The molecular chaperone Hsp90 mediates heme activation of the yeast transcriptional activator Hap1.
Hsp90 plays critical roles in the proper functioning of a wide array of eukaryotic signal transducers such as steroid receptors and tyrosine kinases. Hap1 is a naturally occurring substrate of Hsp90 in Saccharomyces cerevisiae. Hap1 transcriptional activity is precisely and stringently controlled by heme. Previous biochemical studies suggest that in the absence of heme, Hap1 is bound to Hsp90 and other proteins, forming a higher order complex termed HMC (high molecular weight complex), and is repressed. Heme promotes the disruption of the HMC and activates Hap1, permitting Hap1 to bind to DNA with high affinity and to stimulate transcription. By lowering the expression levels of wild-type Hsp90, using a highly specific Hsp90 inhibitor, and by examining the effects of various Hsp90 mutants on Hap1, we show that Hsp90 is critical for Hap1 activation by heme. Furthermore, we show that many Hsp90 mutants exert differential effects on Hap1 and steroid receptors. Notably, mutant G313N weakens Hsp90 steroid receptor interaction but strongly enhances Hsp90-Hap1 interaction and increases Hap1 resistance to protease digestion. Additionally, we found that a heme-independent Hap1 mutant still depends on Hsp90 for high activity. These experiments together suggest that Hsp90 promotes Hap1 activation by inducing or maintaining Hap1 in a transcriptionally active conformation.
Lee
Hee Chul
HC
Department of Biochemistry, New York University School of Medicine, New York, NY 10016, USA.
Hon
Thomas
T
Zhang
Li
L
D015153:Blotting, Western; D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry* / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000378:metabolism
D018841:HSP90 Heat-Shock Proteins; D006418:Heme; D004247:DNA; D001616:beta-Galactosidase; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase