UBL3 modification influences protein sorting to small extracellular vesicles

Abstract

Exosomes, a type of small extracellular vesicles (sEVs), derived from multivesicular bodies (MVBs), mediate cell-to-cell communication by transporting proteins, mRNAs, and miRNAs. However, the molecular mechanism by which proteins are sorted to sEVs is not fully understood. Here, we report that ubiquitin-like 3 (UBL3)/membrane-anchored Ub-fold protein (MUB) acts as a posttranslational modification (PTM) factor that regulates protein sorting to sEVs. We find that UBL3 modification is indispensable for sorting of UBL3 to MVBs and sEVs. We also observe a 60% reduction of total protein levels in sEVs purified from Ubl3-knockout mice compared with those from wild-type mice. By performing proteomics analysis, we find 1241 UBL3-interacting proteins, including Ras. We also show that UBL3 directly modifies Ras and oncogenic RasG12V mutant, and that UBL3 expression enhances sorting of RasG12V to sEVs via UBL3 modification. Collectively, these results indicate that PTM by UBL3 influences the sorting of proteins to sEVs.

Fig. 1

Analysis of UBL3 as a post-translational modification factor. a UBL3-dependent posttranslational modification was detected by immunoprecipitation (IP) with anti-Flag antibodies from MDA-MB-231 cells transfected with Flag-UBL3, followed by western blotting with UBL3 antiserum (right panel). b The tissue extracts from the cerebral cortex of WT and Ubl3 KO mice were immunoprecipitated with anti-UBL3 antibodies. c, f Schematic structures of Flag-tagged wild-type or mutant UBL3. d, g Detection of UBL3 modification in these cells. IP products were boiled without 2-mercaptoethanol (βME-). A portion of the samples was treated with 2-mercaptoethanol (βME+). e, h Subcellular localisation of UBL3 in MDA-MB-231 cells transfected with Flag-UBL3 (wild-type and mutants). Twenty μg per lane

Fig. 2

The localisation of UBL3 to MVBs depends on UBL3 modification. a Representative projected images of MDA-MB-231 cells transfected with EGFP-UBL3 and co-stained with markers for MVB (CD63, n = 10), early endosome (EEA1, n = 10), lysosome (LysoTracker, n = 10), or recycling endosome (Rab11, n = 10). The regions in the dotted box are shown as a single confocal image in the inset. Scale bars, 10 and 1 μm. b Quantitative analysis of EGFP-UBL3 fluorescence intensity in a. *, p < 0.05; ***, p < 0.001 by Kruskal–Wallis/Dunn´s multiple-comparisons test. c Representative images of MDA-MB-231 cells transfected with EGFP-UBL3 (wild-type, n = 5; mutants, n = 5) and co-stained with CD63 values shown as % of total UBL3. Scale bars, 10 and 1 μm. d Immune-EM images of wild-type UBL3 and UBL3Δ1 in MDA-MB-231 cells. Scale bars, 500 nm (left and right panels) and 200 nm (middle panel). e Quantification of the numbers of gold colloids per area in d. MVB, n = 10; Plasma membrane, n = 10. *, p < 0.05; ***, p < 0.001 by two-tailed Student’s and Welch’s t-tests

Fig. 3

The level of total proteins in the sEVs is reduced in Ubl3 knockout mice. a The cell lysate (CL) and pellets from the conditioned medium of MDA-MB-231 cells transfected with 3xFlag-UBL3 vectors were blotted with various antibodies. b The presence of UBL3 and its mutants in sEVs. c Electron microscopic analyses of purified sEVs by negative staining. Scale bars, 100 nm. d Upper left panel, protein staining for the sEVs from the serum in WT and Ubl3 KO mice. Lower panels, purified serum sEVs were blotted with anti-tubulin and CD9 antibodies with βME. Right panel, relative intensity of total proteins in serum sEVs. n = 9 pairs. e Total RNA levels in the serum sEVs. WT, n = 5; KO, n = 5. n.s., p > 0.05 by Mann–Whitney test. a,b, βME + condition: Flag, Flotillin-1, GP96, Actinin-4, Calreticulin, GAPDH, and Alix antibodies. βME- condition: CD63, and CD9 antibodies

Fig. 4

UBL3 modification influences the sorting of proteins to the sEV. a, b Heat map of z-scored LFQ intensities of the significantly regulated proteins (ANOVA, FDR = 0.05 & S0 = 1) in all three conditions (3xFlag-UBL3, 3xFlag-UBL3C113/114A or 3xFlag empty vector) revealed the UBL3 interacting proteins (Cluster 1). The colour key denotes normalised protein abundances (z-score). Profiles of all proteins (1447) found in Cluster 1 are shown and 454 of those (31%) with ‘extracellular vesicular exosome’ annotations are highlighted in dark blue. c Volcano plots showing the p-values vs. the log2 protein abundance differences in Flag-UBL3 compared with either Flag-UBL3C113/114A or Flag empty vector. The significance cut-off is based on an FDR = 0.05 and S0 = 1. Disease-related molecules, black colour. HRAS and KRAS, red colour. TUBA1A, 1B, 1C, and 4A, orange colour. TUBB, B2A, B3, B4A, B4B, B6, and B8, blue colour. d sEV pellets were blotted with anti-Ras antibodies. e Images of phosphorylated ERK (pERK) in PKH67-labelled sEV-incorporated MDA-MB-231 cells. Purified sEVs from the conditioned medium of MDA-MB-231 cells transfected with RasG12V and either mock, 3xFlag-UBL3, or 3xFlag-UBL3C113/114A or with mock and 3xFlag-UBL3 were labelled with PKH67 dye (green) and added to MDA-MB-231 cells. Scale bars, 10 μm. f Each plot shows pERK fluorescence in PKH67-labelled sEV-incorporated cells normalised to the average pERK values in the two neighbouring PKH67-labelled sEV-unincorporated cells from each image in e. RasG12V-mock, n = 18; RasG12V-UBL3, n = 19; RasG12V-UBL3C113/114A, n = 19; Mock-UBL3, n = 21. **, p < 0.01; ***, p < 0.001 by Kruskal–Wallis/Dunn's multiple-comparisons test

Fig. 5

Subcellular localisation and sorting to sEV of UBLs. a Representative images of MDA-MB-231 cells transfected either with EGFP-ubiquitin, -SUMO1, -SUMO2 or -UBL3 and co-stained with markers for MVBs (CD63). Scale bars, 10 and 1 μm. b Quantitative analysis of EGFP-ubiquitin, -SUMO1, -SUMO2, and -UBL3 fluorescence intensity in a. EGFP-ubiquitin, n = 10; EGFP-SUMO1, n = 10; EGFP-SUMO2, n = 10; EGFP-UBL3, n = 10; EGFP, n = 10. *, p < 0.05; ***, p < 0.0001 by Kruskal–Wallis/Dunn's multiple-comparisons test. c EGFP-UBL3, but not EGFP, EGFP-ubiquitin, EGFP-SUMO1, or EGFP-SUMO2, was preferentially enriched in the sEVs of the cell culture media. Before sample loading, the samples were boiled with βME

Fig. 6

EGFP and biotinylated protein tagged by UBL3 are sorted to the sEVs. EGFP-UBL3 (left panel) and biotinylated protein tagged by UBL3 (right panel), but not EGFP-UBL3C113/114A or biotinylated protein tagged by UBL3C113/114A were preferentially accumulated in the sEVs of the cell culture media. After the western blot analysis, gels were stained with SYPRO Ruby (lower panel). Before loading, the samples were boiled with βME. The same amounts of protein were loaded on the gels (Cell Lysate, 20 μg per lane; sEVs, 1 μg per lane)