A model suggesting a mutually exclusive role for Ahc1 and Spt7 in SAGA complex assembly.

<p>Co-IP experiments showed that pre-IL-1α binds to the HAT/Core of both the ADA and SAGA complexes. In the TAP/Spt7,<i>ahc1</i>Δ strain, only rarely weak co-precipitation of Spt7-TAP and pre-IL-1α was observed. Ahc1 thus may operate as an exchange factor that facilitates Spt7 binding to the ADA HAT, bringing various non-canonical co-activators and accessory proteins (e.g., IL-1α) and providing the resulting complex with DNA-binding abilities that give rise to a fully functional SAGA complex. Therefore, at least from the point of IL-1α function, ADA might not represent a real HAT complex but rather an intermediate protein complex that is however necessary for the assembly and proper function of the SAGA HAT complex.</p

Nucleotide sequences of the primers used for the <i>loxP-kanMX-loxP</i> and <i>loxP-Leu2-loxP</i> gene disruption cassette amplification.

<p>Nucleotide sequences of the primers used for the <i>loxP-kanMX-loxP</i> and <i>loxP-Leu2-loxP</i> gene disruption cassette amplification.</p

The interleukin-1α precursor suppresses hypersensitivity of <i>snf1</i>Δ strain to 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of the His3 imidazoleglycerol-phosphate dehydratase.

<p>This suppressive property of pre-IL-1alpha (pre) is more profound on SD media containing glycerol/ethanol as a carbon source (SDgly) in case of the <i>snf1-108</i> strain carrying incomplete <i>SNF1</i> deletion. Mature interleukin-1alpha (Mat) is not able to rescue the 3-AT hypersensitivity of both <i>snf1</i>Δ and <i>snf1-108</i> strains and was used as a control. We did not observe any differences in growth between strains producing pre-IL-1alpha and mature IL-1alpha on SD agar plates which did not contain and/or contain only a minute amount of 3-AT. This is an example of 3 independent biological experiments.</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

The structure of IL-1αNTP resembles the C-terminal portion of the catalytical subunit of the eukaryotic AMP-activated protein kinase.

<p>(A) Prediction of the 3-D structure of the first N-terminal 112 amino acid residues of the IL-1α precursor (IL-1αNTP). Acidic amino acid residues are depicted in red. (B) The 3-D structure of the INL domain of the yeast Snf1 protein kinase (PDB ID: 3T4N). (C) A superimposition of IL-1αNTP (blue) and the C-terminal INL domain of the yeast Snf1 protein kinase (green). (D) A superimposition of the INL domains of yeast Snf1 (green, PDB ID: 3T4N) and rat AMP-activated protein kinase (grey, PDB ID: 2V92). Acidic amino acid residues are depicted in red tones. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041801#pone.0041801.s003" target="_blank">File S1</a> for cordinates of IL-1αNTP prediction and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041801#pone.0041801.s004" target="_blank">File S2</a> for primary sequences of all proteins used in this analysis.</p

The SAGA and ADA complex subunits co-purify as a part of the IL-1α precursor-binding complex.

<p>Co-immunoprecipitation experiments with an anti-Flag antibody using yeast strains from a TAP tag library transformed with IL-1α expression vectors revealed that both of the HAT complexes bound pre-IL-1α (pre) but not mature IL-1α (Mat). Control cells (ctrl) carry the empty plasmid pYX212. Western blotting was performed using an anti-CBP antibody that recognizes the TAP tag at the C-terminus of the respective HAT complex subunits. For each line of the IP experiment, 1.4 mL of the cell lysate prepared from 5.10⁸ yeast cells in average was used. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.</p

Disruption of the SAGA and ADA complexes confirmed binding of the IL-1α precursor to the HAT/Core module and suggested the mutually exclusive role of Spt7 and Ahc1 in SAGA complex assembly.

<p>Co-immunoprecipitation was performed using yeast lysates with an anti-Flag antibody that recognizes the Flag tag at the N-terminus of the IL-1α precursor. Western blotting was performed with an anti-CBP antibody which identifies the TAP tag at the C-terminus of the respective HAT complex subunits. (A) Gcn5 does not bind to pre-IL-1α, and because it is not required for SAGA or ADA complex integrity, its deletion has no effect on Ahc1 or Spt8 co-immunoprecipitation with the IL-1α precursor (pre). Deletion of the AHC2 gene doesn’t impair co-IP of pre-IL-1α with Gcn5, Spt8 and Spt7. (B) The disruption of the ADA HAT complex did not affect the co-immunoprecipitation of Gcn5 and Spt8 with IL-1α. However, the interaction between Spt7 and the IL-1α precursor was significantly weakened. In experiments with TAP/Spt7,<i>ahc1</i>Δ strain, we received either no or very low signal (the latter is depicted) of TAP-tagged Spt7, with a success rate 3∶1, respectively. (C) The disruption of the SAGA complex abolished the interaction between Spt8 and the IL-1α precursor but had no effect on Ahc1 binding to the IL-1α precursor. Control cells (ctrl) carry the empty plasmid pYX212. For each line of the IP experiment, 3.5 mL of cell lysate prepared from 20.10⁸ yeast cells in average was used, except TAP/Spt8,<i>gcn5</i>Δ, TAP/Spt8,<i>ahc1</i>Δ, TAP/Spt8,<i>spt7</i>Δ and TAP/Gcn5,<i>ahc1</i>Δ strains, where 1.3 mL of cell lysates from 9.10⁸ yeast cells each were applied. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.</p

Subcellular localization of pre-IL-1α and IL-1αMat in <i>Saccharomyces cerevisiae.</i>

<p>The IL-1α precursor (pre-IL-1α) is exclusively localized in the nucleus of yeast cells, which is in contrast to the observed cytoplasmic localization of mature IL-1α (IL-1αMat). Control cells (ctrl) carry the empty pUG36 vector. The cell nuclei are stained with DAPI.</p