SBA1 Encodes a Yeast Hsp90 Cochaperone That Is Homologous to Vertebrate p23 Proteins

Abstract

The Saccharomyces cerevisiae SBA1 gene was cloned by PCR amplification from yeast genomic DNA following its identification as encoding an ortholog of human p23, an Hsp90 cochaperone. The SBA1 gene product is constitutively expressed and nonessential, although a disruption mutant grew more slowly than the wild type at both 18 and 37°C. A double deletion of SBA1 and STI1, encoding an Hsp90 cochaperone, displayed synthetic growth defects. Affinity isolation of histidine-tagged Sba1p (Sba1 His6 ) after expression in yeast led to coisolation of Hsp90 and the cyclophilin homolog Cpr6. Using an in vitro assembly assay, purified Sba1His6 bound to Hsp90 only in the presence of adenosine 5-O-(3-thiotriphosphate) or adenyl-imidodiphosphate. Furthermore, interaction between purified Sba1His6 and Hsp90 in yeast extracts was inhibited by the benzoquinoid ansamycins geldanamycin and macbecin. The in vitro assay was also used to identify residues in Hsp90 that are important for complex formation with Sba1His6 , and residues in both the N-terminal nucleotide binding domain and C-terminal half were characterized. In vivo analysis of known Hsp90 substrate proteins revealed that Sba1 loss of function had only a mild effect on the activity of the tyrosine kinase v-Src and steroid hormone receptors.

The Assembly of Progesterone Receptor-hsp90 Complexes Using Purified Proteins

“…He showed that deletion of the p23 gene of Saccharomyces cerevisiae had a minimal effect on GR action, but it did sensitize the GR to inhibition by the GA-related drug, macbecin II (42). Another report by Fang et al (43) showed that the deletion of yeast p23 resulted in only a mild reduction in cell growth or the hsp90-dependent activities of the tyrosine kinase v-Src and the androgen receptor. Our results show a marked dependence upon p23 for the generation of hormone binding activity.…”

Sti1 and Cdc37 Can Stabilize Hsp90 in Chaperone Complexes with a Protein Kinase

“…Furthermore, comparison of the results presented here with those previously reported using heterologous clients reveals some common trends. Deletion of CPR6 or SBA1, for example, results in a only a slight defect in v-Src or nuclear receptor signaling, whereas deletion of CPR7 or YDJ1 resulted in strong signaling defects (Kimura et al, 1995;Dey et al, 1996;Duina et al, 1996;Warth et al, 1997;Fang et al, 1998). By correlation, cochaperones that are essential for viability are also likely to play essential roles in the folding pathway; examples here are Cdc37 and Cns1.…”

“…Whether the cochaperones are required for this reaction is not clear, because purified Hsp70 and Hsp90 can together facilitate folding of GR in vitro, whereas cochaperones such as Hop stimulate the reaction (Morishima et al, 2000;Rajapandi et al, 2000). Furthermore, deletion of yeast Hop (STI1), or p23 (SBA1) does not result in a slow growth phenotype except under stress conditions (Nicolet and Craig, 1989;Chang et al, 1997;Bohen, 1998;Fang et al, 1998).…”

“…Whether the cochaperones are required for this reaction is not clear, because purified Hsp70 and Hsp90 can together facilitate folding of GR in vitro, whereas cochaperones such as Hop stimulate the reaction (Morishima et al, 2000;Rajapandi et al, 2000). Furthermore, deletion of yeast Hop (STI1), or p23 (SBA1) does not result in a slow growth phenotype except under stress conditions (Nicolet and Craig, 1989;Chang et al, 1997;Bohen, 1998;Fang et al, 1998).Another question is whether the scheme described above for nuclear receptors reflects a set of general principles by which Hsp90 functions and whether it applies to other client types. Folding of protein kinases, for example, requires a cochaperone called Cdc37, that does not associate with GR or PR, although it does function in activation of androgen receptor and a viral reverse transcriptase (Fliss et al, 1997;Rao et al, 2001;Wang et al, 2002).…”

“…Levels of FIG1 and HHF1 mRNA were determined by quantitative real-time rtPCR relative to ARP5, whose mRNA levels do not change upon pheromone treatment (Roberts et al, 2000; our unpublished results). The cochaperones chosen for study have all been characterized previously as Hsp90 binding proteins, and include Cpr6, Cpr7, Hch1, Sba1, Sse1, and Sti1 (Duina et al, 1996;Chang et al, 1997;Bohen, 1998;Fang et al, 1998;Liu et al, 1999;Panaretou et al, 2002;Lotz et al, 2003). Ydj1, a cochaperone of Hsp70 was also included because it affects the activity of Hsp90 clients (Bohen et al, 1995).…”