Figure 4: TRIM31 interacts with PE in an Atg7-independent manner.

(a) Colocalization of NBD–PE with TRIM31-Myc in HeLa cells. Intracellular NBD–PE labelling using delivery of liposomes consisting 80% 18:1–16:0 PC and 20% 18:1–12:0 NBD–PE. Cells were exposed to liposomes, washed with PBS, and maintained in complete media for 2 h. The C-terminal glycine-exposed form of LC3, Myc-LC3-Gly, was used as a positive control. (b) Colocalization of NBD–PE with endogenous TRIM31 in HT-29 cells. Cells were exposed to NBD–PE liposomes and fixed. Cells were then immunostained with an anti-TRIM31 antibody. Scale bars, 5 μm. (c) Crosslinking experiment of purified GST-tagged TRIM31-wild-type B-Box (GST-TRIM31-WT) with liposomes consisting of 20% NBD–PE/80% POPC or 20% NBD-sphingosine/80% POPC. Liposomes were incubated with the cross-linker AMAS (0.5 mM), and then glycine was added to terminate the crosslinking reaction. GST-TRIM31-WT was added to the reaction buffer, and sample reactions were terminated with the addition of 5 × SDS loading buffer. The LAS image analyser was used to detect NBD fluorescence (upper) while Coomassie blue staining was used to visualize protein levels (bottom). (d) The ability of the GST-tagged TRIM31-wild-type B-Box (2 μg ml⁻¹) to bind PE or POPC was analysed by a protein-lipid binding assay on nitrocellulose. Nitrocellulose membranes were spotted with 100 pmol of phospholipid. (e) TRIM31-Myc colocalizes with NBD–PE in puncta independent of Atg7. Wild-type or Atg7^−/− cells stably expressing TRIM31-Myc were labelled with liposomes containing 20% NBD–PE and 80% PC and chased in complete media for 1 h, followed by immunostaining with anti-Myc antibody. Scale bars, 5 μm. Quantification is shown in the right graph (mean±s.d.). All data are representative of at least three independent experiments.