FIG. 7.
Complementation of yhp23 by human p23 in yeast cells. (A) Overexpression of HA-p23 suppresses the G400V ER phenotype. An HA epitope-tagged human p23 (HA-p23) was subcloned into the yeast expression vector pRS316GPD. The W303a yeast strain expressing G400V ER and an ERE–β-galactosidase reporter gene was transformed with expression vectors containing no insert (vector), HA-p23, or HA-yhp23. G400V ER transcriptional activity was measured by liquid β-galactosidase assay in galactose-containing medium with 1 nM 17-β-estradiol. Equal amounts of whole-cell lysates from the strain containing vector, HA-p23, and HA-yhp23 were analyzed by immunoblotting by using anti-HA antibody as described in Materials and Methods (bottom panel). (B) Human p23 partially complements the loss of yhp23 with respect to ER signaling. yhp23 KO yeast strain expressing wt ER and an ERE-responsive β-galactosidase reporter gene were transformed with expression vectors containing no insert (vector), HA-p23, or HA-yhp23. ER transcriptional activity was determined by liquid β-galactosidase assay in raffinose medium containing 0.1 nM 17-β-estradiol. Immunoblot analysis for HA-p23 and HA-yhp23 was performed as in panel A and demonstrates that HA-p23 and HA-yhp23 are expressed at similar levels (bottom panel).