Interactions of the Mcm1 MADS box protein with cofactors that regulate mating in yeast

Abstract

The yeast Mcm1 protein is a member of the MADS box family of transcriptional regulatory factors, a class of DNA-binding proteins that control numerous cellular and developmental processes in yeast, Drosophila melanogaster, plants, and mammals. Although these proteins bind DNA on their own, they often combine with different cofactors to bind with increased affinity and specificity to their target sites. To understand how this class of proteins functions, we have made a series of alanine substitutions in the MADS box domain of Mcm1 and examined the effects of these mutations in combination with its cofactors that regulate mating in yeast. Our results indicate which residues of Mcm1 are essential for viability and transcriptional regulation with its cofactors in vivo. Most of the mutations in Mcm1 that are lethal affect DNA-binding affinity. Interestingly, the lethality of many of these mutations can be suppressed if the MCM1 gene is expressed from a high-copy-number plasmid. Although many of the alanine substitutions affect the ability of Mcm1 to activate transcription alone or in combination with the alpha 1 and Ste12 cofactors, most mutations have little or no effect on Mcm1-mediated repression in combination with the alpha 2 cofactor. Even nonconservative amino acid substitutions of residues in Mcm1 that directly contact alpha 2 do not significantly affect repression. These results suggest that within the same region of the Mcm1 MADS box domain, there are different requirements for interaction with alpha 2 than for interaction with either alpha1 or Ste12. Our results suggest how a small domain, the MADS box, interacts with multiple cofactors to achieve specificity in transcriptional regulation and how subtle differences in the sequences of different MADS box proteins can influence the interactions with specific cofactors while not affecting the interactions with common cofactors.

FIG. 1.

Role of Mcm1 in a regulatory network for control of mating type functions. In a cells, Mcm1 activates a-specific gene expression. In α cells, Mcm1 activates α-specific gene expression in complex with α1 and represses a-specific genes in complex with α2. In the presence of a pheromone, Mcm1 combines with Ste12 in both haploid cell types to activate high-level expression of genes involved in cell cycle arrest and mating. Representative target genes in each class are shown.

FIG. 2.

Model of Mcm1 bound to DNA and positions of mutations that produce a lethal phenotype. (A) The views of the Mcm1 dimer binding to its site are derived from the coordinates of the crystal structure of the α2-Mcm1-DNA ternary complex (44). The two views represent a 90° rotation around the vertical axis. A cartoon of the Mcm1 dimer is shown with the DNA as a stick figure at the bottom of the structure. One monomer is in gray, and the other is in black. The positions of the secondary structures that form the different layers of the dimer are shown. NT ext., N-terminal extension. (B and C) Positions of alanine substitutions that fail to complement an mcm1 null mutation. A space-filling model of the Mcm1 dimer is shown in which the relative orientation of the models is roughly the same as that in panel A. Positions at which alanine substitutions cause a lethal phenotype in low copy numbers but result in viability at high copy numbers are gray. Positions at which alanine substitutions cause a lethal phenotype at low and high copy numbers are black. Many of these residues are buried in the interior of the dimer and are not visible from the surface. Panel C shows the lethal alanine substitutions in Mcm1 with a view of the dimer from the bottom of the structure seen through the DNA. Residues in the N-terminal extension were removed from this view to show the lethal residues that contact the DNA.

FIG. 3.

In vivo transcription assays of mcm1 alanine mutations. Alanine substitutions were made at each position in Mcm1, and the mutants were assayed for expression of lacZ reporter constructs by autonomous activation at a P(PAL) site (A), in complex with α1 at an α1-Mcm1 binding site from the STE3 promoter (B), in complex with Ste12 at a PRE site from the STE2 promoter (C), and for repression in complex with α2 at an α2-Mcm1 binding site from the STE6 promoter (D). Data are expressed as fold decreases with respect to wild-type activity. A value of 1 indicates wild-type activity, whereas large bars indicate significant decreases in activity. The solid bars represent mutations resulting in viability at low copy numbers; open bars represent mutations that cause inviability at low copy numbers but allow viability at high copy numbers. The fold decreases in activity are indicated for mutations that are above the top axis. Values above the horizontal lines were considered significant. Each bar represents the average of three transformants with standard deviations that usually varied by less than 10%. The X's indicate those mutations that are lethal when expressed at low or high copy numbers. Positions L50 and F77 were not tested. The asterisks in panel D denote those residues in Mcm1 that directly contact α2 in the crystal structure of the α2-Mcm1-DNA ternary complex (44).

FIG. 4.

Position in Mcm1 of alanine substitutions that affect transcriptional regulation with its cofactors. The positions of alanine substitutions that affect autonomous activation (A), activation in complex with α1 (B), activation in complex with Ste12 (C), and repression in complex with α2 (D) are black. Shaded residues are those that lie above the line in the corresponding panels in Fig. 3. The views shown are in the same orientation as those in Fig 2A and B. The positions of the residues in the linker region of α2 that contact Mcm1 are shown as stick figures in panel D.

FIG. 5.

DNA binding of Mcm1 alanine mutant proteins in combination with α1. Shown is the affinity of binding to the STE3 α1-Mcm1 site of wild-type (WT) Mcm1 (lanes 3 to 6) and mutant Mcm1 proteins with the V34A (lanes 7 to 10), K40A (lanes 11 to 14), F48A (lanes 15 to 18), Y70A (lanes 19 to 22), S73A (lanes 23 to 26), and S73R (lanes 27 to 30) mutations in the presence of α1. All of the Mcm1 proteins contain the entire MADS box domain (residues 1 to 97) and are titrated as fivefold dilutions from a concentration of 2 × 10⁻⁹ M (lanes 3, 7, 11, 15, 19, 23, and 27). Lane 1 contains wild-type Mcm1 in the absence of α1. Lanes 2 to 30 contain 100 ng of partially purified α1. The positions of Mcm1 and the Mcm1-α1 complex are indicated.

FIG. 6.

DNA binding of mcm1-encoded alanine mutant proteins in combination with α2. Shown is the affinity of binding to the STE6 α2-Mcm1 site of wild-type (WT) Mcm1 (lanes 3 to 6) and mutant proteins with the F48A (lanes 7 to 10), E49A (lanes 11 to 14), Y70A (lanes 15 to 18), and S73A (lanes 19 to 22) mutations (A) and those with the S51A (lanes 1 to 4), V52A (lanes 5 to 8), V69A (lanes 9 to 12), V81A (lanes 13 to 16), and R87A (lanes 17 to 20) mutations (B) in the presence of α2. The Mcm1 proteins were diluted by fivefold dilutions from a concentration of 4 × 10⁻¹⁰ M (lanes 1, 3, 7, 11, 15, and 19) in the presence of 4.7 × 10⁻⁷ M α2. Lane 1 in panel A shows binding by wild-type Mcm1 in the absence of α2, and lane 2 shows binding by α2 in the absence of Mcm1. The positions of α2, Mcm1, and the Mcm1-α2 complex are indicated.

FIG. 7.

DNA binding of radical double-mutation mcm1-encoded proteins in combination with α2. (A) Binding affinity of wild-type (WT) Mcm1 (lanes 1 to 4) and mutant proteins with the R87F/V69F (lanes 5 to 8), R87F/S73R (lanes 9 to 12), and R87A/S51A (lanes 13 to 16) mutations to the STE6 α2-Mcm1 site. The proteins were diluted by fivefold dilutions from a concentration of 4 × 10⁻¹⁰ M (lanes 1, 5, 9, and 13). (B) Binding affinity of wild-type Mcm1 (lanes 3 to 6) and mutant proteins with the R87F/V69F (lanes 7 to 10), R87F/S73R (lanes 11 to 14), and R87A/S51A (lanes 15 to 18) mutations in the presence of 4.7 × 10⁻⁷ M α2. The proteins were diluted by fivefold dilutions from a concentration of 4 × 10⁻¹⁰ M (lanes 3, 7, 11, and 15). In panel B, lane 1 shows the position of wild-type Mcm1 binding in the absence of α2 and lane 2 shows the position of α2 binding in the absence of Mcm1.

FIG. 8.

Mating pheromone halo assays of the wild-type (WT) and mcm1 mutant strains. The wild-type strain and the indicated mcm1 mutant strains in a MATa background (A) or a MATα background (B and C) were patched onto lawns of MATα (A and C) and MATa (B) pheromone-sensitive strains. The presence of a zone of growth inhibition around the patched cells indicates that they produce a pheromone of the mating type opposite to that of the cells on the lawn.