PMC

Multiple recombinant Gal11 regions interact with Gcn4p in vitro. A, schematic representation of Gal11 indicating regions (labeled Gcn4) shown previously to interact with Gcn4 in vitro () (upper bar), locations of GAL11 deletions analyzed here (middle bar), and the recombinant Gal11 segments tested here for binding to Gcn4 (three lower bars labeled with amino acid coordinates). B, ³⁵S-labeled Gal11 fragments synthesized in rabbit reticulocyte lysate were incubated with equal amounts of GST, GST-Gcn4p (WT), or GST-Gcn4-Ala₁₀ (10Ala) expressed in E. coli and immobilized on glutathione-Sepharose resin. The bound fractions and 20% of the input ³⁵S-labeled Gal11 fragments were separated by SDS-PAGE, stained with GelCode Blue stain reagent (Pierce), and subjected to autoradiography or phosphorimaging analysis. Arrows indicate the full-length Gal11 polypeptides, which exhibit the predicted electrophoretic mobilities.

Ala substitutions of surface-exposed KIX residues displaying the largest chemical shift changes on interaction with Gcn4 impair activation by Gcn4 in vivo and Gcn4 binding to the Mediator tail subdomain in vitro. A, complementation of the 3AT^S/Gcn⁻ phenotypes of gal11Δ by the indicated plasmid-borne myc-GAL11 alleles was determined as described in the legend for . B, expression of the UAS_GCRE-CYC1-lacZ reporter in transformants of strain IJY3 harboring the indicated myc-GAL11 alleles was assayed by measuring β-galactosidase-specific activities in WCEs as described in the legend for . The means ± S.E. (error bars) were calculated from two independent assays on 4–6 different transformants for each construct (n = 8–12). C, WCEs from transformants of sin4Δ strain IJY7 harboring the indicated myc-GAL11 alleles were incubated with GST-Gcn4 (WT) immobilized on glutathione-Sepharose resin, as described in the legend for .

NMR analysis of the interactions between the Gal11 KIX domain (residues 2–100) and the Gcn4 activation domain (residues 2–151). A, assigned 800-MHz two-dimensional ¹⁵N-¹H HSQC spectrum of ¹⁵N,²H-labeled Gal11 KIX domain. B, representative regions of overlaid HSQC spectra of free ¹⁵N,²H-Gal11 KIX (red contours) and ¹⁵N,²H-Gal11 KIX in the presence of increasing amounts of unlabeled Gcn4 activation domain, corresponding to Gcn4/KIX molar ratios of 0.2 (green), 0.4 (blue), and 0.8 (magenta). C, the plot of amide chemical shift changes (see under “Experimental Procedures”) of Gal11 KIX observed upon the addition of the Gcn4 activation domain. D, ribbon representation of Gal11 KIX (Protein Data Bank (PDB) entry 2K0N) with residues displaying amide chemical shift changes. Δδ ≥ 0.10 ppm is highlighted in red. E, left, surface representation of the ribbon diagram in panel D, and right, the same surface representation rotated by 90° about the axis indicated.

The KIX domain and regions encompassing Δ5 and Δ8 contribute independently to Myc-Gal11 and Rpb3 (pol II) recruitment in vivo. A–C, recruitment of Myc-Gal11 to the ARG1 UAS by Gcn4 was measured by ChIP analysis in transformants of gal11Δ strain HQY1037 harboring the indicated myc-GAL11 alleles, as described in the legend for . D–G, pol II recruitment to P_ARG1-HIS3 was measured by ChIP analysis of transformants of HQY1037 as described in the legend for except using antibodies against Rpb3p and primers to amplify the ARG1 TATA element or 5′ end of the HIS3 coding region. Rpb3 occupancies calculated as in described in the legend for were corrected by subtracting the Rpb3 occupancy measured in transformants harboring empty vector in place of a myc-GAL11 allele and normalizing the resulting values for the corresponding corrected value measured for the WT myc-GAL11 strain. At least three independent cultures and two PCR amplifications for each immunoprecipitation were performed for each strain to yield the means ± S.E. (error bars) (n = 6–16) plotted in the figure.

Combining deletion of the Gal11 KIX domain (Δ1Δ2) with Δ5 or Δ8 exacerbates Gcn⁻ phenotypes without affecting Myc-Gal11 expression or Mediator integrity. A, complementation of the 3AT^S/Gcn⁻ phenotypes of gal11Δ by the indicated plasmid-borne myc-GAL11 alleles was determined as described in the legend for . B, expression of the UAS_GCRE-CYC1-lacZ reporter in transformants of strain IJY3 harboring the indicated myc-GAL11 alleles was assayed by measuring β-galactosidase-specific activities in WCEs after culturing strains in SC-URA and LEU and treating with sulfometuron methyl at 0.5 μg/ml for 6 h. β-Galactosidase activities were corrected by subtracting the activity measured in transformants harboring empty vector in place of a myc-GAL11 allele and normalizing the resulting values for the corresponding corrected value measured for the WT myc-GAL11 strain to yield the plotted values. The means ± S.E. (error bars) were calculated from two independent assays on three different transformants for each construct (n = 6). C, expression of the indicated myc-GAL11 alleles was determined by Western blot analysis of the transformants in A, as described in the legend for . Two different amounts of extract, differing by a factor of 3, were loaded in successive lanes for each strain. D, integrity of Mediator was analyzed by coimmunoprecipitation analysis of the transformants in A, as described in the legend for .

Three segments in the Gal11 N-terminal region function additively to promote binding to Gcn4 in vitro and recruitment to ARG1 in vivo of the Mediator tail in sin4Δ cells, and B-box mutation WQV mimics the Gcn⁻ phenotype of Δ5 in SIN4 cells. A and B, WCEs from transformants of sin4Δ strain IJY6 harboring the indicated myc-GAL11 alleles were incubated with equal amounts of GST, GST-Gcn4 (WT), and mutant GST-Gcn4-Ala₁₀ (10Ala) proteins immobilized on glutathione-Sepharose resin. Bound fractions and 10% of the input yeast WCEs were subjected to Western analysis with antibodies against Snf6 or Spt3, Myc antibodies to detect Myc-Gal11, and HA antibodies to detect HA-Med2. C and D, ChIP analysis of Myc-Gal11 occupancy of the ARG1 UAS in transformants of sin4Δ strain IJY6 harboring the indicated myc-GAL11 alleles, conducted as described in the legend for . At least three independent cultures and two PCR amplifications for each immunoprecipitation were performed for each strain to yield the means ± S.E. (error bars) (n = 6–16) plotted here. E, coimmunoprecipitation analysis of the Mediator tail subdomain in transformants of gal11Δ sin4Δ MED2-HA strain IJY6 containing the indicated myc-GAL11 alleles. WCEs were immunoprecipitated with HA antibodies, and immune complexes were subjected to Western analysis with c-Myc antibodies to detect Myc-Gal11, HA antibodies to detect HA-Med2, or Rpb3 antibodies. I, 5% of input WCE; P, total immunoprecipitate; S, 10% of supernatant. F, complementation of the 3AT^S/Gcn⁻ phenotype of gal11Δ by the indicated myc-GAL11 alleles in transformants of strain IJY3, as described in the legend for . G, ChIP analysis of Myc-Gal11 occupancy of the ARG1 UAS in transformants of SIN4 strain IJY3 harboring the indicated myc-GAL11 alleles, as described in the legend for . At least three independent cultures and two PCR amplifications for each immunoprecipitation were performed for each strain to yield the means ± S.E. (error bars) (n = 6–16) plotted here.

Deletion of segments in the N-terminal third of Myc-Gal11 reduce its recruitment to the ARG1 UAS in vivo without affecting Myc-Gal11 expression or Mediator integrity. A, coimmunoprecipitation analysis of Mediator integrity. WCEs from transformants of IJY3 (gal11Δ MED2-HA), IJY4 (gal11Δ PGD1-HA), or HQY1037 (gal11Δ) containing GAL11 or the indicated myc-Gal11 alleles were immunoprecipitated (IP) with Myc antibodies. Immune complexes were subjected to Western analysis with the appropriate antibodies to detect the proteins listed on the left, Myc antibodies to reveal Myc-Gal11, and HA antibodies to reveal HA-Med2 and HA-Pgd1. I, 5% of the input WCE; P, total pellet fraction from immunoprecipitations; S, 10% of supernatant fraction. B and C, ChIP analysis of Myc-Gal11 recruitment to the ARG1 UAS by Gcn4. Transformants of HQY1037 harboring the indicated myc-GAL11 alleles and IJY1 (gcn4Δ) containing WT myc-GAL11 were cultured at 30 °C in SC-URA medium and treated with sulfometuron methyl to induce Gcn4. ChIP analysis was performed using Myc antibodies. ARG1 UAS and POL1 coding sequences were quantified in the immunoprecipitated and input chromatin samples by PCR in the presence of [³³P]dATP using primers listed in . PCR products were resolved by PAGE and either visualized by autoradiography (as illustrated in panel B) or quantified with a PhosphorImager. The ratios of ARG1 to POL1 signals in immunoprecipitated samples were normalized for the corresponding ratios for input samples, and the resulting occupancy values were normalized to the occupancy measured for the WT myc-GAL11 strain to yield the relative Gal11 occupancies. At least three independent cultures and two PCR amplifications for each immunoprecipitation were performed for each strain to yield the means ± S.E. (error bars) (n = 6–16) plotted in C.

Identification of regions in GAL11 required to complement the 3AT^S/Gcn⁻ phenotype of gal11Δ and a summary of their roles in Gal11 expression and Mediator integrity. A, schematic of Gal11 domain organization (upper bar) and deletions made in a myc-Gal11 allele (lower bar). The region of similarity to the Qr domain of mammalian SRC-1 is indicated above the top bar. The criterion used for designating the KIX and regions C-II, C-III, C-IV, and C-V as conserved blocks was the occurrence of sequence identity or conservative replacements in all four fungal orthologs at >50% of the residues in the block (see “Results” for further details). The color scheme for panels A–D is explained below under Keys. B, summary of the effects of deletions in myc-Gal11 on complementation of the 3AT^S phenotype of gal11Δ arg1Δ P_ARG1 HIS3 strain HQY1037, with relative growth determined as in panel F and scored on a scale from 0 to 10, with 10 and 0 corresponding to growth with wild-type or no myc-Gal11 allele, respectively. C, summary of the effects of deletions on expression of Myc-Gal11 protein determined as in panel E. D, summary of the effects of deletions on Mediator integrity determined as in A. E, transformants of HQY1037 harboring WT myc-Gal11 or the indicated deletion alleles were cultured to mid-logarithmic phase in SC-URA medium and treated with sulfometuron methyl at 0.5 μg/ml for 30 min to induce Gcn4. WCE extracts were prepared by trichloroacetic acid extraction and subjected to Western blot analysis using antibodies against the Myc epitope to detect Myc-Gal11 and antibodies against Gcd6 and Gcn4. Two different amounts of extract, differing by a factor of 3, were loaded in successive lanes for each strain. F, transformants described in E were cultured in SC-URA medium, and serial dilutions were spotted on SC-URA containing 15 mm 3AT and incubated for 3 days at 30 °C.