Fig. 8

Tra1 promotes DUB module assembly into SAGA. a, b LC-MS/MS analysis of SAGA complexes purified from mutants defective in Tra1-SAGA interaction, including tra1Δ (n = 2) and β-estradiol-treated tti2-CKO mutants (-Tti2, n = 2, grown as indicated in Fig. 2a), as well as four distinct mutants that disrupt the hinge region (Hinge). These include tra1-Sptra2, spt20–290, spt20-HITΔ and spt20-FIEN mutants, labelled from dark to light green, respectively. Relative LFQ intensity ratios of Sgf73 (a) and Ubp8 (b) to the bait, Spt7, from independent experiments or mutants are plotted individually. c Silver staining of SAGA complexes purified from WT (sgf73) or sgf73Δ mutants, using Ada1 as the bait. Data are representative of three independent experiments. d Silver staining and western blotting of SAGA complexes purified upon Tra1 synthesis (neo-Tra1) from a strain in which the DUB subunit Sgf11 is MYC-tagged. spt7-TAP RI-tra1 sgf11-MYC cells were grown to exponential phase and harvested at different time points after β-estradiol addition, as indicated (hours). SAGA was purified from a tra1Δ strain as a control for the complete loss of Tra1 from SAGA and from a non-tagged strain (no TAP) as a control for background. Silver staining reveals Spt7 and Tra1, which migrate around 150 and 400 kDa, respectively. Anti-HA and anti-MYC western blotting of Spt7-TAP and Sgf11-MYC in a fraction of the input (Input) and in TAP eluates (TAP) is shown below. An anti-tubulin antibody and Ponceau red staining are used as loading controls. Data are representative of four independent experiments, quantified and averaged in (e). e Quantification of the ratio of Tra1 to Spt7 from silver stained gels (top) and of the ratio of Sgf11-MYC to Spt7-TAP from western blots (bottom). Signal intensities were quantified from four independent experiments (n = 4), except at 24 h (n = 2). Each data point was plotted individually. f Working model for the last steps of SAGA assembly. The core subunits (Spt7, Ada1 and TAFs), the HAT module (Gcn5, Ada2, Ada3 and Sgf29) and Spt20 form a pre-assembled complex. The Hsp90 cochaperone TTT promotes the maturation of nascent Tra1, which is then anchored to SAGA by the HIT domain of Spt20. Consequently, Tra1 stabilises the interaction of the DUB module (Sgf73, Ubp8, Sgf11 and Sus1) with SAGA to form a mature, fully active multifunctional transcriptional co-activator complex. Source data are provided as a Source Data file