Fig. 5: UBE2J2 activity modulates ubiquitination cascade outcome.

a Ubiquitination of RNF145 and UBE2J2. Fluorescently labelled ^DL800RNF145‑ALFA (P/L  =  1:32,000) and UBE2J2_DL680 (P/L  =  1:32,000) were co‑reconstituted in PC/PE liposomes with the indicated saturated fatty acid (SFA) content. Samples collected at the indicated time points were analyzed by reducing SDS–PAGE and fluorescence scanning. Quantification of ubiquitinated RNF145 ((b), n = 10) and UBE2J2 ((c), n = 7 for 10%, n = 6 for 60% SFAs) from reactions as in (a). Solid lines connect means, error bars mean ± s.d. Each replicate is an independent protein reconstitution into liposomes. d Quantification of RNF145-ALFA ubiquitination with soluble UBE2J2 (sUBE2J2_1-226). ^DL800RNF145-ALFA was reconstituted in liposomes with indicated SFA content (P/L = 1:32,000) and incubated with E1, ubiquitin, ATP and 0.5 μΜ sUBE2J2_1-226 (n = 2). e Ubiquitination of SQLE by MARCHF6 and UBE2J2. ^DL800SQLE, MARCHF6 and UBE2J2_DL680 were co-reconstituted in PC/PE liposomes with the indicated SFA content (each at P/L = 1:32,000). Samples were collected over time and analyzed as in (a). f Quantification of ubiquitinated SQLE from (e) (n = 2). g Quantification of SQLE ubiquitination by MARCHF6 and soluble UBE2J2. ^DL800SQLE and MARCHF6 were co-reconstituted in PC/PE liposomes with the indicated SFA content (each at P/L = 1:16,000); sUBE2J2 was added at the indicated concentrations. h RNF145–UBE2J2 interaction analysis. ^DL800RNF145-ALFA, UBE2J2_DL680 and Syb2_A488 were co-reconstituted in liposomes with indicated SFA content (each at P/L = 1:32,000). Immunoprecipitation (IP) was performed with biotinylated anti-ALFA nanobody, eluted with SUMO protease Ulp1*. Co-reconstitution was estimated from IP of intact liposomes; protein-protein interactions were assessed by IP from detergent-solubilized liposomes. In: Input, UB, unbound, E elution i Quantification of UBE2J2_DL680 co-purifying with RNF145-ALFA from solubilized liposomes in (h). Co-purified UBE2J2_DL680 was normalized to eluted RNF145-ALFA and UBE2J2_DL680 input. Error bars, mean ± s.d. Statistical significance was assessed using a paired-sample, two-sided t-test (*p  =  0.00283, n = 6). In (b, c, d, f, g, and i), each replicate represents an independent protein reconstitution into liposomes. Detailed liposome compositions are given in Table 1. Source data are provided as a Source Data file.