Mutated Yeast Heat Shock Transcription Factor Exhibits Elevated Basal Transcriptional Activation and Confers Metal Resistance

Andrew K Sewell,Fumihiko Yokoya,Wei Yu,Terumi Miyagawa,Tetsuo Murayama,Dennis R Winge

doi:10.1074/jbc.270.42.25079

Andrew K Sewell, Fumihiko Yokoya + Show 4 more

Open Access

https://doi.org/10.1074/jbc.270.42.25079

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Abstract

Cadmium-resistant Saccharomyces cerevisiae strain 301N exhibits high basal as well as cadmium-induced expression of the CUP1 metallothionein gene. Since regulation of CUP1 is usually restricted to copper ions, our goal was to identify the factor responsible for the high metallothionein levels in strain 301N. The gene responsible for the observed phenotype is a spontaneously mutated heat shock transcription factor gene (HSF1). A double, semidominant HSF1 mutant with substitutions at codons 206 and 256 within the DNA-binding domain of the heat shock factor (HSF) confers two phenotypes. The first phenotype is elevated transcriptional activity of the HSF mutant (HSF301), which results in constitutive thermotolerance. A second HSF301 phenotype is enhanced binding affinity for the heat shock element (HSE) within the CUP1 5'-sequences, resulting in high basal transcription of metallothionein. The CUP1 HSE is a minimal heat shock element containing only two perfectly spaced inverted repeats of the basic nGAAn block. Cells containing HSF301 are resistant to cadmium salts. The single R206S mutation is responsible for the high affinity binding to the CUP1 HSE. In addition, the R206S HSF substitution exhibits constitutive transcriptional activation from a consensus HSE (HSE2). The F256Y substitution in HSF attenuates the effects of R206S on the consensus HSE2, but not on the CUP1 HSE.

Highlights

All cells are capable of coping with changes in their environment, such as exposure to elevated temperatures, toxins, and oxidants
We demonstrate that the regulatory factor conferring constitutive expression of CUP1 and cadmium metalloregulation in strain 301N is heat shock factor (HSF)
The cadmium tolerance observed in S. cerevisiae strain 301N was found to arise from a semidominant mutation within the DNA-binding domain of HSF

Summary

Introduction

All cells are capable of coping with changes in their environment, such as exposure to elevated temperatures, toxins, and oxidants. In response to certain stress conditions, activation of stress gene expression occurs, resulting in an elevated synthesis of stress proteins, commonly called heat shock proteins (hsp)1 [1, 2]. Yeast HSF is a trimeric protein reported to bind HSE sequences constitutively at low temperature [5, 15,16,17]. Yeast HSF contains domains that function as constitutive transcriptional activation domains when fused to heterologous DNA-binding domains [22, 23]. Since these transactivation domains are not constitutive in HSF at low temperature, it appears that the normal mode of action of HSF is to hinder the effectiveness of these domains. The regulatory domains of HSF can even repress the activity of a heterologous transcriptional activation domain fused in place of its own C-terminal activation domain [24]

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