Figure 5: TRIM31 interacts with PE in a palmitoyl-dependent manner.

(a) Multiple sequence alignment of the B-box domain containing a predicted palmitoylation site composed of the conserved CXXC motif. The wild-type TRIM31 CXXC motif (TRIM31-WT) was mutated to AXXA (TRIM31-AXXA). Blue box, conserved hydrophobic amino acids; yellow box, conserved cysteine residues. The RING, B-box and coiled-coil domains are depicted in green, pink and blue, respectively. (b) Crosslinking experiment of GST-tagged recombinant proteins with 80% 18:1–16:0 PC and 20% 18:1–12:0 NBD–PE liposomes. GST-TRIM31-wild-type B-Box (GST-TRIM31-WT), GST-TRIM31-AXXA-mutated B-Box (GST-TRIM31-AXXA), or GST-glycine-exposed LC3 (GST-LC3-Gly) was incubated with liposomes, and then samples were analysed by SDS–PAGE. The LAS image analyser was used to detect NBD fluorescence (upper) while Coomassie blue staining was used to visualize protein levels (bottom). GST-LC3-Gly was used as a positive control. (c) Colocalization of wild-type (TRIM31-WT) or AXXA-mutated TRIM31-Myc (TRIM31-AXXA) with NBD–PE. HeLa cells were transfected with TRIM31-WT-Myc or TRIM31-AXXA-Myc, labelled with NBD–PE liposomes for 1 h, washed twice with PBS, and incubated for 1 h in complete media. Scale bars, 5 μm. Quantification is shown in the right graph (mean±s.d.). *P<0.01 (Student’s t-test). (d) HeLa cells expressing mRFP-GFP-tagged wild-type TRIM31 (mRFP-GFP-TRIM31-WT) or AXXA-mutated TRIM31 (mRFP-GFP-TRIM31-AXXA) were treated with LPS (100 ng ml⁻¹) for 4 h. Scale bar, 5 μm. (e) Palmitoylation is involved in forming a punctate structure of TRIM31. HeLa cells stably expressing TRIM31-Myc were left untreated or treated with 2-BP (20 μM) for 12 h. TRIM31-Myc was stained using anti-Myc antibody and analysed by IFA. Blue, DAPI. Scale bar, 5 μm. (f) Palmitoylation is essential for TRIM31–PE interaction. HeLa cells expressing TRIM31-Myc were left untreated or treated with 2-BP (20 μM) for 12 h and then labelled with 20% NBD–PE/80% POPC liposomes. TRIM31-Myc was immunostained with anti-Myc antibody and analysed by IFA. Blue, DAPI. Scale bar, 5 μm. (g) HeLa cells expressing mRFP-GFP-TRIM31 were incubated for 9 h in 2-BP (25 μM) and then stimulated with LPS (100 ng ml⁻¹) for 3 h. Scale bar, 5 μm. All data are representative of at least two independent experiments.