medium, and subjected to stress of high temperature (42°C, 30 min) or high osmolarity. (B) Growth assay of cells cultured under high-salt or high-temperature conditions. Cells were grown to early log phase, adjusted to OD610=1, and 3 μl of tenfold serial dilutions were dropped onto YPD plates with or without 0.5 M NaCl. Subsequently, one plate containing 0.5 M NaCl and one without additional NaCl were incubated at 28°C, and one plate without additional NaCl was subjected to high-temperature stress (37°C, 24 h) before incubation at 28°C. Cell growth was scored after 2 days. Growth of Cbhog1Δ strains expressing ScHog1-Venus, ScHog1(K52R)-Venus, ScHog1(T174A/Y176F)-Venus, or ScHogg1-Venus-CAAXRas2 was compared to that of the Cbhog1Δ strain expressing CbHog1-Venus and the Cbhog1Δ strain alone. (C) Growth of the Cbhog1Δ strain expressing ScHog1(1-350) was compared to that of the Cbhog1Δ strain expressing CbHog1-Venus and the Cbhog1Δ strain alone. Growth conditions were the same as in B. Scale bars: 2 μm.
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