Article Title: The importomer is a peroxisomal membrane protein translocase
Figure Lengend Snippet: Pex5 mediates Pex14 integration independently of PTS1 import by binding to the Pex14 NTD (A) (above) Schematic depicting Pex14’s domain organization. NTD: N-terminal domain, TMD: transmembrane domain, CTD: C-terminal domain. (below) Amino acid alignment of the indicated NTD region from the following Pex14 homologs: Saccharomyces cerevisiae (sc), Caenorhabditis elegans (ce), Homo sapiens (hs) and Arabidopsis thaliana (at). Absolutely conserved residues are highlighted in blue. (B) Solution NMR structure of the conserved region of the human Pex14 NTD bound to a Pex5-derived peptide (PDB2w84, Neufeld et al., 2009 ). The key interacting phenylalanine F35 (corresponding to F19 in yeast) on Pex14 is highlighted. (C) In vitro translation of mRNAs encoding the indicated Pex14 variants in the presence of S35-labeled methionine and recombinant FLAG-Pex5 was followed by anti-FLAG immunoprecipitation. Input, flowthrough (FT), and 2x-loaded immunoprecipitation (2xIP) samples were resolved by SDS-PAGE and analyzed by autoradiography (Autorad) and immunoblotting (IB) with anti-FLAG. (D) Representative confocal micrographs (maximum intensity projections) of logarithmically growing cells of the indicated genotypes expressing EmCitrine-Pex14-FLAG variants and the peroxisomal matrix marker mTurquoise2-PTS1. (E) Extracts from logarithmically-growing cells of the indicated genotypes expressing EmCitrine-Pex14-FLAG or the indicated deletion variants were resolved by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (F) Cells expressing the indicated FLAG-Pex14-MYC variant (WT or F19A) and Pex13-HA were grown to post-log phase before being subjected to spheroplasting and gentle lysis. Following centrifugation of lysates, membranes were resuspended in buffer and treated with proteinase K (doses: 0, 31 and 125 ng/μL) for 5 minutes at 37°C as indicated. Following proteinase K quenching, samples were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with the indicated antibodies. Arrow, full length proteins; arrowhead, protected fragments; asterisk, proteinase K-independent degradation product; double asterisk, proteinase K-dependent partially digested intermediate. (G) As in (D), except cells had the indicated PEX5 locus genotypes and also expressed the peroxisomal membrane marker Pex11-mCherry. (H) Proteinase K protection analysis of membranes derived from cells with the indicated PEX5 locus genotypes expressing FLAG-Pex14-MYC and Pex13-HA was carried out with or without Triton X-100 as in (F). Arrow, full length proteins; arrowhead, protected fragments; asterisk, proteinase K-independent degradation product; double asterisk, proteinase K-dependent partially digested intermediate.
Article Snippet: Where indicated, membranes were incubated in 1% Triton X-100 for 30 min on ice followed by treatment with proteinase K (Roche 3115879001) at the indicated concentrations at 37°C for 5 min. To quench proteinase K activity, samples were then treated with PMSF (5 mM) at 4°C for 10 min, before being diluted with pre-boiling 5/4× SDS-PAGE loading buffer (62.5 mM Tris-Cl pH 6.8, 2.5% SDS, 0.125% bromophenol blue, 12.5% glycerol, 125 mM β-mercaptoethanol) to a final loading buffer concentration of 1x, and further boiled for 10 min.
Techniques: Binding Assay, Nuclear Magnetic Resonance, Derivative Assay, In Vitro, Labeling, Recombinant, Immunoprecipitation, SDS Page, Autoradiography, Expressing, Marker, Variant Assay, Lysis, Centrifugation