The Gcn4p activation domain interacts specifically in vitro with RNA polymerase II holoenzyme, TFIID, and the Adap-Gcn5p coactivator complex

Abstract

The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathione S-transferase (GST)-Gcn4p fusion protein to components of three different coactivator complexes in Saccharomyces cerevisiae cell extracts, including subunits of transcription factor IID (TFIID) (yeast TAFII20 [yTAFII20], yTAFII60, and yTAFII90), the holoenzyme mediator (Srb2p, Srb4p, and Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p). The binding to these coactivator subunits was completely dependent on the hydrophobic clusters in the Gcn4p activation domain. Alanine substitutions in single clusters led to moderate reductions in binding, double-cluster substitutions generally led to greater reductions in binding than the corresponding single-cluster mutations, and mutations in four or more clusters reduced binding to all of the coactivator proteins to background levels. The additive effects of these mutations on binding of coactivator proteins correlated with their cumulative effects on transcriptional activation by Gcn4p in vivo, particularly with Ada3p, suggesting that recruitment of these coactivator complexes to the promoter is a cardinal function of the Gcn4p activation domain. As judged by immunoprecipitation analysis, components of the mediator were not associated with constituents of TFIID and Adap-Gcn5p in the extracts, implying that GST-Gcn4p interacted with the mediator independently of these other coactivators. Unexpectedly, a proportion of Ada2p coimmunoprecipitated with yTAFII90, and the yTAFII20, -60, and -90 proteins were coimmunoprecipitated with Ada3p, revealing a stable interaction between components of TFIID and the Adap-Gcn5p complex. Because GST-Gcn4p did not bind specifically to highly purified TFIID, Gcn4p may interact with TFIID via the Adap-Gcn5p complex or some other adapter proteins. The ability of Gcn4p to interact with several distinct coactivator complexes that are physically and genetically linked to TATA box-binding protein can provide an explanation for the observation that yTAFII proteins are dispensable for activation by Gcn4p in vivo.

FIG. 1

Locations and relative importance of hydrophobic clusters in the Gcn4p activation domain. A diagram of Gcn4p protein is depicted with the DNA-binding (b) and dimerization (ZIP) domains at the extreme C terminus shown in α-helical conformation, as has been predicted from X-ray crystallography (20), and with the rest of the protein shown as a rectangular box. The critical hydrophobic residues identified in our previous studies (18, 36) are shown above the sequence at the appropriate positions in the protein by the single-letter code. The critical residues are grouped in seven hydrophobic clusters, symbolized by shaded ovals. The size and height of the lettering for each cluster is proportional to the reduction in GCN4 function seen in response to Ala substitutions at that site. The functions of these hydrophobic clusters are redundant; thus, several must be inactivated simultaneously to destroy GCN4 function. Mutation of F97 and F98 (cluster 5) and W120, L123, and F124 (cluster 7) produced the only situation where GCN4 function in vivo was greatly impaired by substitutions in only two of the seven clusters. At the other extreme, mutation of clusters 1 to 4 produced the only situation where a substantial amount of GCN4 function occurred with only three clusters left intact (18, 36).

FIG. 2

Binding of yTAF_II90 and yTAF_II60 in cell extracts to a GST-Gcn4p fusion protein is dependent on the critical hydrophobic residues in the Gcn4p activation domain. (A and C) Aliquots of bacterial extracts containing approximately 25, 50, or 75 μg of total protein from strains expressing different GST fusion proteins were mixed with the appropriate amount of a control bacterial extract lacking a GST fusion protein (to bring the amount of bacterial protein in each reaction mixture to ca. 75 μg) and an aliquot of yeast extract containing 500 to 1,000 μg of protein from strains YBY40-8 and YBY181 expressing HA-yTAF_II90 (A) or HA-yTAF_II60 (C) as described in Materials and Methods. GST-GCN4 contains the wild-type Gcn4p activation domain, whereas GST–gcn4–14 Ala contains the 14 Ala substitutions in hydrophobic clusters 1 to 3 and 5 to 6 shown in Table 1. The GST fusion proteins were precipitated from the reaction mixtures with glutathione-Sepharose 4B resin, and the precipitated proteins were resolved by SDS-PAGE and subjected to immunoblot analysis with monoclonal antibodies against the HA epitope to detect the HA-tagged proteins (upper blots) with an enhanced-chemiluminescence system to detect immune complexes. Subsequently, the blots were stripped and reprobed with polyclonal antibodies against Gcn4p to detect the GST-Gcn4p proteins (lower blots). (B and D) The same yeast cell extracts were incubated with bacterial extracts containing GST-VP16 (bearing the wild-type VP16 activation domain), GST-VP16 Δ456 (bearing the truncated activation domain), or GST alone and processed exactly as described for panels A and C, except that the immunoblots were probed only with anti-HA antibodies.

FIG. 3

Additive effects of mutations in hydrophobic clusters 5 to 7 of the activation domain in GST-Gcn4p fusion proteins on binding of yTAF_II20, yTAF_II60, and yTAF_II90 in cell extracts. A fixed amount of yeast extract (containing 1,500 μg of protein) prepared from strain DPY213 expressing HA-yTAF_II130 was incubated with three different amounts of bacterial extracts for each GST-Gcn4p fusion containing ca. 5, 10, and 20 μg of total bacterial protein and the appropriate amounts of a control bacterial extract lacking a GST fusion protein to bring the total amount of bacterial protein in each reaction mixture to 23 μg. GST-Gcn4p fusion proteins contained the wild-type Gcn4p activation domain (+) (lanes 1 to 3, 14 to 16, and 27 to 29) or mutant activation domains with alanine substitutions in the hydrophobic clusters shown in brackets across the top of the figure. The designations are those adopted for Fig. 1. The GST fusion proteins were precipitated, resolved by SDS-PAGE, and subjected to immunoblot analysis with monoclonal anti-HA antibodies (to detect HA-yTAF_II130) and then with polyclonal antibodies against the proteins indicated to the left of each panel. An enhanced-chemiluminescence system was used to detect the immune complexes. Lanes 13, 26, and 39 contain 1/20 of the input (In) amount of yeast extract employed in each of the binding reaction mixtures.

FIG. 4

Quantitation of the effects of mutations in the hydrophobic clusters in GST-Gcn4p fusions on binding to yTAF_II20, -60, and -90; Ada3p; and Srb2p, -4p, and -7p in yeast cell extracts. The amounts of the different coactivator proteins that were precipitated with each GST-Gcn4p fusion protein were determined by densitometric scanning of the immunoblots shown in Fig. 3, 5, 6, and 7. The band intensities measured for the three binding reaction mixtures with different quantities of the GST fusion protein were summed and averaged for each mutant GST-Gcn4p construct, and these averages were divided by the corresponding value determined for the wild-type GST-Gcn4p analyzed in parallel. The resulting ratios were plotted on the y axis for each mutant GST-Gcn4p protein listed on the x axis. The designations are those adopted for Table 1.

FIG. 5

Additive effects of mutations in hydrophobic clusters 5 to 7 of the activation domain in GST-Gcn4p fusion proteins on binding of Srb2p, Srb4p, and Srb7p in yeast cell extracts. A fixed amount of yeast extract (containing 1,500 μg of protein) prepared from strain DPY213 expressing HA-yTAF_II130 was incubated with three different amounts of bacterial extracts for each GST-Gcn4p fusion containing ca. 5, 10, and 20 μg of total bacterial protein and the appropriate amounts of a control bacterial extract lacking a GST fusion protein to bring the total amount of bacterial protein in each reaction mixture to 23 μg. GST-Gcn4p fusion proteins contained the wild-type Gcn4p activation domain (+) (lanes 1 to 3, 14 to 16, and 27 to 29) or mutant activation domains with substitutions in the hydrophobic clusters shown in brackets across the top. The GST fusion proteins were precipitated and subjected to immunoblot analysis with polyclonal antibodies against the proteins indicated to the left of each panel, exactly as described for Fig. 3. Lanes 13, 26, and 39 contain 1/20 of the input (In) amount of yeast extract employed in each binding reaction mixture.

FIG. 6

Additive effects of mutations in hydrophobic clusters 1 to 3 of the activation domain in GST-Gcn4p fusion proteins on binding of yTAF_II20, -60, and -90 and Srb2p, -4p, and -7p in yeast cell extracts. A fixed amount of yeast extract (containing 1,500 μg of protein) prepared from strain DPY213 expressing HA-yTAF_II130 was incubated with three different amounts of bacterial extracts for each GST-Gcn4p fusion containing ca. 2, 4, and 8 μg of total bacterial protein and the appropriate amounts of a control bacterial extract lacking a GST fusion protein to bring the total amount of bacterial protein in each reaction mixture to 9 μg. Shown are the GST-Gcn4p fusion proteins containing the wild-type Gcn4p activation domain (+) (lanes 1 to 3 and 14 to 16) or mutant activation domains with substitutions in the hydrophobic clusters shown in brackets across the top. The GST fusion proteins were precipitated and subjected to immunoblot analysis with polyclonal antibodies against the proteins indicated to the left of each panel, exactly as described for Fig. 3. Lanes 13 and 23 contain 1/20 of the input (In) amount of yeast extract employed in each binding reaction mixture.

FIG. 7

Additive effects of mutations in hydrophobic clusters 1 to 3 and 5 to 7 of the activation domain in GST-Gcn4p fusion proteins on binding of Ada3p in yeast cell extracts. (A) A fixed amount of yeast extract (containing 1,500 μg of protein) prepared from strain SY6-2 expressing HA-Ada3p was incubated with three different amounts of bacterial extracts for each GST-Gcn4p fusion containing ca. 5, 10, and 20 μg of total bacterial protein and the appropriate amounts of a control bacterial extract lacking a GST fusion protein to bring the total amount of bacterial protein in each reaction mixture to 23 μg. (B) Aliquots of yeast extract containing 1,500 μg of protein from strain SY6-2 were incubated with three different amounts of bacterial extracts for each GST-Gcn4p fusion containing ca. 2, 4, and 8 μg of total bacterial protein and the appropriate amounts of a control bacterial extract lacking a GST fusion protein to bring the total amount of bacterial protein in each reaction mixture to 9 μg. Shown are the GST-Gcn4p fusion proteins bearing the wild-type Gcn4p activation domain (+) (lanes 1 to 3, 14 to 16, and 27 to 29 in panel A and lanes 1 to 3 and 14 to 16 in panel B) or the mutant activation domains, with substitutions in the hydrophobic clusters shown in brackets across the top of each panel. The GST fusion proteins were precipitated and subjected to immunoblot analysis with monoclonal anti-HA antibodies to detect HA-Ada3p or polyclonal antibodies against the other proteins indicated to the left of each panel, exactly as described for Fig. 3. Lanes 13, 26, and 39 (A and B) contain 1/20 of the input (In) amount of yeast extract employed in each binding reaction mixture.

FIG. 8

yTAF_II proteins in purified TFIID did not interact specifically with GST-Gcn4p. Components of purified TFIID were tested for the ability to interact specifically with GST-Gcn4p fusion proteins by mixing a fixed amount of purified TFIID with two different amounts of bacterial extract containing GST-Gcn4p fusion proteins bearing the wild-type activation domain or mutant activation domains with alanine substitutions in the hydrophobic clusters (numbered as shown in Fig. 1). The GST fusion proteins were precipitated and subjected to immunoblot analysis with polyclonal antibodies against the proteins indicated to the right of each panel, exactly as described for Fig. 3. (A and B) TFIID immunoaffinity purified with anti-TBP antibodies was incubated at a fixed concentration (60 ng per reaction mixture) with 100 or 200 μg of bacterial extracts containing GST proteins and the appropriate amounts of a control bacterial extract lacking a GST fusion protein to bring the total amount of bacterial protein in each reaction mixture to ca. 300 μg. Lane 1 contained GST alone. Lanes 2 to 7 contained GST-Gcn4p fusion proteins with either wild-type (+) or mutant activation domains, with the clusters in which there were mutations being indicated by numbers in brackets across the top of the figure. Lanes 8 and 9 contained binding assay mixtures with the bacterial extract containing wild-type GST-Gcn4p and 1,200 μg of whole-cell extract (WCE) from yeast strain DPY213. Lane 10 contained 2/3 of the amount of TFIID used in the binding reaction mixtures in lanes 1 to 7 (40 ng), and lane 11 contained 1/30 of the yeast WCE used in the binding reaction mixtures shown in lanes 8 and 9. (C to G) TFIID bearing HA-yTAF_II130 immunoaffinity purified with anti-HA antibodies was incubated at 300 ng per reaction mixture with 40 or 80 μg of bacterial extracts containing GST proteins and the appropriate amounts of a control bacterial extract lacking a GST fusion protein to bring the total amount of protein in each reaction mixture to ca. 80 μg. Lane 1 contained GST alone, and lanes 2 to 9 contained GST-Gcn4p fusion proteins with wild-type (+) or mutant activation domains (with cluster numbers indicated in brackets across the top of the blots). Lane 10 contained one-sixth of the TFIID used in the binding reaction mixtures in lanes 1 to 9. Lanes 12 and 13 contained a binding assay mixture with the bacterial extract containing wild-type GST-Gcn4p and 600 μg of WCE from strain DPY213. Lane 14 contained 1/15 of the yeast WCE used in the binding reaction mixtures in lanes 12 and 13. Lane 11 contained no sample.

FIG. 9

Binding of yTAF_II20, -60, and -90 and Srb2p in yeast extracts to wild-type GST-Gcn4p is not dependent on Ada2p, Ada3p, or Srb10p. Aliquots of yeast extract containing 1,500 μg of protein from pairs of isogenic mutant and wild-type strains were incubated with two different amounts of bacterial extracts containing 3 and 6 μg of protein and the appropriate amounts of a control bacterial extract lacking a GST fusion protein to bring the total amount of bacterial protein in each reaction mixture to 215 μg. The GST-Gcn4p fusion proteins contained either the wild-type activation domain (+; lanes 3 to 4 and 7 to 8) or the {5, 6, 7}⁻ mutant activation domain (lanes 5 to 6 and 9 to 10). The GST fusion proteins were precipitated and subjected to immunoblot analysis with polyclonal antibodies against the proteins indicated to the left of each panel, exactly as described for Fig. 3. Lanes 1 and 12 contain 1/20 of the input (In) amount of yeast extract employed in each binding reaction mixture without incubation; lanes 2 and 11 contain the same amounts of yeast extract after incubation under reaction conditions in the absence of a GST fusion protein. (A) Yeast extracts derived from strains H1511 (ADA2) and KNY104 (ada2Δ); (B) yeast extracts derived from strains H1511 (ADA3) and KNY105 (ada3Δ); (C) yeast extracts derived from strains Z719 (SRB10) and Z687 (srb10Δ); (D) yeast extracts derived from strains H2451 (SRB2) and RMY10 (srb2Δ).

FIG. 10

Coimmunoprecipitation analysis of components of TFIID, mediator, and Adap-Gcn5p complexes in yeast cell extracts. Aliquots of cell extracts containing 1,250 μg of total protein were immunoprecipitated with mouse monoclonal anti-HA antibodies from strains DPY107 (HA-MOT1), DPY213 (HA-yTAF_II130), YBY40-8 (HA-yTAF_II90), and SY6-2 (HA-ADA3). (A) The proteins in 100% of the immune complexes (lanes P), 40% of the supernatants (lanes S), and 20% of the washes from each immunoprecipitation (lanes W) were resolved by SDS-PAGE and subjected to immunoblot analysis with antibodies against the proteins indicated to the left of each blot, except for the Mot1p blot, which was probed with rabbit polyclonal anti-HA antibodies. (B) Proteins in 10% of the input extracts (lanes In), 100% of the immune complexes (lanes P), and 20% of the supernatants (lanes S) were analyzed by immunoblotting as described for panel A, again by probing the last blot at the bottom with polyclonal anti-HA antibodies to detect HA-Mot1p or HA-Ada3p.

FIG. 11

Model summarizing the in vitro interactions between the Gcn4p activation domain and three different coactivator complexes. A dimer of Gcn4p is depicted bound to a Gcn4p binding site located upstream from the TATA element in a Gcn4p-regulated promoter. The activation domain (AD) of Gcn4p bearing seven hydrophobic clusters (filled circles) is shown interacting independently with the mediator complex of RNA Pol II holoenzyme (containing SRB-encoded proteins, TFIIF, Gal11p, and Sug1p) (46) and the Sptp-Adap-Gcn5p complex (see the text for references). The interaction with TFIID (66, 68) may be indirect and may occur only in the context of an Sptp-Adap-Gcn5p–TFIID composite complex. Asterisks indicate subunits of the complexes in cell extracts that bound to recombinant GST-Gcn4p proteins in a manner that depended on the hydrophobic clusters in the Gcn4p activation domain. The Sptp-Adap-Gcn5p and TFIID complexes are shown physically interacting with one another based on our observations that fractions of yTAF_II20, -60, and -90 in cell extracts specifically coimmunoprecipitated with HA-Ada3p and that a fraction of Ada2p coimmunoprecipitated with HA-TAF_II90. TBP is shown as a component of TFIID but also as physically interacting with the Adap-Gcn5p complex and holoenzyme (see the text for details and additional references). DBD, DNA-binding domain.