Fig. 1: E2 enzymatic activity is low in ER-like membranes.
From: UBE2J2 sensitizes the ERAD ubiquitination cascade to changes in membrane lipid saturation
a Schematic of ubiquitin loading assay. Fluorescently labeled E2s were incubated at 37 °C with ubiquitin-loaded E1 (Uba1) in the presence of ATP and free ubiquitin. UBE2J1 and UBE2J2 both possess an N-terminal UBC domain connected to the C-terminal transmembrane segment by a disordered region. The red star marks the position of the fluorescent label. b UBE2J2DL680, reconstituted into ER-like liposomes (protein: lipid ratio 1:32,000) or dissolved in detergent, was subjected to the reaction described in (a). Samples were collected at indicated time points, separated by non-reducing SDS-PAGE, and analyzed by fluorescence scanning. Where indicated, ATP was omitted (-ATP) or the 10 min sample was reduced with 2% (v/v) 2-mercaptoethanol (R). c Quantification of ubiquitin-loaded UBE2J2DL680 from reactions as in (b). Lines connect mean values. For reactions with more than three replicates, error bars indicate the mean ± standard deviation (n = 7, detergent; n = 3, ER-like liposomes). Each replicate for liposome conditions represents an independent protein reconstitution, and for detergent conditions an independently set up reaction. d Ubiquitin loading assay as in (b), but with UBE2J1DL680. e Quantification of ubiquitin-loaded UBE2J1DL680 from reactions as in (d) (n = 3, both conditions). R reduced. ER-like liposomes contained 60 mol% POPC, 20 mol% DOPE, 10 mol% DOPS, 10 mol% cholesterol. Complete lipid compositions are given in Table 1. Source data are provided as a Source Data file.
