Extended Data Fig. 8: Characterization of the KPWE motif in the N-terminus of PEX13.
From: PEX39 facilitates the peroxisomal import of PTS2-containing proteins
a, ScanProsite analysis of generic [R/K]-P-W-[E/Q] motifs in yeast and humans. The UniProtKB sequence databases of yeast or human were searched for proteins harboring the short linear motif [R/K]-P-W-[E/Q] using the webtool ScanProsite (https://prosite.expasy.org/scanprosite/). Hits were then compared with proteins listed as yeast and human PEX7 interactors in the BioGRID database. b, Recombinant FLAG-PEX7 and NtPEX13, alone or mixed together, were analyzed by SEC. Collected fractions were analyzed by SDS-PAGE and Coomassie staining (top). The corresponding area of the chromatogram is also shown (bottom). The elution volumes of the PEX7-NtPEX13 complex, the individual recombinant proteins, and molecular weight standards (Ferritin, BSA, RNAse A) are indicated. The asterisk indicates a proteolytic product of NtPEX13. c, Confidence measures for modeling the PEX7-PEX13 interaction for human and yeast orthologs. Predicted aligned error plots (top) and predicted LDDT (local distance difference test) plots (bottom) of human (left) and yeast (right) AlphaFold models. To predict human and yeast protein complexes, full-length PEX7 and the N-terminus of PEX13 (residues 1-55) were used. d, 35S-ACAA1 in vitro import assays in the presence of increasing concentrations of NtPEX13 or NtPEX13(4A). After incubation, reactions were treated with trypsin and organelles were isolated by centrifugation and analyzed by SDS-PAGE and autoradiography. Precursor and mature ACAA1 indicated by open and solid red arrowheads, respectively. I, input (5% of the reticulocyte lysate containing the 35S-ACAA1 used in each reaction). e, 35S-PEX5(C11K) in vitro import assays in the presence or absence of NtPEX13. After incubation, organelle pellet (OP) and cytosolic supernatant (S) fractions were isolated by centrifugation and analyzed by SDS-PAGE and autoradiography. Unmodified 35S-PEX5(C11K) (“PEX5”) and monoubiquitinated 35S-PEX5(C11K) (“Ub-PEX5”) are indicated. AMP-PNP inhibits the ATPases PEX1/PEX6 and thereby inhibits the transfer of monoubiquitinated 35S-PEX5(C11K) from peroxisomes into the cytosolic supernatant115. I, input [5% of the reticulocyte lysate containing the 35S-PEX5(C11K) used in each reaction].
