Recruitment of the ArgR/Mcm1p repressor is stimulated by the activator Gcn4p: a self-checking activation mechanism

Abstract

Transcription of the arginine biosynthetic gene ARG1 is repressed by the ArgR/Mcm1p complex in arginine-replete cells and activated by Gcn4p, a transcription factor induced by starvation for any amino acid. We show that all four subunits of the arginine repressor are recruited to ARG1 by Gcn4p in cells replete with arginine but starved for isoleucine/valine. None of these proteins is recruited to the Gcn4p target genes ARG4 and SNZ1, which are not regulated by ArgR/Mcm1p. Mcm1p and Arg80p were found in a soluble complex lacking Arg81p and Arg82p, and both Mcm1p and Arg80p were efficiently recruited to ARG1 in wild-type cells in the presence or absence of exogenous arginine, and also in arg81Delta cells. By contrast, the recruitment of Arg81p and Arg82p was stimulated by exogenous arginine. These findings suggest that Gcn4p constitutively recruits an Mcm1p/Arg80p heterodimer and that efficient assembly of a functional repressor also containing Arg81p and Arg82p occurs only in arginine excess. By recruiting an arginine-regulated repressor, Gcn4p can precisely modulate its activation function at ARG1 according to the availability of arginine.

Fig. 1.

Gcn4p recruits the ArgR/Mcm1p complex to the promoter at ARG1 but not at SNZ1 or ARG4. (A) GCN4 strains containing myc-tagged alleles of MCM1 (SY337), ARG80 (SY373), ARG81 (SY375), and ARG82 (SY377) were transformed with empty vector or 2 μm plasmid pHQ1239 harboring GCN4-HA. gcn4Δ strains with myc-tagged MCM1 (SY339), ARG80 (SY374), ARG81 (SY376), or ARG82 (SY378) were transformed with empty vector. Cells were grown to an OD₆₀₀ of ≈1.0 in synthetic complete-Ura medium (containing 0.5 mM Arg), treated for 2 h with SM (0.6 μg/ml), and cross-linked with HCHO. ChIP assays were conducted by using anti-myc antibodies and PCR primers specific for the ARG1 UAS or POL1 ORF. The ARG1_UAS signal in the immunoprecipitate (IP) was normalized for the corresponding POL1 signal and plotted in the histogram. (B and C) The same IP samples described in A were analyzed by using primers to amplify the SNZ1 and ARG4 UAS elements. (D and E) The ARG80-myc or ARG81-myc strains described in A were grown and induced with SM, and WCEs were subjected to Western analysis with antibodies against myc, Mcm1p, or Gcd6p, as indicated on the left. Three different amounts of each extract were loaded in adjacent lanes to ensure that Western signals were dose-dependent.

Fig. 2.

Hydrophobic clusters in the Gcn4p activation domain are required for recruitment of the ArgR/Mcm1p complex. (A and B) The gcn4Δ myc-tagged strains described in Fig. 1 transformed with s.c. plasmids harboring GCN4-HA (p2382) or gcn4–14Ala-HA (pSY285) (A), or with h.c. plasmids containing GCN4 (pHQ1303) or gcn4–14Ala (pHQ1304) (B), were grown in the presence of arginine and SM and subjected to ChIP analysis as in Fig. 1. (C) GST, GST-Gcn4p (GST-WT), and GST-Gcn4p containing 10 Ala mutations in the activation domain (GST-10Ala) were expressed in Escherichia coli, immobilized on glutathione Sepharose, beads and added to WCEs from the GCN4 myc-tagged strains described in Fig. 1. Proteins bound to the beads were subjected to Western analysis with antibodies against myc or Snf5p. (D) gcn4Δ strain 249 transformed with h.c. plasmids harboring GCN4-myc (pHQ1293) and MCM1 (pED40) were cultured in synthetic complete-Ura-Leu and induced with SM. WCEs were immunoprecipitated with myc antibodies. Ten percent of the input samples (Input), 100% of the immunoprecipitates (Ppt), and 10% of the supernatant (Sup) fractions were subjected to Western analysis with myc antibodies or goat polyclonal antibodies against Mcm1p.

Fig. 4.

Evidence for Arg81p-independent recruitment of an Arg80p/Mcm1p heterodimer. (A) GCN4 strains with MCM1-myc (SY337), ARG80-myc (SY373), or ARG81-myc (SY375), and the arg81Δ ARG80-myc strain SY384, were grown in YPD, and WCEs were immunoprecipitated with myc antibodies and subjected to Western analysis by using myc antibodies or polyclonal Mcm1p antibodies to probe the blots. (B) Transformants of GCN4 ARG80-myc strain SY373 and GCN4 arg81Δ ARG80-myc strain SY384 harboring episomal GCN4-HA (pHQ1239), and gcn4Δ ARG80-myc strain SY374 containing empty vector, were grown in the presence of SM and arginine and subjected to ChIP analysis, as described in Fig. 1. (C) GCN4 strains harboring episomal GCN4-HA (pHQ1239), or gcn4Δ strains containing empty vector, and harboring the myc-tagged alleles indicated at the top, were cultured in the presence of SM in medium lacking or containing 0.5 mM arginine, as indicated at the bottom, and subjected to ChIP analysis. (D) GCN4 ARG81-myc arg80Δ strain, SY381 grown in the presence (+) or absence (–) of 0.5 mM arginine, was subjected to ChIP analysis.

Fig. 3.

Constitutive recruitment of Mcm1p/Arg80p and arginine-stimulated recruitment of Arg81p/Arg82p by Gcn4p. (A) GCN4 strains harboring the myc-tagged alleles indicated at the top and episomal GCN4-HA (pHQ1239) were subjected to ChIP assays exactly as in Fig. 1, except for the indicated arginine concentrations in the media, and the presence or absence of SM, as indicated in the histograms. (B) Transformants of gcn4Δ strain 249 containing s.c. GCN4-myc plasmid (pSK-1) or empty vector YCp50 were subjected to ChIP analysis after growing in the presence (+) or absence (–) of 0.5 mM arginine. (C) The arg11Δ strains containing myc-tagged MCM1 (SY590) or ARG81 (SY589) and episomal GCN4-HA (pHQ1239) were cultured with 2 mM (+) or 7.5 μM(–) arginine and subjected to ChIP analysis. (D) The ARG81-myc strain SY589 was grown in the presence or absence of arginine as indicated and subjected to Western analysis as described in Fig. 1 D and E. Lanes 2 and 4 were loaded with 50% of the amounts of extract loaded in lanes 1 and 3, respectively.