Fig. 6: Btn2 SUMOylation opposes K48-Ubiquitin chain buildup when INQ-degradation is blocked.
From: Dynamics of DNA damage-induced nuclear inclusions are regulated by SUMOylation of Btn2
a WT, apj1Δ, hsp104Δ, and apj1Δhsp104Δ cells were treated with MMS for two hours followed by a washout into MMS-free media. Shown are Rpd3-GFP foci percentages in these backgrounds after 2 h of treatment and after one and two hours of washout from three independent biological replicates per time point. b Intensities of INQ foci were measured for strains in (a) and both sets of data reveal epistasis between Btn2 SUMOylation and Hsp104-mediated refolding while indicating that these two pathways may operate in parallel to Apj1-mediated degradation. c Fractionation in WT, apj1Δ, hsp104Δ, and apj1Δhsp104Δ cells treated with MMS and western blot for levels of total Ubiquitin (Ub), K48-linked Ubiquitin chains, and SUMO/Smt3 chains. d Deleting Slx5 causes an increase in Btn2 mono-SUMOylation and polySUMOylation. NiPD was performed in WT and slx5Δ cells treated with MMS. e Slx8-GFP INQ foci levels in WT, apj1Δ, hsp104Δ, and apj1Δhsp104Δ cells with Btn2-WT/Btn2-6KR following MMS treatment. f Fractionation in WT, apj1Δ, hsp104Δ, and apj1Δhsp104Δ cells with or without slx5Δ. All cells were treated with MMS and probed for levels of total Ub, K48-Ub chains, and Smt3 chains. g Rpd3-GFP foci percentages in slx5Δ cells harboring Btn2-WT/Btn2-6KR following the same experiment design and washout in (a). Total ubiquitin, K48-Ubiquitin, and SUMO levels quantified with respect to Btn2-WT MMS cells are indicated below the blot for (c, f). All error bars represent ±SEM, n = 3 biologically independent replicates, >100 cells each. ****p < 0.0001, ***p < 0.0002, **p < 0.002, *p < 0.03, ns, p > 0.1, One-way ANOVA for (b), Fisher’s test for (a, e, g). Source data are provided as a Source Data file.
