Fig. 6: Cholesterol modulates auto-regulation of the E3 ubiquitin ligase RNF145.

a Ubiquitination of RNF145 with a soluble UBE2J2 (sUBE2J2_1-226). Fluorescently labeled ^DL800RNF145-ALFA was reconstituted in ER-like liposomes with or without 10 mol% cholesterol (P/L = 1:32,000) and was incubated with E1, ubiquitin, ATP and 2 μΜ sUBE2J2_1-226. Samples were analyzed by reducing SDS-PAGE and fluorescence scanning at indicated time points. b Quantification of ubiquitinated RNF145 from (a) (n = 3). Error bars indicate mean ± s.d. Statistical significance between conditions at each time point was assessed using Welch’s two-sample, two-sided t-test: p = 0.02561 (5 min, *), p = 0.00057 (10 min, **), p = 0.03553 (20 min, ***), p = 0.06854 (40 min, n.s.). c Ubiquitination of RNF145 with UBE2J2. Proteins were co-reconstituted in liposomes with 60% SFA content, with or without 10 mol% cholesterol (P/L = 1:32,000 for both proteins). Samples were analyzed as above. d Quantification of ubiquitinated RNF145 from (c), n = 3 (with cholesterol), n = 10 (without cholesterol). Error bars indicate the mean ± s.d. Statistical significance at each time point was determined using Welch’s two‑sample, two‑sided t‑test: p = 0.000134 (5 min, *), p = 0.00071 (10 min, **), p = 0.00011 (20 min, ***), p = 0.000024 (40 min, ****). e Analysis of RNF145 oligomerization. ^DL800RNF145-ALFA was co-reconstituted with Syb2_A488 and RNF145_ΔRING-sfGFP (the C-terminal cytoplasmic part including the RING domain was replaced with superfolder GFP) in (liposomes of indicated SFA content ±10% cholesterol (P/L = 1:32,000). After solubilization, proteins were precipitated with anti-GFP or anti-ALFA nanobodies. Samples were analyzed by SDS-PAGE and fluorescence scanning. The experiment under these exact conditions was performed once; similar assays were repeated multiple times under related conditions and yielded consistent results. f Quantification of co-purified RNF145 upon immunoprecipitation of RNF145-ALFA or RNF145_ΔRING-sfGFP from the experiment in (e). The co-purifying RNF145 variant was normalized to nanobody-precipitated RNF145 and to the input. In (b and d), 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.