Intragenic suppression of Gal3C interaction with Gal80 in the Saccharomyces cerevisiae GAL gene switch - PubMed (original) (raw)

Comparative Study

Intragenic suppression of Gal3C interaction with Gal80 in the Saccharomyces cerevisiae GAL gene switch

Cuong Q Diep et al. Genetics. 2006 Jan.

Abstract

Gal4-mediated activation of GAL gene transcription in Saccharomyces cerevisiae requires the interaction of Gal3 with Gal80, the Gal4 inhibitor protein. While it is known that galactose and ATP activates Gal3 interaction with Gal80, neither the mechanism of activation nor the surface that binds to Gal80 is known. We addressed this through intragenic suppression of GAL3C alleles that cause galactose-independent Gal3-Gal80 interaction. We created a new allele, GAL3SOC, and showed that it suppressed a new GAL3C allele. We tested the effect of GAL3SOC on several newly isolated and existing GAL3C alleles that map throughout the gene. All except one GAL3C allele, D368V, were suppressible by GAL3SOC. GAL3SOC and all GAL3C alleles were localized on a Gal3 homology model that is based on the structure of the highly related Gal1 protein. These results provide evidence for allosterism in the galactose- and ATP-activation of Gal3 binding to Gal80. In addition, because D368V and residues corresponding to Gal80-nonbinder mutations colocalized to a domain that is absent in homologous proteins that do not bind to Gal80, we suggest that D368 is a part of the Gal80-binding surface.

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Figures

Figure 1.

Figure 1.

Newly isolated GAL3 mutations constitutively activate the GAL gene switch.(A) Galactose-independent interaction between GSTGal3 and Gal80 in vitro, as determined by a pull-down assay. Yeast whole-cell extracts containing GSTGal3 and Gal80 were incubated at 4° for 4 hr with glutathione sepharose, washed three times, and then boiled and separated on SDS–PAGE and analyzed by Western blot. GSTGAL3 and GAL80 were expressed under the ADH2 promoter in pMPW60 and pMPW82, respectively. (B) Constitutive in vivo expression of the integrated P_GALHIS3_ reporter gene determined by a colony-growth assay. Strain Sc781 carrying the indicated plasmid was grown in liquid culture to late log phase and adjusted to the same number of cells. Dilutions (10-fold) were spotted on selective agar plates and incubated for 3–6 days. Wild-type and GAL3 mutants were expressed under their native promoter in pTEB16. (C) Constitutive expression of the endogenous MEL1 gene determined by an α-galactosidase enzymatic-activity assay. Strain Sc781 carrying the indicated plasmid was grown in liquid culture to an OD600 of 0.5, and the cell-free culture media was removed and assayed for α-galactosidase activity. Three independent transformants were assayed, and the average and standard deviation were reported. Wild-type and GAL3 mutants were expressed under their native promoter in pTEB16.

Figure 2.

Figure 2.

The suppression of constitutivity of _GAL3_-S509D by GAL3_-D68S.(A) Expression of the integrated P_GALHIS3 reporter gene in the yeast strain Sc781 determined by a colony-growth assay. (B) Expression of the endogenous MEL1 gene in the yeast strain Sc781 determined by an α-galactosidase enzymatic-activity assay. (C) Interaction between Gal3 and Gal80 in vitro as determined by a pull-down assay.

Figure 3.

Figure 3.

Specificity in suppression of GAL3C alleles.(A) Expression of the integrated P_GALHIS3_ reporter gene in the yeast strain Sc781 determined by a colony-growth assay. (B) Expression of the endogenous MEL1 gene in the yeast strain Sc781 determined by an α-galactosidase enzymatic-activity assay. (C) Interaction between Gal3 and Gal80 in vitro as determined by a pull-down assay. (D) Protein levels of the Gal3-V396A and Gal3-V396A/D68S mutant proteins not tagged with GST, as determined by a Western blot.

Figure 3.

Figure 3.

Specificity in suppression of GAL3C alleles.(A) Expression of the integrated P_GALHIS3_ reporter gene in the yeast strain Sc781 determined by a colony-growth assay. (B) Expression of the endogenous MEL1 gene in the yeast strain Sc781 determined by an α-galactosidase enzymatic-activity assay. (C) Interaction between Gal3 and Gal80 in vitro as determined by a pull-down assay. (D) Protein levels of the Gal3-V396A and Gal3-V396A/D68S mutant proteins not tagged with GST, as determined by a Western blot.

Figure 3.

Figure 3.

Specificity in suppression of GAL3C alleles.(A) Expression of the integrated P_GALHIS3_ reporter gene in the yeast strain Sc781 determined by a colony-growth assay. (B) Expression of the endogenous MEL1 gene in the yeast strain Sc781 determined by an α-galactosidase enzymatic-activity assay. (C) Interaction between Gal3 and Gal80 in vitro as determined by a pull-down assay. (D) Protein levels of the Gal3-V396A and Gal3-V396A/D68S mutant proteins not tagged with GST, as determined by a Western blot.

Figure 4.

Figure 4.

Multiple sequence alignment of Gal3 and related proteins and the predicted secondary structure of the Gal3 homology model. The multiple sequence alignment was generated using the T-Coffee server. The predicted secondary structure of the Gal3 homology model is represented by arrows (β-sheets) and rectangles (α-helices). The N-terminal domain is blue while the C-terminal domain is red. The four-helical insertion domain is represented by vertical red lines. (•) GAL3C alleles, (○) KlGAL1 constitutive mutants, and (□) Klgal1 interaction-defective mutants. MVK_ Mj is mevalonate kinase of Methanococcus jannaschii, GAL1_ Kl is GAL1 of Kluyveromyces lactis, GAL3_ Sc and GAL1_ Sc are GAL3 and GAL1, respectively, of Saccharomyces cerevisiae.

Figure 5.

Figure 5.

Locations of residues corresponding to mutations affecting binding to Sc/_Kl_Gal80 ona Gal3 homology model.(A) Location of residues shown in space-filling representation. Residues colored cyan are the Gal3 equivalent residues of Klgal1 reg−kin+ mutations. The positions of GAL3C mutations included in the suppression analyses are red. The two views are approximately orthogonal, due to a rotation around the _y_-axis in a counterclockwise direction. (B) Ribbon diagram in the same orientations as in A. In addition to the coloring adopted in A, residues in yellow are the Gal3-equivalent positions of _Kl_Gal1 constitutive mutations, residues in blue are the Gal3-equivalent positions of _Kl_gal1 reg−kin− mutations, and the position of GAL3SOC is green. Residues that are described in detail in the text have been labeled. For clarity, the labeling of other residues has been omitted.

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