Abstract
The ATP-dependent molecular chaperone Hsp90 is an essential and abundant stress protein in the eukaryotic cytosol that cooperates with a cohort of cofactors/cochaperones to fulfill its cellular tasks. We have identified Aha1 (activator of Hsp90 ATPase) and its relative Hch1 (high copy Hsp90 suppressor) as binding partners of Hsp90 in Saccharomyces cerevisiae. By using genetic and biochemical approaches, the middle domain of Hsp90 (amino acids 272-617) was found to mediate the interaction with Aha1 and Hch1. Data base searches revealed that homologues of Aha1 are conserved from yeast to man, whereas Hch1 was found to be restricted to lower eukaryotes like S. cerevisiae and Candida albicans. In experiments with purified proteins, Aha1 but not Hch1 stimulated the intrinsic ATPase activity of Hsp90 5-fold. To establish their cellular role further, we deleted the genes encoding Aha1 and Hch1 in S. cerevisiae. In vivo experiments demonstrated that Aha1 and Hch1 contributed to efficient activation of the heterologous Hsp90 client protein v-Src. Moreover, Aha1 and Hch1 became crucial for cell viability under non-optimal growth conditions when Hsp90 levels are limiting. Thus, our results identify a novel type of cofactor involved in the regulation of the molecular chaperone Hsp90.
Highlights
The 90-kDa heat shock protein (Hsp90)1 is a highly conserved, abundant, and constitutively expressed homodimeric molecular chaperone of the eukaryotic cytosol
We found that Hch1 shares sequence homology to a short 151-aa protein present in Candida albicans (Swiss-Prot accession number O93993) and to the N-terminal half of the 350-aa S. cerevisiae protein Aha1, which is conserved from yeast to mammals (Swiss-Prot accession numbers Q9P782 for Schizosaccharomyces pombe, 336 aa; Q93168 for Caenorhabditis elegans, 343 aa; Q9LHL7 for Arabidopsis thaliana, aa; Q9V9Q4 for Drosophila melanogaster, aa; Q8R3E6 for Mus musculus, 338 aa; and O95433 for Homo sapiens, 338 aa)
The results presented here demonstrate that Aha1 and Hch1, in remarkable contrast, associate with the middle domain of Hsp90, whereas C- and N-terminal domains of the molecular chaperone are dispensable
Summary
The 90-kDa heat shock protein (Hsp90) is a highly conserved, abundant, and constitutively expressed homodimeric molecular chaperone of the eukaryotic cytosol. It is involved in the folding and conformational regulation of a limited subset of proteins. Several Hsp90-associated cofactors contain tetratricopeptide repeat motifs, a degenerate 34-aa sequence, that mediate binding to the C-terminal EEVD motif of the molecular chaperone [10]. Recent crystal structures of the N-terminal domain of Hsp90 [11, 12] have identified a conserved binding site for ATP. We demonstrate binding of the novel cofactors Aha and Hch to the middle domain of Hsp, currently a poorly investigated region in the molecular chaperone. Deletion of the genes encoding Aha and Hch in Saccharomyces cerevisiae impaired activation of the heterologous Hsp90-dependent substrate protein v-Src and interfered with cell viability under non-optimal conditions
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