1. Biochemistry and Chemical Biology
  2. Cell Biology
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Synergy between the small intrinsically disordered protein Hsp12 and trehalose sustain viability after severe desiccation

  1. Skylar Xantus Kim
  2. Gamze Çamdere
  3. Xuchen Hu
  4. Douglas Koshland  Is a corresponding author
  5. Hugo Tapia  Is a corresponding author
  1. University of California, Berkeley, United States
Research Article
Cite this article as: eLife 2018;7:e38337 doi: 10.7554/eLife.38337
5 figures and 4 additional files

Figures

Figure 1 with 1 supplement
Trehalose and Hsp12 are necessary and sufficient for desiccation tolerance.

(A) Yeast cells were grown to saturation (5 days), air-dried for 2, 15, or 30 days at 23°C, 60% relative humidity (RH), then rehydrated and assessed for viability by counting colony forming units (CFU). Desiccation tolerance of wild type, tps1∆, hsp12∆ and tps1∆hsp12∆ cells. (B) Yeast cells (nth1∆) were grown to mid-exponential phase (OD <0.5) in minimal media lacking histidine. Cells were then transferred to either minimal media lacking histidine (SC-His, 0% trehalose) or minimal media lacking histidine with trehalose (SC-His, 2% trehalose) for 1 hr. Cells were collected, washed, and air dried for 2 d at 23°C, 60% relative humidity (RH), then rehydrated and assessed for viability by counting colony forming units (CFU). Yeast cells are ± Trehalose transporter (AGT1) and ± Hps12 (p423-GPD-Hsp12).

https://doi.org/10.7554/eLife.38337.002
Figure 1—figure supplement 1
Trehalose and Hsp12 heat tolerance.

(A) Cells (wild type, hsp12∆, tps1∆, or tps1∆ hsp12∆) were grown to midexponential phase (OD <0.5) in non-selective media (YP, 1% yeast extract and 2% peptone) containing 2% glucose at 30C. Cells were plated and grown at either 30 or 37°C, or grown for one hour at 34°C (pre-heat shock) before growing at either 30 or 37°C. (B) Cells (wild type, hsp12∆, tps1∆, or tps1∆ hsp12∆) were grown to midexponential phase (OD <0.5) in non-selective media (YP, 1% yeast extract and 2% peptone) containing 2% glucose at 30°C. Cells were then heat-shocked for 30 min at temperatures ranging from 42–60°C, followed by plating and growing at 30°C. Cells were also grown at 34°C prior to heat shock.

https://doi.org/10.7554/eLife.38337.003
Figure 2 with 1 supplement
Hsp12 proteostasis activities can act synergistically with, or independent of trehalose.

(A–B) (A) Enzymatic activity after drying of CS in presence of varying concentrations of Hsp12. CS enzymatic activity was measured by examining activity-induced changes in absorbance at 412 nm after drying compared to measurements before drying. (B) Citrate synthase desiccation-induced aggregation in the presence of varying concentrations of Hsp12. Absorbance at 340 nm. Citrate Synthase (0.12 mg). Absorbance measured after drying at 36°C for 24 hr followed by 5 hr at 23°C, then rehydration in water. Measurements after drying compared to measurements before drying. (C–D) (C) Enzymatic activity after drying of CS in presence of varying concentrations of trehalose. CS enzymatic was measured by examining activity-induced changes in absorbance at 412 nm after drying compared to measurements before drying. (D) Citrate synthase desiccation-induced aggregation in the presence of varying concentrations of trehalose. Absorbance at 340 nm. Citrate Synthase (0.12 mg). Absorbance measured after drying at 36°C for 24 hr followed by 5 hr at 23°C, then rehydration in water. Measurements after drying compared to measurements before drying.

https://doi.org/10.7554/eLife.38337.005
Figure 2—figure supplement 1
Synergistic protection by trehalose and Hsp12

(A–B) Luciferase activity and aggregation as a reading of proteostasis of logarithmically growing and desiccated cells. (A) Yeast strains (nth1∆, +AGT1, ±Hps12) were transformed with a temperature-sensitive firefly luciferase-fusion protein expressed from the constitutive (GPD) promoter (p426-GPD-FFL) or empty vector (pEV, p426-GPD). These strains were grown to mid-log phase in media lacking uracil or media lacking uracil with trehalose (YPD, 2% trehalose) for 1 hr. Desiccated samples were air-dried for 2 days followed by rehydration in YPD or SC-HIS + cycloheximide (10 μl/ml, to block new FFL protein synthesis). Luciferase activity was measured in vivo by addition of 0.5 mM D-Luciferin to equal number of intact cells. Light emission was measured immediately with a luminometer. Percentage of activity was normalized to WT saturated ‘wet’ sample. (B) Cells were prepared as in A. Total cellular protein was extracted, and aggregated proteins were sedimented by high-speed centrifugation. The amounts of luciferase in the total protein fraction (total) and in high-speed supernatants (soluble) were determined by SDS-PAGE followed by immunoblotting with antiserum recognizing luciferase. (C–D) Citrate synthase desiccation-induced activity and aggregation in the presence of constant concentrations of trehalose and varying concentrations of Hsp12. Absorbance at 340 nm. Citrate Synthase (0.12 mg) and trehalose (33 μg/ml). (B) CS aggregation was measured by examining absorbance measured after drying at 36°C for 24 hr followed by 5 hr at 23°C, then rehydration in water. Measurements after drying compared to measurements before drying.

https://doi.org/10.7554/eLife.38337.006
Hsp12 and trehalose can act synergistically in membrane proteostasis.

(A–B) Prion propagation as a measure of in vivo protein propagation after desiccation. (A) [PSI+] prion propagation before, and after desiccation. To assess [PSI+] prion state, cells were plated on media lacking adenine (SC-ADE) compared to non-dried controls. (B) [GAR+] prion propagation before, and after desiccation. To assess [GAR+] prion state, cells were plated on YP media with 2% glycerol and 0.05% glucosamine compared to non-dried controls.

https://doi.org/10.7554/eLife.38337.008
Hsp12 causes membrane remodeling.

(A) DMPG liposomes (5 mg/ml) where incubated for 1 hr at room temperature in the presence or absence of Hsp12 (25 μg/ml) and/or in the presence or absence of trehalose (2% final). Pellet (p) and supernatant (s) fractions where separated by high-speed centrifugation, and lipid distribution was assessed by SDS-PAGE followed by altered Coomassie staining (10% acetic acid only). (B) DMPG liposomes (5 mg/ml) where incubated for 1 hr at room temperature in the presence or absence of Hsp12 (1.5–5 μg/ml). Pellet (p) and supernatant (s) fractions where separated by high-speed centrifugation, lipid distribution was assessed by SDS-PAGE followed by altered Coomassie staining (10% acetic acid only). (C) Samples for electron microscopy were prepared the same as in A, with Hsp12 at 2.5 μg/ml. Images where taken from samples prior to centrifugation. Samples where spread on glow-discharged EM grids and stained using 2% uranyl acetate.

https://doi.org/10.7554/eLife.38337.010
Figure 5 with 1 supplement
Desiccation protection not a common hydrophilin feature.

(A) Yeast cells were grown to saturation (5 days), air-dried for 2 days at 23°C, 60% relative humidity (RH), then rehydrated and assessed for viability by counting colony forming units (CFU). Desiccation tolerance of wild type vs. 8X∆: hsp12∆ gre1∆ sip18∆ stf2∆ nop6∆ ybr016w∆ yjl144w∆ ynl190w∆ cells. (B) Exponential Phase. Yeast cells (nth1∆, AGT1+) were grown to mid-exponential phase (OD <0.5) in rich media (YPD). Cells were then transferred to either rich media (YPD, 0% trehalose) or rich media with trehalose (YPD, 2% trehalose) for 1 hr. Cells were collected, washed, and air dried for 2 d at 23°C, 60% relative humidity (RH), then rehydrated and assessed for viability by counting colony forming units (CFU). Stationary Phase. Yeast cells are ± AGT1 (trehalose transporter) and ± Stf2 (over-expression from 2μ plasmid, GPD promoter). Yeast cells were grown to saturation (5 days), air-dried for 2 days at 23°C, 60% relative humidity (RH), then rehydrated and assessed for viability by counting colony forming units (CFU). Desiccation tolerance of wild type, tps1∆, stf2∆ and tps1∆stf2∆ cells. (C) Circular dichroism spectroscopy performed on 0.32 mg/ml Hsp12 or 0.32 mg/ml of Stf2 in the presence or absence of 1.2 mg/ml DMPG small unilamellar vesicles (SUVs), measuring from 250 to 190 nm. Units converted to Mean Molar Residue Ellipticity, accounting for concentration and protein size. (D) DMPG liposomes (5 mg/ml) where incubated for 1 hr at room temperature in the presence or absence of Stf2 (1.5–5 μg/ml). Pellet (p) and supernatant (s) fractions where separated by high-speed centrifugation, lipid distribution was assessed by SDS-PAGE followed by altered Coomassie staining (10% acetic acid only). (D) Samples for electron microscopy where the same as in A, with Stf2 at 5 μg/ml. Images where taken from samples prior to centrifugation. Samples where spread on glow-discharged EM grids and stained using 2% uranyl acetate.

https://doi.org/10.7554/eLife.38337.013
Figure 5—figure supplement 1
Hsp12 and Stf2 are expressed to similar levels in growing cells.

(A) Yeast cells (nth1∆) were grown to mid-exponential phase (OD <0.5) in minimal media lacking histidine. Cells were collected, washed, and air dried for 2 d at 23°C, 60% relative humidity (RH), then rehydrated and assessed for viability by counting colony forming units (CFU). Yeast cells expressing empty vector EV, p3V5-Hsp12, pHsp12-3V5, p3V5-Stf2 or pStf2-3V5. (B) Protein was extracted from samples from (A), hydrophilin levels were determined by SDS-PAGE followed by immunoblotting with antiserum recognizing 3V5.

https://doi.org/10.7554/eLife.38337.014

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Additional files

Supplementary file 1

Hydrophilin protein purifaction.

Hsp12 and Stf2 are bacterial derived protein preps. Gel stained with Coomassie Blue demonstrates purity of protein preps.

https://doi.org/10.7554/eLife.38337.018
Supplementary file 2

Synthetic Genetic Array Desiccation Screen – tps1∆.

(A) Three different groups where identified from our SGA - tps1∆ screen. (1) gene deletions that were synthetic lethal with tps1∆: failed to grow completely. (2) gene deletions that lead to desiccation sensitivity with tps1∆, and (3) gene deletions that allowed tps1∆ to grow after 30 days of drying: suppressors. List of genes attached as 1–3. (B) Breakdown of desiccation sensitive tps1Δ double mutants into categories based on cellular function. Each desiccation sensitive double mutant was placed into a category based on their cellular function (GO).

https://doi.org/10.7554/eLife.38337.019
Supplementary file 3

Strain Table.

Strains used in this study.

https://doi.org/10.7554/eLife.38337.020
Transparent reporting form
https://doi.org/10.7554/eLife.38337.021

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