Figure 9. TOPORS is a SUMO1-specific... | Rockefeller University Press

Figure 9.

$TOPORS is a SUMO1-specific ubiquitin E3 ligase required for efficient PML degradation. (A) Total peptide (protein) signal intensity for TOPORS, RNF111, and SUMO1 associated with WT, A216T, and L217F PML bodies before or after exposure for 2 h with 1 μM arsenic. SUMO1 is included for comparison. Columns represent average, and error bars are SEM. Number of purifications, n = 4. P values are derived from Brown–Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons test. Experimental details are shown in Fig. S1. (B) Relative mRNA abundance normalized to TATA binding protein (TBP) expression for RNF4, RNF111, and TOPORS, in U2OS PML−/− +YFP-PML-V WT cells transfected with the indicated siRNAs. (C) Immunoblots for RNF111, TOPORS, and RNF4 from crude extracts taken from U2OS PML−/− +YFP-PML-V WT cells transfected with the indicated siRNAs. (D) Summary of live-cell microscopy analysis of PML body intensity for U2OS PML−/− + YFP-PML-V cells transfected with either non-targeting siRNA (siNT) or the indicated siRNAs for 48 h and exposed to 1 μM arsenic for 24 h; values are average (solid lines) with SEMs (shaded areas) of PML body intensity relative to t = 0. n = 3 fields of view containing multiple cells. (E) Summary of the relative YFP-PML body intensity data shown in D for t = 16 h. P values are derived from Brown–Forsythe and Welch ANOVA tests with an unpaired t test using Welch’s correction. (F) Representative cell images from live-cell microscopy summarized in D (scale bars are 20 μm). (G) Schematic depiction of the primary sequences for TOPORS and RNF4 with RING and SIMs (SIM) indicated. (H) Fluorescent scans of gels fractionating the products of in vitro ubiquitin conjugation reactions using Alexa Fluor 647–labeled linear fusions of 4xSUMO1 and 4xSUMO2 as substrates. E3 ligase activities of lipoyl domain–tagged WT TOPORS (2-574) (“TOPORS”) and untagged WT RNF4 (full-length) are compared with the indicated SIM mutants. Assays were either stopped prior to the addition of ATP (0) or incubated for 5, 10, or 30 min after the addition of ATP. Control samples showing Alexa Fluor 647-4xSUMOs and lacking all ubiquitin conjugation machinery (−) are also included. Unmodified 4xSUMOs (4xS) and ubiquitinated 4xSUMOs (4xS+Ub) are indicated. Gels were also Coomassie-stained (Fig. S7 E). Source data are available for this figure: SourceData F9.$

TOPORS is a SUMO1-specific ubiquitin E3 ligase required for efficient PML degradation. (A) Total peptide (protein) signal intensity for TOPORS, RNF111, and SUMO1 associated with WT, A216T, and L217F PML bodies before or after exposure for 2 h with 1 μM arsenic. SUMO1 is included for comparison. Columns represent average, and error bars are SEM. Number of purifications, n = 4. P values are derived from Brown–Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons test. Experimental details are shown in Fig. S1. (B) Relative mRNA abundance normalized to TATA binding protein (TBP) expression for RNF4, RNF111, and TOPORS, in U2OS PML^−/− +YFP-PML-V WT cells transfected with the indicated siRNAs. (C) Immunoblots for RNF111, TOPORS, and RNF4 from crude extracts taken from U2OS PML^−/− +YFP-PML-V WT cells transfected with the indicated siRNAs. (D) Summary of live-cell microscopy analysis of PML body intensity for U2OS PML^−/− + YFP-PML-V cells transfected with either non-targeting siRNA (siNT) or the indicated siRNAs for 48 h and exposed to 1 μM arsenic for 24 h; values are average (solid lines) with SEMs (shaded areas) of PML body intensity relative to t = 0. n = 3 fields of view containing multiple cells. (E) Summary of the relative YFP-PML body intensity data shown in D for t = 16 h. P values are derived from Brown–Forsythe and Welch ANOVA tests with an unpaired t test using Welch’s correction. (F) Representative cell images from live-cell microscopy summarized in D (scale bars are 20 μm). (G) Schematic depiction of the primary sequences for TOPORS and RNF4 with RING and SIMs (SIM) indicated. (H) Fluorescent scans of gels fractionating the products of in vitro ubiquitin conjugation reactions using Alexa Fluor 647–labeled linear fusions of 4xSUMO1 and 4xSUMO2 as substrates. E3 ligase activities of lipoyl domain–tagged WT TOPORS (2-574) (“TOPORS”) and untagged WT RNF4 (full-length) are compared with the indicated SIM mutants. Assays were either stopped prior to the addition of ATP (0) or incubated for 5, 10, or 30 min after the addition of ATP. Control samples showing Alexa Fluor 647-4xSUMOs and lacking all ubiquitin conjugation machinery (−) are also included. Unmodified 4xSUMOs (4xS) and ubiquitinated 4xSUMOs (4xS+Ub) are indicated. Gels were also Coomassie-stained (Fig. S7 E). Source data are available for this figure: SourceData F9.