Fig. 2

Interactions between the SMC5/6 and SAGA complexes. A Extracts from fission yeast strains MMP21 (Nse4-FLAG) and YLJ507 (Nse4-FLAG and Gcn5-myc) were immunoprecipitated using the anti-myc antibody. The input (I), unbound (U) and bound (B) fractions were separated by 12% SDS-PAGE. The Nse4-FLAG and Gcn5-myc proteins were analysed on a western blot using anti-FLAG-HRP and anti-myc-HRP, respectively. B The yeast two-hybrid (Y2H) system was used to determine individual protein–protein interactions between SMC5/6 and SAGA HAT module subunits. The Gal4AD or Gal4BD domains fused to the full-length Ada2 or Gcn5 subunits were co-transformed together with the fragments of SMC5/6 subunits into the PJ69 cells and grown on the plates without Leu, Trp (-L, W; control plates). The protein–protein interactions between SMC5/6 and SAGA were scored by the growth of the yeast PJ69 transformants on the plates without Leu, Trp and His, containing 3-Amino-1,2,4-triazole (0.5 mM AT or 10 mM AT plates). The fragments were as follows: Gal4BD-Nse2 (aa2-178), Gal4AD-Nse3 (aa1-190), Gal4AD-Nse3 (aa200-307) and Gal4BD-Smc5 CC arm (aa170-225 + 837-910). In control experiments, respective empty pGADT7 (AD) or pGBKT7 (BD) vector was co-transformed with either SAGA or SMC5/6 construct. Note that the Gal4BD-Smc5 CC arm construct self-activated (Smc5-vector combination) and was therefore grown on 10 mM AT plates to assess its binding to Ada2 (Smc5-Ada2 combination). The Nse2 (black), Nse3 (green) and Smc5 (blue) subunits binding either Ada2 or Gcn5 are highlighted within the SMC5/6 rod-shaped structural model (shaded; based on the 7QCD structure from [14]) next to the Y2H results