The Skn7 response regulator of Saccharomyces cerevisiae interacts with Hsf1 in vivo and is required for the induction of heat shock genes by oxidative stress - PubMed (original) (raw)

The Skn7 response regulator of Saccharomyces cerevisiae interacts with Hsf1 in vivo and is required for the induction of heat shock genes by oxidative stress

D C Raitt et al. Mol Biol Cell. 2000 Jul.

Free PMC article

Abstract

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein-protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.

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Figures

Figure 1

Figure 1

(A) Domain structures of Skn7 and Hsf1. (B) Homology between the DNA-binding domains of Skn7 and heat shock factor. The DNA-binding domain of Skn7p (amino acids 87–151) is aligned with the corresponding regions of S. cerevisiae Hsf1 (Hsf1sc), the fission yeast heat shock factor (Hsf1sp), and the human heat shock factor 2 (Hsf2hs). Highly conserved residues that may directly contact the DNA and may have diverged in Skn7 are indicated by asterisks.

Figure 2

Figure 2

skn7Δ cells are sensitive to acute heat stress. Midlog-phase cultures of W303-1a and isogenic skn7Δ cells were grown in YPD at 25°C, and an aliquot was shifted to a test tube placed in a 51°C water bath. Samples were taken at the times indicated, diluted into ice-cold YPD, and immediately plated onto YPD agar to assess cell viability. Survival at 51°C was expressed as a percentage of viable cells relative to cells grown at 25°C. Because the vertical axis is logarithmic, only positive errors are included for clarity.

Figure 3

Figure 3

Purified 6His-Skn7p can specifically bind HSEs in vitro. EMSA was performed with the use of _E. coli_–expressed affinity-purified 6His-Skn7 protein and a probe comprising a double-stranded oligonucleotide corresponding to the 26-base pair HSE2 region of the SSA1 gene. The specificity of binding was assessed by the addition of cold HSE2 probe (HSE: tcgaTTTTCCA

G

AACGTT

C

CATCGGC) at 5-, 10-, and 50-fold molar excess, compared with the addition of a mutated HSE (MUT HSE: tcgaTTTTCCA

A

AACGTT

T

CATCGGC) at 10-, 50-, and 100-fold molar excess. Mutation of the underlined nucleotides in the consensus HSE abolishes Hsf1 binding to the sequence. Polyclonal anti-Skn7 antibody (α-Skn7) at a 1:100 dilution or preimmune serum at the same concentration (Pre Imm) was added to the binding reaction 15 min before the addition of labeled probe. Free probe without the addition of protein migrated off the gel and is indicated (Free).

Figure 4

Figure 4

Skn7 is required for the induction of heat shock gene expression specifically in response to hydrogen peroxide. (A) Northern blot analysis of the effect of skn7Δ mutations on heat shock gene induction by oxidative stress. Total RNA was prepared from midlog-phase cultures of W303-1a and W303-1a skn7Δ grown at 30°C in YPD. Samples for RNA extraction were taken before (time 0) and at the times indicated after the addition of 0.6 mM _t_-butyl hydrogen peroxide. Northern blots were prepared as described (see MATERIALS AND METHODS) and hybridized to probes specific for HSP12, HSP26, and HSP104. (B) Skn7 is not required for the heat shock induction of heat shock gene expression. W303-1a and skn7Δ cells were grown to midlog phase at 25°C, and a portion of the culture was transferred to a 39°C water bath. Cells were harvested at the times indicated, and RNA was extracted for Northern hybridization with the probes specified in A. Quantitation of mRNA was by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA) and was expressed relative to the ACT1 transcript, the abundance of which appeared to be unaffected by oxidative stress.

Figure 5

Figure 5

Genetic interactions between SKN7 and HSF1. (A) Single-copy expression of SKN7 rescues the growth defect of DR20-2b (_hsf1_ts skn7Δ) at 33°C. DR20-2b (_hsf1_ts skn7Δ) was transformed with the CEN vector YCplac111 (vector) or with this vector containing the SKN7 gene (+ SKN7) and was incubated at permissive (25°C) and intermediate (33°C) temperatures on rich medium. (B) High-copy expression of SKN7 rescues the growth defect of the _hsf1_ts strain, MYY385, at 35°C. The _hsf1_ts strain MYY385 was transformed with the 2μ-based plasmid YEp24 containing the SKN7 gene or with the vector alone (vector) and was streaked onto selective medium and incubated at 35°C for 4 d. The wild-type strain MYY290 (HSF1+) was included as a positive growth control.

Figure 6

Figure 6

The double mutant _hsf1_ts skn7Δ is hypersensitive to hydrogen peroxide. Sensitivity to hydrogen peroxide was assayed for W303-1a and isogenic skn7Δ, for MYY290 (HSF1+) and the _hsf1_ts derivative MYY385, and for an _hsf1_ts skn7Δ spore clone (DR20-2b) derived from a cross between skn7Δ and MYY385. Cell suspensions were streaked on a YPD plate (Control) and a YPD plate onto which 1 μl of 7.7 M _t_-butyl hydrogen peroxide was spotted on a disk of Whatman 3MM paper positioned in the center of the plate (+ hydroperoxide). Suppression of hypersensitivity was judged by the ability to grow in the presence of hydrogen peroxide of a YCplac111 (Gietz and Sugino, 1988) plasmid expressing the SKN7 gene (+SKN7) or an _SKN7_D427N mutant (+ _SKN7_-DN) in which the conserved aspartate residue (D427) was mutated to asparagine. The _hsf1_ts skn7Δ strain was also transformed with the YCplac111 vector alone (+ vector); plates were incubated for 2 d at 30°C.

Figure 7

Figure 7

Hsf1 and Skn7 copurify. (A) Western blot analysis with 12CA5 mAb reveals that pGAL-Skn7-HA copurifies with GST-Hsf1. Lane 1, crude extract containing GST vector alone and pGAL-Skn7-HA; lane 2, GST pull-down from extract containing pGAL-Skn7-HA and GST vector alone; lane 3, crude extract containing pGAL-Skn7-HA and GST-Hsf1; lane 4, GST pull-down from cells containing pGAL-Skn7-HA and GST-Hsf1. (B) Lane 1, 20 μg of input protein (HA-Skn7); lane 2, GST pull-down of extract from cells containing empty GST vector and pGAL-Skn7-HA; lane 3, GST pull-down of extract from cells containing GST-Hsf1 vector and pGAL-Skn7-HA and grown in glucose; lane 4, GST pull-down of extract from galactose-grown cells containing pGAL-Skn7-HA and pGST-Hsf1. (C) Nickel-affinity copurification of Hsf1 with 6His-tagged Skn7. Lane 1, immunoprecipitate with anti-Hsf1 antibody from 1 mg of whole cell extract of galactose-grown cells expressing pGAL-SKN7–6His; lane 2, Ni2+-NTA agarose beads plus 1 mg of cell extract that does not contain the 6His-Skn7 protein; lane 3, Ni2+-NTA agarose beads plus 1 mg of galactose-induced extract from cells expressing pGAL-SKN7–6His; lane 4, 20 μg of input galactose-induced extract. (D) Skn7p can interact with itself. Coimmunoprecipitations from cell extracts containing galactose-induced pGAL-Skn7-HA and 6Myc-Skn7 were performed with the use of 9E10 mAb followed by 12CA5 Western blot analysis. HA-Skn7p expression was under the control of the GAL promoter, and integrated 6Myc-Skn7 was under the control of its own promoter. Immunoprecipitations with 1.5 μg of 9E10 were as follows: lane 1, 20 μg of extract from galactose-grown cells; lane 2, immunoprecipitation of extract from glucose-grown cells; lane 3, immunoprecipitation of extract from galactose-grown cells that did not contain the Myc-tagged SKN7; lane 4; immunoprecipitation of extract from galactose-grown cells containing 6Myc-Skn7 and pGAL-Skn7-HA. Western blot analysis was carried out with 12CA5 mAb.

Figure 8

Figure 8

The Skn7 response regulator is localized in the nucleus. An _SKN7_-GFP fusion protein expressed from a CEN plasmid was visualized in cells from a midlog-phase culture (A; Nomarski image). The Skn7p-GFP fusion protein visualized by fluorescence (C) colocalizes with the DAPI signal of nuclear DNA (B).

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References

    1. Alberts AS, Bouquin N, Johnston LH, Treisman R. Analysis of RhoA-binding proteins reveals an interaction domain conserved in heterotrimeric G protein β-subunits and the yeast response regulator protein Skn7. J Biol Chem. 1998;273:8616–8622. - PubMed
    1. Bouquin N, Johnson AL, Morgan BA, Johnston LH. Association of the cell cycle transcription factor Mbp1 with the Skn7 response regulator in budding yeast. Mol Biol Cell. 1999;10:3389–3400. - PMC - PubMed
    1. Brown JL, Bussey H, Stewart RC. Yeast Skn7p functions in a eukaryotic two-component regulatory pathway. EMBO J. 1994;13:5186–5194. - PMC - PubMed
    1. Brown JL, North S, Bussey H. SKN7, a yeast multicopy suppressor of a mutation affecting cell wall β-glucan assembly, encodes a product with domains with homology to prokaryotic two-component regulators and heat shock transcription factors. J Bacteriol. 1993;175:6908–6915. - PMC - PubMed
    1. Christianson TW, Sikorski RS, Dante M, Shero JH, Heiter P. Multifunctional yeast high-copy number shuttle vectors. Gene. 1992;110:119–122. - PubMed

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