Rapid deacetylation of yeast Hsp70 mediates the cellular response to heat stress

Abstract

Hsp70 is a highly conserved molecular chaperone critical for the folding of new and denatured proteins. While traditional models state that cells respond to stress by upregulating inducible HSPs, this response is relatively slow and is limited by transcriptional and translational machinery. Recent studies have identified a number of post-translational modifications (PTMs) on Hsp70 that act to fine-tune its function. We utilized mass spectrometry to determine whether yeast Hsp70 (Ssa1) is differentially modified upon heat shock. We uncovered four lysine residues on Ssa1, K86, K185, K354 and K562 that are deacetylated in response to heat shock. Mutation of these sites cause a substantial remodeling of the Hsp70 interaction network of co-chaperone partners and client proteins while preserving essential chaperone function. Acetylation/deacetylation at these residues alter expression of other heat-shock induced chaperones as well as directly influencing Hsf1 activity. Taken together our data suggest that cells may have the ability to respond to heat stress quickly though Hsp70 deacetylation, followed by a slower, more traditional transcriptional response.

Figure 1

Heat shock alters acetylation of Ssa1. (A) Domain structure of Ssa1. All lysine residues that were found to be deacetylated upon heat shock as deretmined by mass spectrometry are indicated. (B,C) Cartoon representation of Hsp70 in the ADP-bound open conformation (PDB: 2KHO) and in the ATP-bound closed conformation (PDB: 4JNE) showing the NBD (green), SBD (blue) and CTD lid (yellow). The four deacetylated residues are highlighted in red. (D) Conservation of the deacetylated residues in Hsp70. amino acid sequences of Saccharomyces cerevisiae Hsp70 isoforms Ssa1, Ssa2, Ssa3 and Ssa4 as well as sequences for human Hsc70 and Hsp70 were aligned using Snapgene. Amino acids identified as becoming deacetylated upon heat shock are annotated with a red dot. Raw mass spectrometry data are available via ProteomeXchange with identifier PXD015185.

Figure 2

Ssa1 deacetylation alters yeast thermotolerance. (A) Yeast expressing acetylation site mutations were grown to exponential phase, serially diluted fivefold and plated onto YPD plates. Plates were photographed after 3 days incubation at the indicated temperatures. (B) Assessment of acetylation site mutant recovery after acute heat shock. Fresh cultures were heat shocked at 47 °C for the indicated times and then were serially diluted fivefold and plated onto YPD plates. Plates were photographed after 3 days incubation at 30 °C. (C) WT, 4Q and 4R cells were inoculated at an OD₆₀₀ of 0.1 into 96 well format and were shaken in a plate reader at 30 °C. OD₆₀₀ was measured at 1 h intervals. (D) Assessment of [PSI⁺] propagation. Single colonies of cells expressing WT, 4Q and 4R Ssa1 were streaked on YPD and −ADE plates which were then incubated at RT for 5–7 days. [psi⁻] cells were red colonies on YPD and unable to grow −ADE plates; [PSI⁺] cells were white colonies on YPD and viable on −ADE plates; [psi⁻] cells were attained by streaking [PSI⁺] colonies on YPD plates containing 3 mM GdnHCl and incubating for 2–3 days. The curing of [psi⁻] cells was repeated at least twice to obtain a stable [psi⁻] heritage. (E) Growth assay of acetylation site mutants for [PSI⁺] and [psi⁻] cells. Yeast were grown and treated as in (A). (F) Response of acetylation site mutants to stresses that perturb DNA integrity or the yeast cell wall. Cells grown as in (A) were five-fold serially diluted and plated onto media containing the indicated stressors.

Figure 3

Effects of Ssa1 deacetylation on in vivo function. (A) Ability of Ssa1 (WT, 4Q and 4R) to refold Luciferase over a 60 min time course. Activity relative to fully active luciferase was calculated and data shown are the average and SD of three independent experiments. (B) Effects of acetylation of Ssa1 on HSF1 activation. A plasmid containing the HSE-lacZ reporter gene was transformed into G402 cells. Cells were cultured at 30 °C or heat shock at 39 °C for 2 h. The activation of Hsf1 was calculated by measuring the expression of β-galactosidase under Heat Shock Element (HSE) control. Data are the average and SD of three independent experiments. *Represents p < 0.05. (C) Acquired thermotolerance assay of acetylation site mutants. Fresh cultures were pre-treated at 39 °C for 1 h, then cells were heat shocked at 47 °C for the indicated times and then plated on media either containing or lacking 3 mM Gdn-HCl (an inhibitor of Hsp104). (D) Steady state levels of major co-chaperone proteins in acetylation site mutants. WT, 4Q and 4R cells were grown to exponential phase and were either incubated at 30 °C or 39 °C for 2 hours. Cell extracts were obtained, resolved on SDS-PAGE gels and analyzed by immunoblotting with anti-Hsp104, Ssa1, Ydj1 and Hsp26 antibodies. GAPDH was used as a loading control. (E) Quantitation of major co-chaperone in acetylation site mutants. Data shown are the average and SD of three independent experiments.

Figure 4

Acetylation alters the interactome of Ssa1. (A) Scheme for proteomic analysis. Cells expressing 4Q or 4R mutant His₆-Ssa1 were grown to exponential phase, whereupon Ssa1 complexes were affinity purified and digested with trypsin. Peptides from 4Q interactors were isotopically labeled with ¹⁸O, mixed 1:1 with 4R interactor peptides, and analyzed by quantitative LC-MS/MS. (B) Functional classification of the Ssa1 interactome. Ssa1 interactors were categorized by cellular function using Gene Ontology (GO) Slim analysis and plotted against relative affinity for 4Q vs 4R Ssa1. The dotted lines represent a ratio of two-fold. Interactors are colored by relative selectivity to 4Q or 4R Ssa1 as follows: red (significant selectivity for 4Q), green (significant selectivity for 4R), black (equal binding to 4Q and 4R). (C) Venn diagram of Ssa1 interactors observed in 4Q and 4R interactomes after applying statistical filters. (D) Gene Ontology (GO) term analysis of 4Q and 4R interactomes. Interactors were categorized by cellular function using GO Slim analysis and relative enrichment compared to occurrence in the non-essential genome was calculated. The top 10 enriched cellular processes and function are shown for both 4Q and 4R interactomes. (E) Analysis of chaperone/co-chaperone interactions of 4Q and 4R Ssa1. A network of the 15 co-chaperones and chaperones that were detected by mass spectrometry as interactors of 4Q and 4R Ssa1 was generated using Cytoscape. The nodes were colored based on relative binding preference for 4Q and 4R as follows: red (selectivity for 4Q), green (selectivity for 4R) and gray (no preference). Raw mass spectrometry data are available via ProteomeXchange with identifier PXD015185.

Figure 5

Acetylation of Ssa1 alters metabolism. (A) Glycolysis pathway highlighting protein interactors of Ssa1 altered by acetylation (4Q, orange) or deacetylation (4R, in green) from the interactome. (B) Assay of oxygen consumption rate. The oxygen consumption rate (OCR) was measured by a seahorse FXe96. Data shown are the average and SD of six independent replicates. *Represents p < 0.01.