Extended Data Fig. 2: Interaction of PEX5 variants with cargo and kinetics of peroxisomal protein import in Xenopus egg extract.

a, Glutathione-conjugated beads were bound to 1 µM GST alone or to the purified recombinant GST-tagged PEX5 variants shown (described in Fig. 1b), and incubated in the presence of 1 µM purified recombinant yeast PEX7 and 1 µM GFP fused at the N terminus to the import signal (residues 1–27) from yeast POT1 (PTS2). Recruitment of the fluorescent cargo to the beads was visualized after 1 h on a spinning-disk confocal microscope as a bright ring around the bead perimeter. Scale bar equals 100 µm. b, Xenopus egg extract was depleted of endogenous PEX5 using beads conjugated to the PEX5-binding domain from PEX14, then supplemented with the chimeric receptor PEX5-21 (described in Fig. 1b) and purified recombinant yeast PEX7. Import was assessed using fluorescent PTS1 and PTS2 cargo: the PTS2 cargo is described in panel a, whereas the PTS1 cargo consisted of GFP fused at the C terminus to the SKL peroxisome targeting signal. Reactions were imaged at the indicated times on a spinning-disk confocal microscope. The fluorescence intensity of the resulting puncta (corresponding to imported cargo) was then measured, converted into GFP concentrations as described in the Methods, and plotted as a function of time. Error bars span the range of at least 30 imaged fields; lines represent linear fits to the data generated by least-squares regression. The corresponding import rates (±the standard error) are specified beside the graph. Scale bars equal 5 µm. c, As in panel a, using GST alone or the indicated GST-tagged PEX5 variants (described in Fig. 1b), in the presence or absence of purified recombinant yeast PEX7 and the fluorescent cargo described in panels a and b. Source numerical data are provided.

Source data