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Abstract
Cells may sense heat shock via the accumulation of thermally misfolded proteins. To explore this possibility, we determined the effect of protein misfolding on gene expression in the absence of temperature changes. The imino acid analog azetidine-2-carboxylic acid (AZC) is incorporated into protein competitively with proline and causes reduced thermal stability or misfolding. We found that adding AZC to yeast at sublethal concentrations sufficient to arrest proliferation selectively induced expression of heat shock factor-regulated genes to a maximum of 27-fold and that these inductions were dependent on heat shock factor. AZC treatment also selectively repressed expression of the ribosomal protein genes, another heat shock factor-dependent process, to a maximum of 20-fold. AZC treatment thus strongly and selectively activates heat shock factor. AZC treatment causes this activation by misfolding proteins. Induction of HSP42 by AZC treatment required protein synthesis; treatment with ethanol, which can also misfold proteins, activated heat shock factor, but treatment with canavanine, an arginine analog less potent than AZC at misfolding proteins, did not. However, misfolded proteins did not strongly induce the stress response element regulon. We conclude that misfolded proteins are competent to specifically trigger activation of heat shock factor in response to heat shock.
Highlights
Eukaryotic cells respond to heat shock by the induction of a conserved set of proteins, the heat shock proteins, via transcriptional activation of the corresponding genes (1)
Herskowitz. § Supported by a Ph.D. studentship from the Biotechnology and Biological Sciences Research Council (United Kingdom). ʈ Damon Runyon-Walter Winchell Postdoctoral Fellow. §§ Planted the seeds for the AZC project while a visiting professor in the laboratory of Ira Herskowitz at the University of California at San Francisco. ¶¶ To whom correspondence should be addressed: Div. of Molecular Genetics, Faculty of Biomedical and Life Sciences, University of Glasgow, Anderson College Complex, 54 –56 Dumbarton Rd., Glasgow G11 6NU, UK
In agreement with the results from the microarray analysis described above, we found that 1) expression of the Heat shock elements (HSEs)-containing genes HSP42, SSA4, HSP12, and HSP30 was strongly induced by AZC treatment, comparable with, if not more profoundly than, the peak transient induction of each gene upon temperature upshift; 2) expression of the STREdriven gene CTT1 was, at best, weakly induced by AZC treatment, but was more strongly induced by temperature upshift; and 3) expression of the ribosomal protein genes RPL3, RPL30, and RPS1a was strongly repressed by AZC treatment, comparable with, if not more profoundly than, the peak transient repression caused by temperature upshift
Summary
All chemicals were from Sigma (Dorset, UK). Components of the growth medium were from BD Biosciences and Fisher. DL-AZC was used throughout this work, but L-AZC is the active agent in this racemic mixture.[2] Any given concentration of AZC refers to the concentration of the racemic mixture Liquid media, both rich and minimal, were prepared as described previously (16). AZC and canavanine were dissolved in water to a stock concentration of 500 mM and added to the growth medium to achieve the final concentrations specified in individual experiments. Ethanol was added to the growth medium to a final concentration of 8% (v/v). Cells were grown at 23 °C to A600 nm ϭ 0.2 and added to an equal volume of YPD medium in a conical flask preheated at 36 °C in a water bath. All absorbance measurements were made on a Milton Roy Spectronic 601 spectrophotometer
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