Fig. 1: Development of a single-molecule approach to count Hrd1 membrane protein stoichiometry in reconstituted proteoliposomes. | Nature Communications

Fig. 1: Development of a single-molecule approach to count Hrd1 membrane protein stoichiometry in reconstituted proteoliposomes.

From: Direct observation of autoubiquitination for an integral membrane ubiquitin ligase in ERAD

Fig. 1

a Purified Hrd1 with a sortase A transpeptidation site and streptavidin binding protein tag (Hrd1-LPETGG-SBP) was immobilized on streptavidin resin and eluted into solution using sortase A and GGGC-Cy5 peptide (GGGCCy5) to maximize labeling efficiency. Samples were separated by SDS-PAGE and imaged using Stain free technology to visualize total protein (top panel) and in gel fluorescence for Cy5 (bottom panel). Sortase A elution catalyzes the exchange of a ~5 kDa SBP affinity tag with the GGGCCy5 peptide, resulting in >95% of Hrd1 labeled with Cy5 (HrdCy5). b Schematic of the glycerol density step gradient used to float proteoliposomes to verify proteoliposome integrity. Any un-reconstituted protein would be found at the bottom of the density gradient. c Hrd1Cy5 containing proteoliposomes were separated using a glycerol density gradient as in (b). The gradient fractions were collected and analyzed as in (a). Hrd1Cy5 was reconstituted at 200 nM in 5 mM total lipid (99% DOPC, 0.5% NBD-PC, 0.5% biotinyl-cap-PE). d As in (c), but the gradient fractions were analyzed by fluorescence imaging in tubes. For this experiment, Hrd1Cy5 was reconstituted at 100 nM in 5 mM total lipid. e Schematic of Hrd1Cy5 proteoliposome immobilization using biotinylated lipids on a coverslip surface passivated with a PEG polymer brush. Individual proteoliposomes can be visualized using TIRF excitation. f NBD-PC (LipidNBD, left panels) and Hrd1Cy5 (center panels) were visualized using TIRF excitation. Empty liposomes are shown in the top row and Hrd1Cy5 proteoliposomes are shown in the bottom row. The right panels are overlay images to show colocalization of lipidNBD (cyan) and Hrd1Cy5 (magenta). Each diffraction limited spot corresponds to an individual liposome. Hrd1Cy5 was reconstituted at 100 nM in 5 mM total lipid (N = 3). The inset represents a 3 fold magnified view of the region enclosed within the rectangle and the scale bar is 10 μm. Each panel in this figure is representative of at least three independent biological replicates. Source data are provided as a Source Data file. See also Supplementary Fig. 1.

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