Transcriptional activation by estrogen receptor (ERalpha) and steroid receptor coactivator (SRC1) involves distinct mechanisms in yeast and mammalian cells.

Steroid receptors activate transcription in yeast cells via interactions with endogenous coactivators and/or basal factors. We examined the effects of mutations in the ligand binding domain on the transcriptional activity of ERalpha in yeast. Our results show that mutations in Helix 3 (K366A) and Helix 12 (M547A, L548A) disrupt transcriptional activity of ERalpha in yeast, as previously observed in mammalian cells. However, replacement of a conserved tyrosine residue in Helix 12 with alanine or aspartate (Y541A and Y541D), which renders ERalpha constitutively active in mammalian cells, had only a weak stimulatory effect on ligand-independent reporter activation by ERalpha in yeast. Two-hybrid interaction experiments revealed that a Y541A mutant expressed in yeast was capable of ligand-independent binding to a mammalian coactivator, suggesting that there is a subtle difference in how this mutant interacts with mammalian and yeast cofactors. We also show that the ligand-dependent activities of ERalpha and progesterone receptor (PR) in yeast cells were strongly enhanced by the human p160 protein steroid receptor coactivator (SRC1), but not by CREB-Binding Protein (CBP) or the p300/CBP associated factor (P/CAF). Although the SRC1 activation domains AD1 and AD2 are functional in yeast, deletion of these sequences only partially impaired SRC1 coactivator function in this organism; this is in contrast to similar experiments in mammalian cells. Thus SRC1 sequences involved in recruitment of CBP/p300 and Co-Activator-Associated Arginine Methyltransferase (CARM-1) in mammalian cells are not essential for its function in yeast, suggesting that SRC1 operates via distinct mechanisms in yeast and mammalian cells.