PMC

 Shown is a schematic representation of the hsb-1 locus with the CeHSB-1 coding sequences shown as a solid bar. The numbers represent nucleotide numbering from the sequence of the cosmid K08E7 (accession no. Z77666). The CH116 mutant strain contains a deletion of the entire coding sequences and some of the upstream sequences as indicated by the broken lines and the allele name. The PstI (P)–SalI (S) 2.3-kb fragment from cosmid K08E7 was used for establishing strains that overexpress CeHSB-1.

 The overexpression of HSBP1 results in reduced HSF1 transactivation ability. pHB–CAT (hsp70 promoter fused to the CAT reporter gene), pRSV–luciferase with or without HSBP1 or mutants of HSBP1 (mutant 16/19 and tetra mutant) were transfected into COS7 cells. Cells were harvested either at 48 hr post-transfection (C) or after a 2-hr heat treatment at 42°C and a 4-hr recovery period (H). Transfection efficiencies were normalized to equivalent luciferase activities. The relative CAT activities are indicated with standard deviations observed.

 Interaction of HSBP1 coincides with the attenuation of HSF1 DNA-binding activity. 3T3 cells were treated at 43°C for up to 4 hr (A–C) or treated at 43°C for 1 hr and allowed to recover at 37°C for 1–4 hr (D–F) to allow attenuation of HSF1. The extracts were used for gel mobility shift analysis (A,D), immunoblot analysis, and immunoprecipitations with anti-HSBP1 antibodies. The cell extracts (B,E) and immunoprecipitates (C,F) were separated on an 8% SDS–polyacrylamide gel and then used for immunoblotting analysis with anti-HSF1 and anti-Hsp70 antibodies.

 HSBP1 interacts specifically with HSF1 trimers and leads to the recruitment of Hsp70. 3T3 cells were exposed to 42°C for the indicated times ranging from 15 min to 4 hr (A–C) or treated at 42°C for 1 hr followed by recovery at 37°C for 4 hr (D–F). The cell extracts were used for gel mobility shift analysis (A,D), immunoblot analysis, and immunoprecipitations with anti-HSBP1 antibodies. The cell extracts (B,E) and immunoprecipitates (C,F) were separated on an 8% SDS-polyacrylamide gel and then used for immunoblotting analysis with anti-HSF1 and anti-Hsp70 antibodies.

 HSF1 interacts with HSBP1 in vivo and is a nuclear localized protein. (A) HeLa cells were cotransfected with HA- tagged HSBP1 and non-epitope-tagged HSF1 or mutant HSF1 containing a deletion of HR A. The extracts were used to perform immunoblot analysis using HSF1 antiserum and immunoprecipitations with an anti-HA epitope antibody. The HSBP1 immunoprecipitates were separated by 8% and 16% SDS-PAGE and analyzed for their HSF1 and HSBP1 content, respectively, by immunoblot analysis. (B) The cellular localization of HSBP1 was determined by transient transfection of the HA-tagged HSBP1 in HeLa cells. The cells were fixed and immunofluoresence staining was performed with an anti-HA epitope monoclonal antibody. (C) Hoechst staining revealed the nuclear staining of the transfected cell and also adjacent nontranfected cells.

 Overexpression of CeHSB-1 results in a block of the activation of the stress response in C. elegans. (A,B) The PC72 reporter strain containing integrated copies of the hsp16::lacZ fusion was treated at 33°C for 1 hr and stained for β-galactosidase activity. lacZ activity was observed in intestinal, pharyngeal, nerve ring, and body wall muscle cells [intestinal (i) and body wall muscle (bm) cells are indicated by arrows]. Animals were photographed at 10× (A,C) and 40× (B,D) magnification. (C,D) The unc-54::hsb-1 fusion was used to overexpress CeHSB-1 specifically in body wall muscle cells of the PC72 strain. These animals were also stained for β-galactosidase activity after heat shock. Intestinal cells are stained (arrow), but there is reduced staining of body wall muscle cells.

 Isolation of HSBP1, a novel and conserved protein that interacts with the trimerization domain of HSF1. (A) The sequence of the human HSBP1 cDNA. HSBP1 was isolated based on its ability to interact with the trimerization domain of mHSF1. (B) Comparison of the deduced amino acid sequences of the C. elegans, human, and canine HSBP1. The identities among the three proteins are indicated by the shaded regions. Asterisks indicate the two amino acid difference between the human and the canine HSBP1 proteins. (C) Hydrophobic repeats of HSBP1. Analysis of the amino acid sequence of HSBP1 revealed the presence of potential hydrophobic repeats indicated by the a and d positions.

 Interactions between HSF1 and HSBP1 are mediated through hydrophobic repeats. The amino acid sequences of the hydrophobic repeats of HSF1 and HSBP1 with the amino acids forming the repeats indicated by a and d positions. The sites of the mutations to lysines (K) are shown. Various LexA fusions were tested for their ability to interact with HSBP1 in the yeast two-hybrid system. The β-galactosidase enzyme activities along with the standard deviations observed are indicated. HSBP1 interacts with LexA fusion with the trimerization domain of mHSF1 but not with the trimerization domain of mHSF2 (HSF2 HR-A/B). To assess which hydrophobic residues of the trimerization domain of HSF1 contribute to the interaction with HSBP1, we used a panel of LexA fusions with specific mutations in the trimerization domain of HSF1 as indicated above. Mutants 16/19, 45/48, and the Tetra mutant (containing all four substitutions) of HSBP1 were also tested for their ability to interact with the trimerization domain of mHSF1 fused to LexA.

 Coexpression of HSBP1 and HSF1 in reticulocyte lysates blocks in vitro activation of HSF1 DNA-binding activity. (A) mHSF1 RNA was cotranslated with increasing amounts of HSBP1 RNA using the rabbit reticulocyte lysate system, and the lysates were incubated at 4°C, 37°C, and 43°C for 1 hr. The DNA-binding activity of mHSF1 was analyzed by gel mobility shift assay. The amount of HSF1 DNA-binding activity obtained upon cotranslation with HSBP1 is indicated in percent activity relative to the amount in the case of translation of HSF1 alone. (B) An aliquot of the in vitro translation reaction in A was supplemented with [³⁵S]methionine, analyzed by SDS-PAGE, and autoradiography. Quantification of HSF1 and HSBP1 synthesis revealed a ratio of 1:25 (HSF1/HSBP1) and 1:70 in the 1× RNA and 3× RNA reaction, respectively.