Genetic and biochemical analysis of p23 and ansamycin antibiotics in the function of Hsp90-dependent signaling proteins.

The ubiquitous molecular chaperone Hsp90 acts in concert with a cohort of associated proteins to facilitate the functional maturation of a number of cellular signaling proteins, such as steroid hormone receptors and oncogene tyrosine kinases. The Hsp90-associated protein p23 is required for the assembly of functional steroid aporeceptor complexes in cell lysates, and Hsp90-binding ansamycin antibiotics disrupt the activity of Hsp90-dependent signaling proteins in cultured mammalian cells and prevent the association of p23 with Hsp90-receptor heterocomplexes; these observations have led to the hypotheses that p23 is required for the maturation of Hsp90 target proteins and that ansamycin antibiotics abrogate the activity of such proteins by disrupting the interaction of p23 with Hsp90. In this study, I demonstrate that ansamycin antibiotics disrupt the function of Hsp90 target proteins expressed in yeast cells; prevent the assembly of Sba1, a yeast p23-like protein, into steroid receptor-Hsp90 complexes; and result in the assembly of receptor-Hsp90 complexes that are defective for ligand binding. To assess the role of p23 in Hsp90 target protein function, I show that the activity of Hsp90 target proteins is unaffected by deletion of SBA1. Interestingly, steroid receptor activity in cells lacking Sba1 displays increased sensitivity to ansamycin antibiotics, and this phenotype is rescued by the expression of human p23 in yeast cells. These findings indicate that Hsp90-dependent signaling proteins can achieve a functional conformation in vivo in the absence of p23. Furthermore, while the presence of p23 decreases the sensitivity of Hsp90-dependent processes to ansamycin treatment, ansamycin antibiotics disrupt signaling through some mechanism other than altering the Hsp90-p23 interaction.

Hsp90 mutants disrupt glucocorticoid receptor ligand binding and destabilize aporeceptor complexes.

In order to attain competence to respond to hormone, certain steroid hormone receptors must be assembled into hetero-oligomeric aporeceptor complexes, containing Hsp90 and other proteins. Members of the Hsp90 gene family are highly conserved, strongly expressed, and required for viability in eukaryotic organisms. To elucidate the role of Hsp90 in the activity of steroid hormone receptors in vivo, four Hsp90 mutatns, which cause defects in glucocorticoid receptor (GR) signaling, but support the viability of Saccharomyces cerevisiae, were previously isolated (Bohen, S. P., and Yamamoto, K. R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11424-11428). In this study, I characterize the effects of the Hsp90 mutants on GR ligand response, ligand binding activity, and aporeceptor complex stability. The mutants fall into two classes. Three of the Hsp90 mutants cause defects in GR ligand binding in vivo and form aporeceptor complexes that are unstable in vitro, relative to those containing wild-type Hsp90. The other mutant affects GR signaling, but aporeceptor complexes with this mutant are not defective for ligand binding or stability. These findings indicate that the binding of Hsp90 to GR in the aporeceptor complex is insufficient to induce a high ligand affinity conformation, rather the high ligand affinity to GR requires a specific interaction with Hsp90, which is altered by certain Hsp90 mutants.

LEM1, an ATP-binding-cassette transporter, selectively modulates the biological potency of steroid hormones.

The rat glucocorticoid receptor confers hormone-dependent transcriptional enhancement when expressed in yeast, thereby enabling the genetic identification of nonreceptor proteins that function in the hormone signal-transduction pathway. We isolated a yeast mutant, lem1, with increased sensitivity to dexamethasone and triamcinolone acetonide; responsiveness to a third agonist, deoxycorticosterone, is unaffected. Cloning of wild-type LEM1 revealed a putative transport protein of the ATP-binding cassette family. Dexamethasone accumulation is increased in lem1 cells, suggesting that wild-type LEM1 decreases dexamethasone potency by exporting this ligand. LEM1 appears to affect certain steroids and not others. We propose that transporters like LEM1 can selectively modulate the intracellular levels of steroid hormones. Differential activities of such transporters in mammalian cells might regulate hormone availability and thereby hormone signaling in a cell-type specific manner.

Isolation of Hsp90 mutants by screening for decreased steroid receptor function.

The 90-kDa heat shock protein Hsp90 represents a highly conserved strongly expressed gene family; in Saccharomyces cerevisiae, Hsp90 proteins are essential for cell viability. Hsp90 interacts with certain cellular proteins, including steroid hormone receptors, tyrosine and serine/threonine kinases, and other heat shock proteins, but its biological functions are not understood. The unliganded glucocorticoid receptor must interact with Hsp90 to acquire competence for high-affinity hormone binding and subsequent transcriptional regulation. By screening in yeast for defects in glucocorticoid receptor function, Hsp90 mutants were isolated. Four such mutants are described, all of which interact with the glucocorticoid receptor but display distinct defects in ligand responsiveness and differences in growth and resistance to high temperature.