A role for the Hsp40 Ydj1 in repression of basal steroid receptor activity in yeast.
In addition to its roles in translocation of preproteins across membranes, Ydj1 facilitates the maturation of Hsp90 substrates, including mammalian steroid receptors, which activate transcription in yeast in a hormone-dependent manner. To better understand Ydj1's function, we have constructed and analyzed an array of Ydj1 mutants in vivo. Both the glucocorticoid receptor and the estrogen receptor exhibited elevated activity in the absence of hormone in all ydj1 mutant strains, indicating a strict requirement for Ydj1 activity in hormonal control. Glucocorticoid receptor containing a mutation in the carboxy-terminal transcriptional activation domain, AF-2, retained elevated basal activity, while mutation of the amino-terminal transactivation domain, AF-1, eliminated the elevated basal activity observed in ydj1 mutant strains. This result indicates that the source of activity is AF-1, which is normally repressed by the carboxy-terminal hormone binding domain in the absence of hormone. Chimeric proteins containing the hormone binding domain of the estrogen or glucocorticoid receptor fused to heterologous activation and DNA binding domains also exhibited elevated activity in the absence of hormone. Thus, Ydj1 mutants appear to increase basal receptor activity by altering the ability of the hormone binding domain of the receptor to repress nearby activation domains. We propose that Ydj1 functions to present steroid receptors to the Hsp90 pathway for folding and hormonal control. In the presence of Ydj1 mutants that fail to bind substrate efficiently, some receptor escapes the Hsp90 pathway, resulting in constitutive activity.
Johnson
J L
JL
Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Craig
E A
EA
D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D017930:Genes, Reporter; D016391:Genes, src / Q000235:genetics; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins; D015151:Immunoblotting; D016296:Mutagenesis; D010641:Phenotype; D010957:Plasmids; D017434:Protein Structure, Tertiary; D011960:Receptors, Estrogen / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D011987:Receptors, Steroid / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000378:metabolism
D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011960:Receptors, Estrogen; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C070319:YDJ1 protein, S cerevisiae; D001616:beta-Galactosidase