Fig. 3: Reconstitution and visualization of Hrd1 autoubiquitination within individual proteoliposomes.

a Hrd1^Cy5 proteoliposomes were incubated with ubiquitin^Cy3, recombinant ubiquitin machinery, +/− ATP for the indicated times. The reactions were separated by SDS-PAGE and imaged using in gel fluorescence (for ubiquitin^Cy3 and Hrd1^Cy5) or after staining with coomassie blue. b Schematic of the ubiquitination experiment with immobilized Hrd1^Cy5 proteoliposomes. c Hrd1^Cy5 proteoliposomes were immobilized on passivated coverslip surfaces after ubiquitination for 60 min (as in (a)) and visualized under TIRF excitation. Ubiquitin^Cy3 (left panels) and Hrd1^Cy5 (middle panels) images were overlaid with white showing colocalization (right panels). The scale bar is 10 μm. d Quantification of colocalization for ubiquitin^Cy3 and Hrd1^Cy5 signals from ubiquitination assay in (c). e Ubiquitin^Cy3 intensities for Hrd1^Cy5 proteoliposomes incubated with ubiquitination machinery +/− ATP. Lines indicate the mean fluorescence intensity. f As in (d), but separated by Hrd1 oligomeric sizes. For proteoliposomes with a specific number of photobleaching steps, the data is represented as the fraction of colocalized spots to the total number of spots. g Ubiquitin^Cy3 intensity at individual foci separated by Hrd1 oligomer size in the presence of ubiquitination machinery and ATP in (as in (c)). Lines indicate the mean fluorescence intensity. h Schematic of unlabeled polyubiquitination detection using a tandem ubiquitin binding element labeled with Cy3 (TUBE^Cy3) at Hrd1^Cy5 proteoliposomes. i Hrd1^Cy5 proteoliposomes were immobilized on passivated coverslip surfaces after ubiquitination (using unlabeled ubiquitin) and visualized under TIRF excitation. TUBE^Cy3 was added to the slide surface at 10 pM and incubated for 1 min before imaging TUBE^Cy3 (left panels) and Hrd1^Cy5 (middle panels, overlay in the right panels). The scale bar is 10 μm. j) Quantification of colocalization of TUBE^Cy3 and Hrd1^Cy5 signals from the ubiquitination assay in the absence/presence of ATP (in panel (f)) separated by proteoliposomes with a specific number of photobleaching steps. Imaging panels in (a, c, i) and the data presented in (g) are representative of at least three independent experiments. The summary data in (d–f, j) combine at least three independent reconstitution experiments. Source data are provided as a Source Data file. See also Supplementary Fig. 3.