SBA1 ENCODES A YEAST HSP90 COCHAPERONE THAT IS HOMOLOGOUS TO VERTEBRATE P23 PROTEINS

The Saccharomyces cerevisiae SBA1 gene was cloned by PCR amplification from yeast genomic DNA following its identification as encoding an or tholog of human p23, an Hsp90 cochaperone. The SBA1 gene product is co nstitutively expressed and nonessential, although a disruption mutant grew more slowly than the wild type at both 18 and 37 degrees C. A dou ble deletion of SBA1 and STI1, encoding an Hsp90 cochaperone, displaye d synthetic growth defects. Affinity isolation of histidine-tagged Sba 1p (Sba1(His6)) after expression in yeast led to coisolation of Hsp90 and the cyclophilin homolog Cpr6. Using an in vitro assembly assay, pu rified Sba1(His6) bound to Hsp90 only in the presence of adenosine 5'- O-(3-thiotriphosphate) or adenyl-imidodiphosphate. Furthermore, intera ction between purified Sba1(His6) and Hsp90 in yeast extracts was inhi bited by the benzoquinoid ansamycins geldanamycin and macbecin. The in vitro assay was also used to identify residues in Hsp90 that are impo rtant for complex formation with Sba1(His6), and residues in both the N-terminal nucleotide binding domain and C-terminal half were characte rized. In vivo analysis of known Hsp90 substrate proteins revealed tha t Sba1 loss of function had only a mild effect on the activity of the tyrosine kinase v-Src and steroid hormone receptors.