The C-terminal hydrophobic repeat ofSchizosaccharomyces pombe heat shock factor is not required for heat-induced DNA-binding

Abstract

The C‐terminal hydrophobic repeat (CTR) of heat shock transcription factor (HSF) has been proposed to regulate DNA binding by intramolecular interactions with the leucine zipper motifs present in the HSF trimerization domain. Schizosaccharomyces pombe provides a useful model organism for the study of the regulation of HSF DNA binding because, unlike Saccharomyces cerevisiae, S. pombe hsf is highly heat shock inducible for DNA binding and contains a clear homology to the CTR. We examined the role that the CTR plays in the regulation of S. pombe hsf by constructing isogenic strains bearing deletion and point mutations in the chromosomal copy of hsf. Surprisingly, we found that point mutation of key hydrophobic amino acids within the CTR, as well as full deletion of it, yielded factors that show normal binding at normal growth temperatures and full levels of heat‐induced binding. Deletion of the CTR did, however, slightly lower the temperature required for maximal activation. In contrast, a large deletion of the C‐terminus, which removes close to a third of the coding sequence, was deregulated and bound DNA at control temperature. Several of the deletion mutants were significantly reduced in their level of expression, yet they showed wild‐type levels of DNA binding activity following heat shock. These experiments demonstrate that appropriate regulation of the DNA binding activity of S. pombe hsf is not solely dependent upon the CTR, and imply that a feedback mechanism exists that establishes proper levels of DNA binding following heat shock despite mutations that significantly alter levels of total hsf. © 1998 John Wiley & Sons, Ltd.

A Nuclear Localization Signal Is Essential for Stress-induced Dimer-to-Trimer Transition of Heat Shock Transcription Factor 3

“…Furthermore, substitution of isoleucine at amino acid 371 or leucine at amino acid 375 in the HR-C with glutamic acid made cHSF3 constitutively a trimer (data not shown). Although there is controversy about the role of HR-C (19,20), our data conclusively showed that this structure plays a role in maintaining cHSF3 in an inactive oligomeric state.…”

“…With the exception of HSFs in budding yeasts and mammalian HSF4, HSFs also have another carboxyl-terminal hydrophobic repeat (HR-C) (17,18). Although a consensus has yet to be reached (19,20), HR-C is suggested to maintain HSF in an inactive state by interacting intramolecularly with the HR-A/B (21)(22)(23). HSF1 is also negatively regulated in trans by molecular chaperone complexes (24 -27).…”

Mutations in the Schizosaccharomyces pombe heat shock factor that differentially affect responses to heat and cadmium stress

“…We previously created a series of deletion and point mutations in S. pombe hsf+ and then used the two-step gene replacement method to substitute the genomic copy of hsf+ with epitope-tagged versions of the wild type and mutant genes (Saltsman et al 1998). In all strains, the hsf allele included the DNA binding and trimerization domains and was expressed from the endogenous promoter.…”