Extended Data Fig. 5: Testing interactions of the RFs and the roles of the plug and pore size.
From: A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel
a, Density map of the ligase complex, viewed from the side. The RF tower, TM5 of Pex12, and the central connector linking this TM to RF12 are shown in color. The right panel shows a magnified view of the boxed area. The conserved Pro residues in the central connector are indicated. b, Purified RF2 and RF12 were mixed and subjected to gel filtration. The upper panel shows the absorbance profile. Fractions between the broken lines were analyzed by SDS-PAGE and Coomassie blue staining (lower panel). c, As in b, but with a mixture of RF2 and RF10. d, As in b, but with a mixture of RF10 and RF12. e, As in b, but with a mixture of RF2, RF10, and RF12. f, Wild-type (WT) S. cerevisiae ligase complex and a complex containing mutant Pex2 (C237S), both containing Pex12 with an N-terminal streptavidin-binding peptide (SBP) tag, were expressed in P. pastoris. The complex was isolated with streptavidin beads and the bound material analyzed by SDS-PAGE and Coomassie blue staining. g, The expression levels of FLAG-tagged Pex2 or Pex12 mutants that reduce the pore size (see Fig. 3d) were determined by immunoblotting with FLAG antibodies. Blotting for Sec61α served as a loading control. The results in b–g are representative of three biological repeats. For gel source data, see Supplementary Fig. 1. h, Wild-type (WT) Pex10 or a mutant lacking the putative plug (Δplug) were expressed from the endogenous promoter in S. cerevisiae cells lacking Pex10 (pex10Δ). Controls were performed with pex10Δ cells and cells expressing only an antibiotic resistance gene (mock). Peroxisomal protein import was determined by the reduction in the formation of a fluorescent pigment (PDV). Fluorescence was measured in cell lysates and the data were normalized, setting the fluorescence of pex10Δ cells as 100% and that of WT cells as 0%. The bar graph shows the individual data points, the mean and S.E.M. from three biological repeats. Statistical significance between the wild type and mutants was calculated by one-way analysis of variance, **P < 0.01. See also Source Data file
