Fig. 3: Importin-β2-mediated nuclear import of Golgin45 plays an important role in Golgin45 SUMOylation.

a Domain structure of Golgin45. Tankyrase-1, Rab2-GTP, and GRASP55 bind to the N-terminus, coiled coil domain, and the C-terminus of Golgin45, respectively. The putative NLS on Golgin45 for importin β2 is noted with an arrow indicating a key residue R375. b R375A mutation greatly reduced the binding between Importin β2 and Golgin45. Purified His-tagged importin β2 was incubated with GST, GST-Golgin45 336–385 and GST-Golgin45 336–385 R375A and the bound fractions were analyzed by western blots using anti-importin β2 antibody. c Importin β2 was co-immunoprecipitated by Golgin45, but not R375A mutant. HeLa cells were tranfected with mCherry, mCherry-Golgin45 (mCh-G45), or mCherry-Golgin45 R375A (mCh-G45 R375A) for 18 h. Cells were lysed, incubated with anti-RFP beads, and analyzed with indicated antibodies. d mCherry-Golgin45, but not the R375A mutant, localized to the nucleus. HeLa cells overexpressing mCherry-Golgin45 or mCherry-Golgin45 Δ397–400, mCherry-Golgin45 R375A or mCherry-Golgin45 R375A Δ397–400 mutant were fixed and stained with anti-GM130 antibody. Images were acquired using Zeiss LSM880 confocal microscope. Statistical analysis was performed by one-way ANOVA to quantify the fluorescent intensity ratio of nucleus-(e) or Golgi-(f) localized Golgin45 over the total mCherry-Golgin45. g–j R375A mutation decreases SUMOylation of Golgin45. HeLa cells expressing mCherry-Golgin45, or mCherry-Golgin45-R375 mutant with SUMO1 (g) or SUMO3 (i) and UBC9 were lysed and SUMOylation of Golgin45 were analyzed by western blotting. Relative SUMOlation level of Golgin45 was analyzed by one-way ANOVA with Tukey’s multiple comparisons (h, j). n = 3 independent experiments. *P < 0.05.****P < 0.0001.