Global transcript and phenotypic analysis of yeast cells expressing Ssa1, Ssa2, Ssa3 or Ssa4 as sole source of cytosolic Hsp70-Ssa chaperone activity

Abstract

Background: Cytosolic Hsp70 is a ubiquitous molecular chaperone that is involved in responding to a variety of cellular stresses. A major function of Hsp70 is to prevent the aggregation of denatured proteins by binding to exposed hydrophobic regions and preventing the accumulation of amorphous aggregates. To gain further insight into the functional redundancy and specialisation of the highly homologous yeast Hsp70-Ssa family we expressed each of the individual Ssa proteins as the sole source of Hsp70 in the cell and assessed phenotypic differences in prion propagation and stress resistance. Additionally we also analysed the global gene expression patterns in yeast strains expressing individual Ssa proteins, using microarray and RT-qPCR analysis.

Results: We confirm and extend previous studies demonstrating that cells expressing different Hsp70-Ssa isoforms vary in their ability to propagate the yeast [PSI+] prion, with Ssa3 being the most proficient. Of the four Ssa family members the heat inducible isoforms are more proficient in acquiring thermotolerance and we show a greater requirement than was previously thought, for cellular processes in addition to the traditional Hsp104 protein disaggregase machinery, in acquiring such thermotolerance. Cells expressing different Hsp70-Ssa isoforms also display differences in phenotypic response to exposure to cell wall damaging and oxidative stress agents, again with the heat inducible isoforms providing better protection than constitutive isoforms. We assessed global transcriptome profiles for cells expressing individual Hsp70-Ssa isoforms as the sole source of cytosolic Hsp70, and identified a significant difference in cellular gene expression between these strains. Differences in gene expression profiles provide a rationale for some phenotypic differences we observed in this study. We also demonstrate a high degree of correlation between microarray data and RT-qPCR analysis for a selection of genes.

Conclusions: The Hsp70-Ssa family provide both redundant and variant-specific functions within the yeast cell. Yeast cells expressing individual members of the Hsp70-Ssa family as the sole source of Ssa protein display differences in global gene expression profiles. These changes in global gene expression may contribute significantly to the phenotypic differences observed between the Hsp70-Ssa family members.

Figure 1

Expression of individual members of the Ssa family in yeast and effects on [ PSI ⁺ ] phenotype. Each member of the Ssa family was expressed as the sole source of Ssa in the yeast strain G402 by plasmid shuffle technique. Cells were streaked from 5-FOA onto YPD and following 3 days incubation at 30°C colonies were diluted in water and 20 μl spots were placed on -ade plates and incubated at 30°C for 3 days. Colour of the mutant strains ranged from white to pink, reflecting the varying degree of ade 2–1 suppression due to the mutation. The extent of ade 2–1 suppression is also reflected as density of growth on -ade plates. [psi-] variants were produced by curing [PSI ⁺] by streaking on 3 mM Gdn-HCl.

Figure 2

Acquired thermotolerance assays for Ssa1-4. Overnight culture was diluted in fresh YPD medium to an OD_600nm = 0.1 and then the cells were grown to exponential phase to a density of 3 × 10⁶ cells/ml. Cells were then re-suspended in fresh medium to a density of 5 × 10⁶ cells/ml. An aliquot (T_-1) was then shifted to ice. The cultures were then incubated at 39°C for 1 hour to induce Hsp104 expression to protect against heat shock. Cells were then incubated at 47°C for 0, 10, 20, 30 and 40 minutes (T₀-T₄) and plated on YPD and 3 mM Gdn-HCl for comparative growth analysis. Representative spots shown in the figure are a neat concentration from a 1 in 5 serial dilution series. The plates were then incubated at 30°C for 3 days and were monitored for cellular thermotolerance. Cells without pre-treatment show virtually no growth at T₁ (data not shown).

Figure 3

Comparison of luciferase activity of [ PSI ⁺ ] and [ psi ^- ] versions of the Ssa family. Overnight cultures were diluted in fresh SC medium lacking uracil to an OD_600nm = 0.1. The cultures were then shifted to 37°C for 30 minutes to induce the expression of Hsp104. After 30 minutes at 37°C, the cultures were shifted to 45°C for 1 hour. Cyclohexamide was added to the cultures after 50 minutes at 45°C to prevent any further synthesis of luciferase during the recovery period. Luciferase activity, expressed as a percentage of pre-heat shock activity, was measured at regular intervals during the recovery period of 45 minutes at 25°C. White [PSI ⁺], and black [psi ^-]. (Bar indicates SEM. Value followed by * are significantly different at p ≤ 0.05).

Figure 4

Comparative growth analysis of the Ssa1-4 in response to SDS. Overnight culture was diluted in fresh YPD medium to an OD_600nm = 0.1 and then the cells were grown to an exponential phase to a density of 3 × 10⁶ cells/ml. Cells were then re-suspended in fresh medium to a density of 5 × 10⁶ cells/ml and transferred to a microtitre plate. Representative spots shown in the figure are a neat concentration from a 1 in 5 serial dilution series. The plates were incubated for 3 days at 30°C.

Figure 5

Comparative growth analysis of Ssa1-4 in response to H ₂ O ₂ . Overnight culture was diluted in fresh YPD medium to an OD_600nm = 0.1 and then the cells were grown to an exponential phase to a density of 3 × 10⁶ cells/ml. Cells were then re-suspended in fresh medium to a density of 5 × 10⁶ cells/ml and transferred to a microtitre plate. Representative spots shown in the figure are a neat concentration from a 1 in 5 serial dilution series. The plates were incubated for 3 days at 30°C.

Figure 6

Comparative transcriptome profiling of the Ssa family. A Venn diagram representation of genes induced (A) or repressed (B) in different ∆ssa strains. Analysis was performed relative to expression levels of G402 expressing SSA1 as sole source of cytosolic Ssa protein.

Figure 7

Comparative overview of microarray and RT-qPCR analysis. Twenty five genes were identified by microarray technique as being primed by yeast G402 strain carrying either Ssa1, 2, 3 or 4 as sole source of Ssa family protein. Both microarray and RT-qPCR analysis was conducted using total RNA extracted from 5-ml cultures of the [psi ^-] yeast strains carrying Ssa1, 2, 3 or 4 grown overnight at 30°C. The 3D-Gene^TM Yeast Oligo chip S.cerevisiae 6 k used for the microarray analysis according to the manufacturer ’s instruction. Relative mRNA expression levels were quantified relative to that of the housekeeping gene ACT1 (YFL039C) by 2^{^-∆∆Ct} method [43]. Bothe the global normalize value of microarray and the relative mRNA expression levels were LOG-transformed to linearize the data and the heat map of was created using TIGR MultiExperiment Viewer (MeV) [41]. Results are based on two experiments, each with three replicates.