Fig. 4: E1 interaction is impaired in membranes with lipid packing defects.
From: UBE2J2 sensitizes the ERAD ubiquitination cascade to changes in membrane lipid saturation
a Ubiquitin loading of UBE2J2 chimeras with UBE2J1 TMS and/or disordered linker, reconstituted in PC/PE liposomes with the indicated saturated fatty acids (SFA) content. b Quantification of (a) (n = 2). *Co-purified UBE2J2 fragment. c Ubiquitin loading of UBE2J2DL680 in liposomes with 10% SFAs, pre-incubated for 10 min with the indicated n-octyl-β-D-glucoside (OG) concentrations before loading. ATP was omitted where indicated (-ATP). d Quantification of (c). e Limited proteolysis assay on UBE2J2DL680 proteoliposomes as in (c). Samples were pre-incubated with the indicated OG concentrations for 10 min before trypsinization (0, 1.9, 5.6 or 16.6 ng/µl) for 30 min at 37 °C. f Linegraph showing the probability of contacts between the UBC domain and the ER membrane from molecular dynamics simulations. The probability distribution can be described by a four component Poisson mixture model (unbound, loosely bound (with a mean of 8 contacts), and two tightly bound conformations (30 and 50 contacts, respectively)). Representative structures for the first three components are shown above. g Contact frequency between the UBC domain and the different membranes (blue, ER-like; green, 10% SFAs; yellow, 60% SFAs). h Contact frequency for the ER-like membrane in (g) mapped onto the UBE2J2 structure. i Ubiquitin loading of wild type (WT) and mutant (F137E) UBE2J2, reconstituted PC/PE liposomes of indicated SFA content. j Quantification of (i) (n = 3, F137E; n = 7, WT 10% SFAs; n = 8, WT 60% SFAs). Error bars, mean ± sd. k Cysteine crosslinking to analyze UBE2J2:E1 interaction. UBE2J2DL680 liposomes with indicated SFA content were incubated with the E1 ± 1,2-bismaleimidoethane (BMOE); in detergent-containing reactions, proteoliposomes were pre-incubated with OG for 10 min before crosslinker addition. Samples were collected at the indicated time points were analyzed by SDS-PAGE and fluorescence scanning. P/L = 1:32,000. In (b, j), each replicate represents an independent protein reconstitution into liposomes. For (c, e, and k), experiments under these exact conditions were performed once; similar assays were repeated multiple times under related conditions and yielded consistent results. Detailed liposome compositions are given in Table 1. Source data are provided as a Source Data file.
