Extended Data Fig. 3: SAGA undergoes Ada3 acetylation-dependent dimerization.
a, b, Glycerol density gradient centrifugation analysis of purified Wt SAGA incubated without (a) or with (b) acetyl-CoA. 50 µg Wt SAGA was incubated with 10 mM acetyl-CoA at 30 °C for 15 min. The reaction products were applied to a 10-50% glycerol density gradient centrifugation. The fractions were analyzed by Western blots with indicated antibodies and silver staining. c, d, Glycerol density gradient centrifugation analysis of SAGA Ada3-3KR treated without (c) or with (d) 10 mM acetyl-CoA. e, Silver staining of purified SAGA monomer and SAGA dimer. The fractions 11-15 in Fig. 3a were pooled and concentrated as SAGA monomer. The fractions 19-23 in Fig. 3b were pooled and concentrated as SAGA dimer. f, Quantification of fractions in Figs. 3a-3d. g, In vivo Co-IP showed that Ada3 acetylation is required for interaction between Ada3-FLAG-containing SAGA and Ada3-Myc-containing SAGA. Ada3-FLAG was immunoprecipitated from cell extract of the diploid strain Ada3-FLAG/Ada3-Myc, Ada3-3KR-FLAG/Ada3-Myc, Ada3-FLAG/Ada3-3KR-Myc, and Ada3-3KR-FLAG/Ada3-3KR-Myc, respectively. The co-IPed Ada3-Myc was detected by anti-Myc. Compared with the interaction between SAGA Ada3-FLAG and SAGA Ada3-Myc, the interaction between SAGA Ada3-3KR-Flag and SAGA Ada3-Myc as well as the interaction between SAGA Ada3-Flag and SAGA Ada3-3KR-Myc were reduced. No obvious interaction was detected between SAGA Ada3-3KR-FLAG and SAGA Ada3-3KR-Myc. h-k, In vivo Co-IP showing the self-association of SAGA complex. For Extended Data Fig. 3a-e, g, shown is the typical example of two biological independent experiments. For Extended Data Fig. 3h-k, shown is the typical example of two biological independent experiments.
