Abstract
Heat shock protein 90 (HSP90), an abundant molecular chaperone in the eukaryotic cytosol, is involved the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. In vertebrates, hsp90 has two isoforms, hsp84 and hsp86. Here we report the cDNA cloning and functional analysis of the rat hsp86. Based on the homology among human hsp90□, horse hsp90□, porcine hsp90 and mouse hsp86, the conserved region in rat hsp86 was amplified by RT-PCR and used as the probe. The full-length rat hsp86 cDNA was obtained from cDNA library screening. The cDNA sequence data contains 2,795 bp and the length of coding region was 2,202 bp (GeneBank accession number AJ428213). Based on a crystal structure of the N-terminal domain of human HSP90 with bound ADP-Mg, several mutants were made in the rat HSP86 ATPase domain. Our results show that the full-length HSP86 has 15-fold higher ATPase activity than the N-termunal ATPase domain only, but the ATPase activity of HSP84 ATPase domain was similar to HSP86 ATPase domain. In addition, we found that the presence of magnesium can increase the ability of ATP hydrolysis of HSP86F Therefore, the results establish that magnesium binding, ATP binding and hydrolysis are required for HSP90 function in vitro.