To address the nature of the interaction between Gal4p, Gal80p, and Gal3p during the galactose induction process, we set out to determine the localization and interactions of each of the regulatory proteins. We and others have previously shown that Gal4p accumulates, albeit at a low level, in the nuclei of cells and that both Gal80p and Gal3p are distributed in both the nucleus and the cytoplasm (10
). The low level of Gal4p within cells and its relatively diffuse localization within the nucleus (28
) made it technically difficult to determine the compositions of protein complexes that form with it on the promoters of genes. We therefore set out to deliberately mislocalize Gal4p to a specific region of the nucleus. This was achieved by fusing a version of Gal4p, tagged with YFP, to the first 150 amino acids of Ulp1p, a protein that has previously been shown to localize to the inner side of the nuclear membrane (16
First, we showed that a fusion of Ulp1p to YFP had no effect on the localization of either Gal80p or Gal3p (). The Ulp1-YFP fusion protein was detected as a discrete circle indicating the position of the nuclear membrane and, by inference, the location of the nucleus itself. The localization of Ulp1-YFP was unaltered in the presence of either raffinose (a noninducing carbon source for the GAL
genes) or galactose (, Ulp1-YFP panels). In cells expressing this protein, the localization of both Gal80p and Gal3p (each tagged with CFP) was found to be both nuclear and cytoplasmic (, CFP panels), as previously reported for cells not harboring an Ulp1-YFP fusion (28
). We have previously shown that these Gal80p and Gal3p fusion proteins function indistinguishably from their untagged counterparts (28
). Both Gal80p-CFP and Gal3p-CFP were produced from their genomic loci, and the production of both proteins was found to increase in the presence of galactose (B and D). However, the increase in production levels was not accompanied by an alteration in the cellular location of either protein (B and D). In addition, in the same cells, the production of the galactokinase Gal1p tagged with mCherry was found to increase from undetectable levels in the presence of raffinose (A and C) to a readily detectable signal in both the nucleus and cytoplasm in cells in the presence of galactose (B and D).
Fig 1 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 (more ...)
Two fusion proteins were constructed in which Gal4p sequences were tethered to the first 150 amino acids of Ulp1p and YFP. One of these fusion proteins contained just the activation domain of Gal4p (amino acids 768 to 881) fused to Ulp1p (residues 1 to 150) and YFP, while the other contained the full-length Gal4p sequence (amino acids 1 to 881). The localization of these proteins was monitored in cells by fluorescence microscopy to detect the YFP tag (). In the presence of raffinose, both proteins were found to be located at the nuclear membrane (A and C), as indicated by their colocation with the nuclear pore complex protein Nup49p, which was tagged in the same cells with mCherry. In the presence of galactose, the fusion protein containing just the activation domain of Gal4p (Ulp1-Gal4AD-YFP) (D) remained largely colocalized with Nup49p, while the full-length fusion protein (Ulp1-Gal4p-YFP) (B) adopted a more punctate appearance. We assume that the spotted manifestation was related to having a transcriptionally active protein tethered to both DNA and the nuclear periphery. Under galactose-containing conditions, the integrity of the nuclear membrane itself appears to remain intact, as the distribution of nuclear pore complexes (B, Nup49p-mCherry panel) was largely unaltered in these cells after switching from raffinose to galactose (A and B). The fusion protein containing the full-length Gal4p sequence was able to support growth of the cells on galactose when it was expressed in yeast cells that contained no other source of Gal4p (see Fig. S1 in the supplemental material), indicating that the fusion retained the ability to function as an active Gal4p molecule.
Fig 2 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 (more ...)
The nuclear membrane-tethered fusion protein containing the Gal4p activation domain was found to localize to the nuclear membrane (, Ulp1-Gal4AD-YFP panel) in a manner similar to that of Ulp1-YFP (), but this protein was able to colocalize Gal80p-CFP to the nuclear membrane (A to C). The localization of Gal80p-CFP to the nuclear membrane by Ulp1-Gal4AD-YFP was observed in both raffinose- and galactose-containing media, suggesting that the complex between Gal4p and Gal80p does not dissociate substantially in the switch between uninduced and induced GAL gene expression. A comparison of the localization of Gal3p-CFP in a yeast strain expressing Ulp1-Gal4AD-YFP showed little difference in the nuclear and cytoplasmic localization of Gal3p in cells grown in the presence of raffinose or in cells that had been grown for a substantial period (>2 h) in galactose-containing medium (D and F). However, at times shortly after the addition of galactose to the cells (~10 min), we observed a brief localization of Gal3p-CFP at the nuclear membrane (E). At later times in galactose-containing medium, this localization dissipated (F). A time-lapse movie showing the transient association of Gal3p with nuclear membrane-tethered Gal4p is shown in Movie S1 in the supplemental material.
Fig 3 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, (more ...)
A similar pattern of Gal80p and Gal3p localization in both raffinose- and galactose-containing media to that observed in was also observed for a fusion protein composed of full-length Gal4p fused to Ulp1 and YFP (see Fig. S2 in the supplemental material). In raffinose, the Ulp1-Gal4p-YFP fusion protein was localized as a relatively discrete circle with similar characteristics to those of the Ulp1-Gal4AD-YFP protein shown in , and Gal80p-CFP colocalized with this protein under these conditions (see Fig. S2A in the supplemental material). In galactose, the Ulp1-Gal4p-YFP fusion protein adopted a more punctate appearance, but mirroring our results with Ulp1-Gal4AD-YFP (), the colocalization of Gal3p (albeit briefly) and Gal80p was still observed (see Fig. S2B, C, E, and F in the supplemental material).
The brief association of Gal3p with a complex of Gal4p and Gal80p may suggest that, for example, Gal80p is altered in some way by the transient association with Gal3p to reduce its inhibitory effect on the transcriptional activity of Gal4p. Evidence for the posttranscriptional modification of Gal80p is very limited. Therefore, we sought to understand how a transient association with a ligand sensor could result in continued and long-term activation of the GAL genes. We have already shown that the galactokinase (Gal1p) is located relatively uniformly in both the nuclei and cytoplasm of yeast cells that are grown in the presence of galactose (B and D). We next asked whether mislocalization of Gal4p was able to influence the localization of Gal1p (). In cells producing Ulp1-Gal4p-YFP as the sole source of Gal4p and grown in galactose for 2 h, we observed colocalization between the YFP signal and Gal80p-CFP (A) but not Gal3p-CFP (B). However, in the same cells, we observed colocalization between the YFP signal and Gal1p-mCherry (A to C). The localization of Gal1p by Ulp1-Gal4p-YFP was not replicated by either Gal7p or Gal10p under similar conditions (see Fig. S3 in the supplemental material). In addition, the deletion of GAL80 abrogated the localization of Gal1p by Ulp1-Gal4p-YFP (D), indicating that the localization of the galactokinase occurred through Gal80p tethered at the nuclear membrane by Gal4p.
Fig 4 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 (more ...)
The association of the galactokinase with Gal80p also occurred at transcriptionally active promoters (E). Chromatin immunoprecipitation of either HA-tagged Gal4p (E, bars 1 and 2) or Gal1p (E, bars 3 and 4) showed a clear increase in the accumulation of the tagged protein at the GAL1
promoter when cells were grown in the presence of galactose. For both Gal4p (9
) and Gal1p, protein levels in cells grown under noninducing conditions were considerably lower than those in cells grown in the presence of galactose (see Fig. S4 in the supplemental material). However, in the absence of Gal80p, galactokinase levels were high in both the presence and absence of galactose (see Fig. S4 in the supplemental material), but the lack of Gal80p means that Gal1p did not become localized to active GAL
promoters (E, bars 5 and 6). We then used chromatin immunoprecipitation to investigate the timing of the interaction of Gal3p with the GAL1
promoter (F). HA-tagged Gal3p was found to associate with the GAL1
promoter at low levels when the cells were grown in medium containing either glucose or raffinose as a carbon source (F, bars 1 and 3). However, when the cells were switched to medium containing galactose, a relatively strong but short-lived association of Gal3p with the GAL1
promoter occurred (F, bars 5, 7, and 9). This interaction could be detected around 10 min after the addition of galactose to cultures (F, bar 5) but not after the cells were incubated for 60 min or longer (F, bar 7). Over this time frame, the association of Gal1p with its own promoter increased (F, bars 6, 8, and 10), suggesting that the brief association of Gal3p with the transcriptionally active DNA-tethered Gal4p-Gal80p complex is replaced by Gal1p shortly after induction.
Therefore, it appears that the transient interaction between Gal4p, Gal80p, and Gal3p that occurs early after the addition of galactose to yeast cells is replaced by a complex between Gal4p, Gal80p, and Gal1p at later times. A prediction of this observation would be that in the absence of Gal1p, the complex between Gal4p, Gal80p, and Gal3p should be more stable following the switch of sugar source from raffinose to galactose. The results of such an experiment are shown in . In the absence of Gal1p, the interaction between Ulp1-Gal4AD-YFP and Gal80p-CFP was observed in the presence of both raffinose and galactose (C and D). A fusion protein composed of just Ulp1p-YFP was unable to drive the localization of Gal80p-CFP to the nuclear membrane (A and B). However, the absence of Gal1p stabilized the association between Ulp1-Gal4AD-YFP and Gal3p-CFP, such that the association between the two proteins at the nuclear membrane was now observed constitutively in the presence of galactose (H), even when cells were exposed to the presence of galactose for extended periods (12 h). Again, an Ulp1p-YFP fusion protein lacking Gal4p sequences was unable to colocalize Gal3p-CFP to the nuclear membrane (E and F). Movie S2 in the supplemental material shows the formation of a stable complex of Gal3p-CFP with nuclear membrane-tethered Gal4p following the addition of galactose to cells lacking Gal1p. In the absence of Gal1p, the interaction between Gal3p and the Gal4p-Gal80p complex is reversible. The removal of galactose from the medium resulted in the loss of Gal3p localization within 60 min (see Movie S3 in the supplemental material). A stabilization of the interaction between Gal3p and full-length Gal4p in the absence of Gal1p was also noted (see Fig. S5 in the supplemental material).
Fig 5 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, (more ...)