Identification of a site of Hsp27 binding with Hsp27 and alpha B-crystallin as indicated by the yeast two-hybrid system.
The small heat-shock proteins (sHsp), including Hsp27 and alphaB-crystallin, usually form large oligomers in cells. It has been suggested that the sHsp form oligomers by binding either a conserved C-terminal amino acid sequence or the less conserved N-terminal region. However, the site of binding has not been precisely determined. We used the yeast two-hybrid system to investigate binding of full-length rat Hsp27 or fragments of Hsp27 to full-length rat Hsp27 or alphaB-crystallin molecules. A series of cDNAs coding for fragments of Hsp27 were generated and ligated with the coding sequence for the binding domain of the yeast Gal4p transcription factor. These cDNAs were each transfected into yeast that had been transfected to express full-length rat Hsp27 or alphaB-crystallin fused with the DNA binding domain of Gal4p. Yeast cells transfected with both plasmids were assayed, by both a beta-galactosidase (beta-gal) filter assay and a quantitative liquid assay, for activation of Gal4p-driven beta-gal expression. Results indicated that the N-terminal domain of Hsp27 consisting of amino acids 1-124 did not bind to Hsp27 or alphaB-crystallin. The predominant Hsp27-Hsp27/alphaB-crystallin binding domain was the conserved C-terminal region consisting of amino acids 141-206, particularly amino acids 141-176.
Department of Anatomy and Cell Biology, University of Michigan Medical School, Ann Arbor 48109-0616, USA.
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D000818:Animals; D001665:Binding Sites / Q000235:genetics; D003459:Crystallins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006822:Hybrid Cells; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D003459:Crystallins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors