Fig. 2: Membrane composition dictates the efficiency of UBE2J2 loading.
From: UBE2J2 sensitizes the ERAD ubiquitination cascade to changes in membrane lipid saturation
a Ubiquitin loading assay, with UBE2J2DL680 reconstituted into liposomes containing 80 mol% PC and 20 mol% PE with indicated saturated fatty acids (SFA) content. b Quantification of assays as in (a); n = 7 (10% SFAs), n = 9 (60% SFAs), n = 3 (50% SFAs). Solid lines connect mean values; for n > 3, error bars represent mean ± s.d., standard deviation. c Kinetic analysis of ubiquitin loading of UBE2J2 by quenched-flow. Data from (b) is included in the plot and used for the fitting procedure. Solid lines represent the fitting result. d Schematic of the kinetic model used for the fitting procedure. The table below shows the result of the fitting procedure with units s-1. e As in (a), but with liposomes varying in di-unsaturated lipid content (P/L = 1:32,000). f Quantification of reactions as in (e). n = 3 (50% SFAs), n = 4 (40% and 35% SFAs). g Lipid packing of protein- and sterol-free liposomes with various SFA contents measured by C-Laurdan fluorescence. Emission spectra were recorded at 37 °C (n ≥ 3); generalized polarization was calculated as described18. h Ubiquitin loading of UBE2J2DL680, reconstituted in PC-only liposomes with indicated SFA content (P/L = 1:32,000). i Quantification of reactions as in (h). Data for PC-only liposomes are shown in blue and green (n = 3); for comparison, data from (b) obtained with PC/PE liposomes are shown black and red. j Ubiquitin loading of UBE2J2DL680, reconstituted in PC/PE liposomes with indicated SFA content with or without 10 mol% cholesterol (P/L = 1:32,000). k Quantification of reactions as in (j). Mean values are connected by solid lines; n = 2 (35% SFAs + cholesterol), n = 4 (35% SFAs, no cholesterol, reproduced from (f)), n = 9 (60% SFAs, no cholesterol, reproduced from (c)), n = 3 (60% SFAs + 10% cholesterol). In (b), (f), (i) and (k), each replicate represents an independent protein reconstitution into liposomes. For n > 3, error bars represent mean ± s.d. Detailed liposome compositions are given in Table 1. Source data are provided as a Source Data file.
