Genetic evidence for sites of interaction between the Gal3 and Gal80 proteins of the Saccharomyces cerevisiae GAL gene switch

Abstract

Galactose-activated transcription of the Saccharomyces cerevisiae GAL genes occurs when Gal3 binds the Gal4 inhibitor, Gal80. Noninteracting variants of Gal3 or Gal80 render the GAL genes noninducible. To identify the binding determinants for Gal3's interaction with Gal80 we carried out GAL3-GAL80 intergenic suppression analyses and selected for new GAL3 mutations that impair the Gal3-Gal80 interaction. We show that a GAL3(C)-D368V mutation can suppress the noninducibility due to a GAL80(S-1)-G323R mutation, and a GAL80-M350C mutation can suppress the noninducibility due to a gal3-D111C mutation. A reverse two-hybrid selection for GAL3 mutations that impair the Gal3-Gal80 interaction yielded 12 single-amino-acid substitutions at residues that are predicted to be surface exposed on Gal3. The majority of the affected Gal3 residues localized to a composite surface that includes D111 and a sequence motif containing D368, which has been implicated in interaction with Gal80. The striking colocalization of intergenic suppressor residues and Gal80 nonbinder residues identifies a Gal3 surface that likely interacts with Gal80.

Figure 1.—

Allele-specific intergenic suppression of GAL80^S-1-G323R by GAL3^C-D368V. (A) Colony-growth assay. The strains Sc724 (genomic GAL80 WT), Sc750 (integrated GAL80^S-0), Sc751 (integrated GAL80^S-1), and Sc787 (GAL80^S-2 carried on pMPW87) were transformed with the empty vector (pRS414), GAL3 WT (pTEB16), GAL3^C-V69E (pCD71), GAL3^C-F237Y (pCL-3C-311), GAL3^C-S509P (pCL-3C-362), GAL3^C-S509L (pCL-3C-371), or GAL3^C-D368V (pCL-3C-322). Cells were grown in selective liquid media until late log phase, spotted onto the appropriate selective agar plates with or without galactose, and incubated for 4 days. Intergenic suppression was determined by colony growth directly coupled to expression of the HIS3 reporter gene driven by the GAL1 promoter integrated in the genome of each strain (P_GALHIS3). (B) GST pull-down assay. Protein extracts were prepared from the strain Sc787 (gal1Δgal3Δgal80Δ) bearing GST (pMPW61), GST-GAL3 (pMPW60), or GST-GAL3^C-D368V (pCLAGC-322) plus either GAL80 (pMPW82) or GAL80^S-1-G323R (pMPW86). One milligram of protein extract was incubated with GT–Sepharose for 2 hr at 4° either in the absence or in the presence of galactose and ATP, washed three times, and then boiled and resolved on standard SDS–PAGE and analyzed by Western blot. GST-Gal3 and Gal80 were detected on the same blot by using a mixture of rabbit polyclonal anti-Gal80 (1:300 dilution) and rabbit polyclonal anti-GST (1:3000 dilution).

Figure 1.—

Allele-specific intergenic suppression of GAL80^S-1-G323R by GAL3^C-D368V. (A) Colony-growth assay. The strains Sc724 (genomic GAL80 WT), Sc750 (integrated GAL80^S-0), Sc751 (integrated GAL80^S-1), and Sc787 (GAL80^S-2 carried on pMPW87) were transformed with the empty vector (pRS414), GAL3 WT (pTEB16), GAL3^C-V69E (pCD71), GAL3^C-F237Y (pCL-3C-311), GAL3^C-S509P (pCL-3C-362), GAL3^C-S509L (pCL-3C-371), or GAL3^C-D368V (pCL-3C-322). Cells were grown in selective liquid media until late log phase, spotted onto the appropriate selective agar plates with or without galactose, and incubated for 4 days. Intergenic suppression was determined by colony growth directly coupled to expression of the HIS3 reporter gene driven by the GAL1 promoter integrated in the genome of each strain (P_GALHIS3). (B) GST pull-down assay. Protein extracts were prepared from the strain Sc787 (gal1Δgal3Δgal80Δ) bearing GST (pMPW61), GST-GAL3 (pMPW60), or GST-GAL3^C-D368V (pCLAGC-322) plus either GAL80 (pMPW82) or GAL80^S-1-G323R (pMPW86). One milligram of protein extract was incubated with GT–Sepharose for 2 hr at 4° either in the absence or in the presence of galactose and ATP, washed three times, and then boiled and resolved on standard SDS–PAGE and analyzed by Western blot. GST-Gal3 and Gal80 were detected on the same blot by using a mixture of rabbit polyclonal anti-Gal80 (1:300 dilution) and rabbit polyclonal anti-GST (1:3000 dilution).

Figure 2.—

Intergenic suppression of a gal3-D111C noninducible mutation by a GAL80-M350C mutation. The strain Sc787 carrying GAL3 and GAL80 on separate plasmids was grown in selective liquid media until late log phase and 10-fold dilutions were spotted on selective agar plates with or without galactose and incubated up to 6 days. Intergenic suppression was determined by colony growth coupled to P_GALHIS3 expression. Row 1, GAL3 WT (pMPW66) + vector (pRS416); row 2, GAL3 WT + GAL80 WT (pMPW82); row 3, GAL3 WT + GAL80-M350C (pCD125); row 4, GAL3-D111C (pCD123) + vector; row 5, GAL3-D111C + GAL80 WT; row 6, GAL3-D111C + GAL80-M350C; row 7, vector (pRS414) + vector; row 8, vector + GAL80 WT; row 9, vector + GAL80-M350C.

Figure 3.—

Impaired two-hybrid interaction of the Gal3-SA variants with Gal80. Yeast reverse and forward two-hybrid interactions of Gal3 variants with Gal80 are shown. The strain ScPD2 expressing DBD-GAL80 (pAKS42) and VP16 (pVP16), GAL3-SA-VP16 (pCD107), or the GAL3 mutant alleles in the GAL3-SA-VP16 background was grown in selective liquid media until late log phase and 10-fold dilutions were spotted on selective agar plates containing galactose and 0.1% 5-FOA (reverse two-hybrid) or galactose without 5-FOA (forward two-hybrid).

Figure 4.—

Physical interaction of GST-Gal3-SA variant proteins with Gal80 in vitro. Protein extracts from the strain Sc787 expressing GAL80 (pMPW82) and GST (pMPW61), GST-GAL3-SA (pCD121), or the GAL3 mutant alleles in the GST-GAL3-SA background were prepared. Approximately 1 mg of protein extracts was incubated with GT–Sepharose for 2 hr at 4°, washed three times, and then boiled and resolved on standard SDS–PAGE and analyzed by Western blot using antibodies against GST and Gal80 together at a 1:200 dilution.

Figure 5.—

Impaired activation of the GAL promoter by the gal3^80NB mutant alleles. The strain Sc781 carrying the vector (pRS414), GAL3 WT (pTEB16), GAL3-SA (pAKS130), or the gal3^80NB mutant alleles in the GAL3-SA background was grown in selective liquid media until late log phase and 10-fold dilutions were spotted on selective agar plates. Activation of the GAL promoter was determined by colony growth coupled to P_GALHIS3 expression.

Figure 6.—

Capacity of gal3^80NB mutant alleles to complement the gal1Δ. The strain Sc754 (gal1Δ) carrying the vector (pRS414), GAL3 WT (pTEB16), GAL3-SA (pAKS130), or the gal3^80NB mutant alleles in the GAL3-SA background was grown in selective liquid media until late log phase and 10-fold dilutions were spotted on selective agar plates. Complementation of the gal1Δ was determined by colony growth on agar plates that contained galactose as the sole carbon source.

Figure 7.—

Structural interpretation of the Gal3^80NB variants. Amino acids corresponding to the Gal3^80NB variants were mapped onto the Gal3 homology model (previously derived) using the program PyMOL. The model was rendered to represent spheres (top) or spheres and ribbons (bottom), with each model having a 90° rotation to the right. Green, amino acids of Gal3^80NB variants that are exposed on the surface of the model; red, amino acids of Gal3^80NB variants that are buried in the hydrophobic core of the model; blue, D368, a residue within the insertion motif; cyan, V69, F237, and S509, residues within the hinge region; magenta, galactose and AMPPNP.

Figure 8.—

The upper lip and insertion motif of Gal3. (A) Multiple-sequence alignment. The alignment was performed using the TCoffee web server under the 3DCoffee mode, which considered secondary structural elements. Green, Gal1-Y274 (hydrogen bonding to galactose) and Gal3-Y266 (corresponds to Gal1-Y274); black bar, upper lip helix of HSK; red, corresponding upper lip helix of Gal1 and Gal3; blue, four helices of the insertion motif; yellow, Gal3-D368 (a residue within the insertion motif). Protein Data Bank codes: 1H72, HSK of Methanococcus jannaschii; 1PIE, galactokinase of Lactococcus lactis; 1S4E, galactokinase of Pyrococcus furiosus; 2AJ4, galactokinase (Gal1) of Saccharomyces cerevisiae. (B) Ribbon and sphere rendering of the Gal3 homology model. Green, Gal3-Y266; red, upper lip helix; blue, insertion motif helices; yellow, Gal3-L103, -D111, and -D368; magenta, galactose and AMPPNP.

Figure 8.—

The upper lip and insertion motif of Gal3. (A) Multiple-sequence alignment. The alignment was performed using the TCoffee web server under the 3DCoffee mode, which considered secondary structural elements. Green, Gal1-Y274 (hydrogen bonding to galactose) and Gal3-Y266 (corresponds to Gal1-Y274); black bar, upper lip helix of HSK; red, corresponding upper lip helix of Gal1 and Gal3; blue, four helices of the insertion motif; yellow, Gal3-D368 (a residue within the insertion motif). Protein Data Bank codes: 1H72, HSK of Methanococcus jannaschii; 1PIE, galactokinase of Lactococcus lactis; 1S4E, galactokinase of Pyrococcus furiosus; 2AJ4, galactokinase (Gal1) of Saccharomyces cerevisiae. (B) Ribbon and sphere rendering of the Gal3 homology model. Green, Gal3-Y266; red, upper lip helix; blue, insertion motif helices; yellow, Gal3-L103, -D111, and -D368; magenta, galactose and AMPPNP.