Ulp1-YFP does not affect the localization of Gal3p-CFP or Gal80p-CFP. Yeast cells in which the genomic GAL1 locus had been modified to produce a version of Gal1p tagged at its carboxyl-terminal end with mCherry also produced Ulp1-YFP and either Gal80p-CFP (A and B) or Gal3p-CFP (C and D) from their native genomic loci. Cells were grown at 30°C in 2% raffinose (A and C) as the sole carbon source or taken from this medium, washed, resuspended in medium containing 2% galactose, and cultured for 2 h at 30°C (B and D). DIC, differential interference contrast. The merged panels are overlays of the YFP, CFP, and mCherry images. Bars = 5 μm.

Influence of Ulp1-Gal4p-YFP fusion proteins on integrity of nuclear pore complexes. Yeast cells produced a carboxyl-terminal mCherry-tagged version of Nup49p and a protein composed of the first 150 amino acids of Ulp1p fused to either full-length Gal4p and YFP (A and B) or amino acids 768 to 881 of Gal4p and YFP (C and D). In the presence of raffinose, the YFP and mCherry signals for both the full-length Gal4p and Gal4 AD fusion proteins are similar and indicate the locations of the nuclear membrane and the nuclear pore complexes, respectively. In the presence of galactose, the transcriptionally active full-length Gal4p fusion protein is punctate in appearance, but this does not affect the localization of nuclear pore complexes. The transcriptionally inactive Ulp1-Gal4AD-YFP fusion does not alter the appearance of the nuclear membrane in the presence of galactose. The merged panels are overlays of the DIC, YFP, and mCherry images. Bars = 5 μm.

Influence of Ulp1-Gal4AD-YFP on localization of Gal3p-CFP and Gal80p-CFP. Cells producing a carboxyl-terminally CFP-tagged version of either Gal80p (A to C) or Gal3p (D to F) also produced a fusion protein composed of the first 150 amino acids of Ulp1p, amino acids 768 to 881 of Gal4p, and YFP (Ulp1-Gal4AD-YFP). Cells were grown in 2% raffinose (A and D) or washed from this medium and resuspended in 1% raffinose and 1% galactose (B, C, E, and F). For cells in the presence of galactose, images were collected either 10 min after the addition of the fresh medium (B and E) or after 120 min (C and F). The merged panels are overlays of the YFP and CFP images. Bars = 5 μm.

Localization of Gal1p to Gal4p. Yeast cells in which the genomic GAL1 locus had been modified to produce a version of Gal1p tagged at its carboxyl-terminal end with mCherry also produced Ulp1-Gal4p-YFP. In addition, cells were modified to either produce Gal80p-CFP (A) or Gal3p-CFP (B) from the native genomic locus. Yeasts producing Gal1p-mCherry and Ulp1-Gal4p-YFP were also analyzed in a wild-type GAL80 background (C) or in a background in which the GAL80 gene had been deleted (D). Cells were grown at 30°C for 2 h in a galactose-containing medium. The merged panels in panels A and B are overlays of the YFP, CFP, and mCherry images, and those in panels C and D are overlays of the YFP and mCherry images. Bars = 5 μm. (E) Differential protein occupancy by the tagged protein at the GAL1 promoter, calculated as the ratio of binding to the UAS in comparison to that to the ORF, as indicated in the diagram above, was analyzed by chromatin immunoprecipitation. Relative occupancy was calculated using the 2^−ΔΔCT method, and the standard deviation was calculated for at least three independent immunoprecipitation experiments. Yeast cells contained either a wild-type GAL80 gene (bars 1 to 4) or a deletion of the gene (bars 5 and 6) and produced either HA-tagged Gal4p (bars 1 and 2) or HA-tagged Gal1p (bars 3 to 6). Cells were grown in the presence of glucose or galactose, as indicated. The occupancy of Gal4p in the presence of galactose was set at 100%, and the error bars indicate standard deviations. (F) Replacement of Gal3p and the GAL1 promoter by Gal1p. The GAL1 promoter occupancies by Gal3p-HA and Gal1p-HA in cells grown in the presence of glucose (bars 1 and 2) or raffinose (bars 3 and 4) or after exposure to galactose for 10 min, 60 min, or 12 h (bars 5 to 10) was calculated by chromatin immunoprecipitation as described for panel E. The occupancy of Gal4p at the UAS in the presence of galactose was set at 100%, and the error bars indicate standard deviations (n = 3).

Localization of Gal3p in cells lacking Gal1p and producing a nuclear membrane-tethered version of Gal4p. ΔGAL1 yeast cells producing either Gal80p-CFP (A to D) or Gal3p-CFP (E to H) and either Ulp1-YFP (A, B, E, and F) or Ulp1-Gal4AD-YFP (C, D, G, and H) were grown in medium containing either raffinose as the sole source of carbon (A, C, E, and G) or a mixture of raffinose and galactose (B, D, F, and H). Images were collected 12 h following the change of medium. The merged panels are overlays of the YFP and CFP images. Bars = 5 μm.

Model for the induction of yeast GAL genes. (A) In the absence of galactose, Gal3p and Gal80p are produced at relatively low levels in the cell, and both proteins may be found in the nucleus and cytoplasm. (B) Galactose entering the cell promotes an interaction between Gal3p and Gal80p. This association occurs, at least in part, at the location of Gal4p and results in the expression of the GAL genes. (C) As Gal1p is produced and accumulates to a high level, the complex between Gal3p and Gal80p is disrupted and replaced by a complex between Gal1p and Gal80p. The Gal1p-Gal80p interaction occurs at the location of Gal4p.