Fig. 2: SUMOylation enhances Golgin45 protein stability by inhibiting TNKS1-dependent PARylation of Golgin45.

a XAV939 treatment increases SUMO1-modified SUMOylation on Golgin45. HeLa cells were transiently transfected with mCherry-Golgin45 and SUMO1 or SUMO3 along with Ubc9 WT or DN, followed by either DMSO or XAV939 (a specific inhibitor of TNKS1/2). The cells were lysed and analyzed by western blotting using antibodies against the indicated proteins. b Relative levels of SUMO1 or SUMO3 conjugated mCherry-Golgin45 are presented as mean ± SD. Statistical analysis of relative SUMOylation was performed by one-way ANOVA with Tukey’s multiple comparisons. n = 3 independent experiments. ns: not significant. ****P < 0.0001. c, d Deletion of TNKS1 binding domain (ΔTBD) increases both SUMO1 and SUMO3 modified SUMOylation of Golgin45. After transfection with the indicate plasmids, HeLa cells were lysed and analyzed by western blotting using antibodies against the indicated proteins. e, f HeLa-Golgin45-KO cells stably expressing mCherry-Golgin45, mCherry-Golgin45-8KR mutant or mCherry-Golgin45-ΔTBD mutant were treated with cycloheximide (50 μg/ml) for the indicated times. Ectopically expressed WT and mutant mCherry-Golgin45 protein levels were analyzed by immunoblotting. Quantification of exogenous protein levels relative to GAPDH expression is shown in (f). Relative protein levels of mCherry-Golgin45 are presented as mean ± SD. Statistical analysis of relative protein level was performed by two-way ANOVA with Dunnett’s post-hoc test for multiple comparisons. n = 3 independent experiments. ***P < 0.001. g SUMOylation of mCherry-Golgin45 inhibits its PARylation. Hela cells transfected with indicate plasmids were lysed and immunoprecipitated using anti-RFP beads, and blotted with either anti-mCherry, anti-myc or anti-PAR. h Golgin45 PARylation is increased for Golgin45 8-KR mutant. HeLa cells expressing GST, GST-Golgin45 or GST-Golgin45-8KR were lysed and pulled down using GST beads, and analyzed by western blotting using anti-GST or anti-PAR antibodies. Representative blots are shown and experiments were repeated three times.