title: Modular control of cross-oligomerization: analysis of superstabilized Hsp90 homodimers in vivo.
abstract: Homo-oligomeric proteins fulfill numerous functions in all cells. The ability to co-express subunits of these proteins that preferentially self-assemble without cross-oligomerizing provides for controlled experiments to analyze the function of mutant homo-oligomers in vivo. Hsp90 is a dimeric chaperone involved in the maturation of many kinases and steroid hormone receptors. We observed that co-expression of different Hsp90 subunits in Saccharomyces cerevisiae caused unpredictable synthetic growth defects due to cross-dimerization. We engineered superstabilized Hsp90 dimers that resisted cross-dimerization with endogenous Hsp90 and alleviated the synthetic growth defect. Superstabilized Hsp90 dimers supported robust growth of S. cerevisiae, indicating that dissociation of Hsp90 dimers could be hindered without compromising essential function. We utilized superstabilized dimers to analyze the activity of ATPase mutant homodimers in a temperature-sensitive yeast background where elevated temperature inactivated all other Hsp90 species. We found that ATP binding and hydrolysis by Hsp90 are both required for the efficient maturation of glucocorticoid receptor and v-Src, confirming the critical role of ATP hydrolysis in the maturation of steroid hormone receptors and kinases in vivo.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
authors.forename: Natalie
authors.initials: N
authors.lastname: Wayne
authors.forename: Yushuan
authors.initials: Y
authors.lastname: Lai
authors.forename: Les
authors.initials: L
authors.lastname: Pullen
authors.forename: Daniel N
authors.initials: DN
authors.lastname: Bolon
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D013696:Temperature; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: D018841:HSP90 Heat-Shock Proteins; D050505:Mutant Proteins; D011965:Receptors, Glucocorticoid; D015688:Oncogene Protein pp60(v-src); D000251:Adenosine Triphosphatases