Fig. 1

TRIM5α and SUMO-modified TRIM5α form a complex with RanGAP1 and RanBP2 at the nuclear pore. a The consensus site for in vitro SUMOylation by PIAS1 is not a target for SUMOylation in human cells. HEK 293T cells were transfected with Flag-tagged human TRIM5α or K10R mutant, previously shown to abrogate PIAS1-mediated SUMOylation in vitro. After 3 days, TRIM5α was immunoprecipitated using α-Flag antibodies and probed with α-SUMO1 or α-Flag antibodies. SUMO-modified TRIM5α is detected at ca. 70 kDa in the SUMO1 blots for both WT and K10R lanes (TRIM5α*S1). Western blots are representative of 3 independent experiments (original blots in Supplementary Figure 2a). b TRIM5α localizes at the nuclear envelope in myotubes. To identify the E3 SUMO ligase responsible for TRIM5α SUMOylation in cells, we searched for cell types in which endogenous TRIM5α did not form typical cytoplasmic aggregates, and probed for proximity with SUMO machinery. In human myotubes, the majority of TRIM5α signal accumulated around the nuclear envelope. Scale bar = 20 μm. c TRIM5α co-localizes with RanGAP1 in myotubes. Super-resolution imaging of human myotubes shows TRIM5α co-localization with Ran-specific GTPase activating protein RanGAP1, present on cytoplasmic filaments of the nuclear pore, but not with Nup153, which is found in the nuclear basket. Donor 1: neonatal myoblasts (CHQ), donor 2: adult myoblasts (160 M). Scale bar = 2 μm. d TRIM5α co-localizes with RanGAP1 in HeLa and HEK 293T cells. Proximity ligation assay was performed in HeLa and 293T cells, only HeLa are shown as an example. TRIM5α×RanGAP1 PLA signals were detected at a frequency of 4 and 1.5 spots/cell, respectively, and localized to the nuclear membrane in 55% of cases in both cells types, confirming that TRIM5α co-localizes with RanGAP1 at the nuclear envelope in these cells. Scale = 10 μm. e TRIM5α and SUMO-modified TRIM5α form a complex with RanGAP1 and RanBP2 in HeLa cells. RanGAP1 was immunoprecipitated from HeLa cytoplasmic extracts and precipitates were probed for TRIM5α and SUMO1. RanGAP1 immunoprecipitates contained RanBP2, unmodified TRIM5α (55 kDa), and a ~70 kDa TRIM5α species that also appeared in SUMO1 blots identifying it as SUMO1-modified TRIM5α (TRIM5α*S1). SUMOylated RanGAP1 (~90 kDa) is present at very low levels in cytoplasmic extracts and is therefore not detected here. Western blots are representative of 2 independent experiments (original blots in Supplementary Figure 2b)