FIG. 6.

ER and yhp23-GFP colocalize within the nucleus of yeast cells. To examine yhp23 subcellular localization in yeast cells, a yhp23-GFP fusion protein was constructed. Yeast strains were created that express yhp23-GFP (A) or GFP (C) either alone or in combination with G400V ER (B and C), wt ER (D and E), or GR (F). Cells were grown in galactose-raffinose-containing medium in the absence or the presence of 17-β-estradiol. Cells were fixed, permeabilized, and incubated with the appropriate receptor primary antibody, a corresponding Texas-red-conjugated secondary antibody, and the DNA in the nucleus was stained with Hoechst dye H334211. The GFP, Texas red, and Hoechst fluorescent signals were visualized by using a Zeiss Axioplan 2 fluorescence microscope. Note that yhp23-GFP is expressed throughout the cytoplasm in the absence of ER expression. In the presence of coexpressed G400V ER as well as wt ER, yhp23-GFP becomes localized to the nucleus. Incubation of the wt ER strain in 1 μM 17-β-estradiol results in the redistribution of yhp23-GFP from the nucleus to the cytoplasm, thereby reversing the ER-yhp23-GFP colocalization observed in the absence of estradiol. α-S.R., anti-steroid receptor primary antibody.