Journal: bioRxiv

Article Title: The importomer is a peroxisomal membrane protein translocase

doi: 10.1101/2020.05.01.072660

Figure Lengend Snippet: Pex14 carries an N-terminal translocation signal that is dispensable for peroxisomal targeting (A) Schematic showing EmCitrine- and FLAG-tagged Pex14 and mutants lacking indicated domains or numbered N-terminal amino acid sequences. (B) Extracts from logarithmically-growing cells expressing EmCitrine-Pex14-FLAG or the indicated deletion variants were resolved by SDS-PAGE and analyzed by immunoblotted (IB) with the indicated antibodies. (C) Representative confocal micrographs (maximum intensity projections) of logarithmically growing cells expressing EmCitrine-Pex14-FLAG or the indicated variants and the peroxisomal matrix marker mTurquoise2-PTS1. (D) Representative confocal micrographs (maximum intensity projections) of logarithmically growing cells expressing GFP or Pex14N58-GFP and the peroxisomal membrane marker Pex11-mCherry. (E) Cells expressing Pex14N58-GFP were grown to post-log phase before being subjected to spheroplasting and gentle lysis. Following centrifugation of lysates, membranes were resuspended in buffer with or without Triton X-100, treated with proteinase K (doses: 0, 31, 125, 500 and 2000 ng/μL) for 5 minutes at 37°C. Following proteinase K quenching, samples were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with the indicated antibody. Arrow, full length Pex14N58-GFP; arrowhead, GFP-fragment resistant to proteinase K digestion even in the presence of Triton X-100. (F) Proteinase K protection analysis of membranes derived from cells expressing FLAG-Pex14ΔTMD-MYC was carried out as in (E). Arrow, full length FLAG-Pex14ΔTMD-MYC; asterisk, proteinase K-independent degradation product. (G) Proteinase K protection analysis of membranes derived from cells expressing either FLAG-GFP-Pex14-MYC or FLAG-GFP-Pex14ΔN50-MYC in addition to Pex13-HA was carried out as in (E). Proteinase K doses: 0, 31 and 125 ng/μL. Arrows, full length proteins; arrowheads, fragments resistant to proteinase K digestion.

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: Translocation Assay, Expressing, SDS Page, Marker, Lysis, Centrifugation, Derivative Assay

Journal: bioRxiv

Article Title: The importomer is a peroxisomal membrane protein translocase

doi: 10.1101/2020.05.01.072660

Figure Lengend Snippet: Pex14 becomes arrested in a pre-integrated state on peroxisomes in importomer mutants (A) Schematic illustrating the strategy for detecting an integration defective mutant using a novel in vivo protease assay. The site-specific HRV 3C protease (scissors) is expressed in the cytosol and its cleavage site (star) is engineered into the matrix domain of Pex14. When Pex14 is integrated, the cleavage site is sequestered from the protease. When Pex14 is pre-integrated, the cleavage site becomes accessible to cleavage by the protease. (B) Wild-type (WT) cells or the indicated gene deletions expressing Pex14(hrv)-FLAG under the PEX14 promoter from the URA3 locus and cytosolic HRV 3C protease under the GAL1 promoter were grown logarithmically for 15 hours in 2%/2% galactose/raffinose followed by 2 hours in 2% glucose. Extracts were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with an anti-FLAG antibody. Arrow, full length Pex14(hrv)-FLAG; arrowhead, cleaved Pex14(hrv)-FLAG. (C) Representative confocal micrographs (maximum intensity projections) of logarithmically growing cells expressing Pex14-EmCitrine and the peroxisomal membrane and matrix markers Pex11-mCherry and the mTurquoise2-PTS1, respectively. (D) Distributions of Pex14-EmCitrine density at Pex11-mCherry-defined peroxisomes in the images shown representatively in (C). Medians are represented as horizontal lines. Violin plot tails were cut off at 1 arbitrary units (AUs) for clarity of presentation. 2865, 3204, 2703 and 808 peroxisomes were analyzed for WT, pex5Δ, pex4Δ and pex15Δ strains, respectively. (E) Cells expressing FLAG-Pex14-MYC 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.

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: Mutagenesis, In Vivo, Protease Assay, Expressing, SDS Page, Lysis, Centrifugation

Journal: bioRxiv

Article Title: The importomer is a peroxisomal membrane protein translocase

doi: 10.1101/2020.05.01.072660

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

Journal: bioRxiv

Article Title: The importomer is a peroxisomal membrane protein translocase

doi: 10.1101/2020.05.01.072660

Figure Lengend Snippet: Yeast Pex14 has an N-terminal matrix domain and is directly targeted to peroxisomes (A) Strategy for defining membrane protein topology using protease protection. (B) Cells expressing FLAG-Pex14-MYC were grown to post-log phase before being subjected to spheroplasting and gentle lysis. Following centrifugation of lysates, membranes were resuspended in buffer with or without Triton X-100 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 FLAG-Pex14-MYC; arrowhead, protected fragment; asterisk, proteinase K-independent degradation product; double asterisk, proteinase K-dependent partially digested intermediate. (C) Strategy for assessing the direct targeting of Pex14. 1) Post-translationally targeted Pex14 dissociates from its RNC prior to membrane targeting (-Tether), whereas a large C-terminal EmCitrine tag (yellow) tethers the ribosome to the Pex14 nascent chain during membrane targeting. 2) PEX14 mRNAs are visualized by FISH (FISH probes, magenta). (D) Representative confocal micrographs (maximum intensity projections) of fixed, permeabilized spheroplasts derived from logarithmically growing cells with the indicated PEX14 locus genotype were stained with anti- PEX14 mRNA FISH probes. All cells expressed the peroxisomal membrane marker Pex11-mTurquoise2. (E) Distributions of Pex11-mTurquoise2 density at computationally defined PEX14 mRNA puncta in the images shown representatively in (E). Peroxisome-localized PEX14 mRNA puncta were defined as having a Pex11-mTurquoise2 density ≥ 2100 arbitrary units (AUs). 147, 125 and 195 PEX14 mRNA puncta were analyzed for PEX14, pex14Δ ::EmCitrine-Pex14 and pex14Δ ::Pex14-EmCitrine strains, respectively. (F) Bar graph of data from (E) showing the fraction of PEX14 mRNA puncta defined as peroxisomal.

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: Expressing, Lysis, Centrifugation, SDS Page, Fluorescence In Situ Hybridization, Derivative Assay, Staining, Marker

Journal: bioRxiv

Article Title: The importomer is a peroxisomal membrane protein translocase

doi: 10.1101/2020.05.01.072660

Figure Lengend Snippet: Translocation of the Pex14 NTD without unfolding (A) “Piggy-backing” strategy for testing if Pex14 NTD can undergo membrane translocation in a folded state. One half of a coiled-coil (-) is fused to the Pex14 N58 and the other half (+) to GFP. The appearance of GFP in the matrix implies that it underwent peroxisomal targeting and translocation as part of a coiled-coil complex. (B) Representative confocal micrographs (maximum intensity projections) of logarithmically growing cells expressing CC(+)-GFP and CC(-)-FLAG-Pex14N58 as indicated under TDH3 promoters. Cells also expressed the peroxisomal membrane marker Pex11-mCherry. (C) Cells expressing CC(+)-GFP and CC(-)-FLAG-Pex14N58 were grown to post-log phase before being subjected to spheroplasting and gentle lysis. Following centrifugation of lysates, membranes were resuspended in buffer with or without Triton X-100, treated with proteinase K (doses: 0, 125 and 500 ng/μL) for 5 minutes at 37°C. Following proteinase K quenching, samples were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with the indicated antibodies. Arrow, full length proteins; arrowhead, GFP-fragment resistant to proteinase K digestion even in the presence of Triton X-100.

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: Translocation Assay, Expressing, Marker, Lysis, Centrifugation, SDS Page

Journal: bioRxiv

Article Title: The importomer is a peroxisomal membrane protein translocase

doi: 10.1101/2020.05.01.072660

Figure Lengend Snippet: Evidence for conservation of importomer-mediated Pex14 integration in humans (A) Extracts from HEK293T cells of the indicated genotypes expressing FLAG-Pex26-HA (introduced via lentiviral transduction and expressed under control of the UbC promoter) were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with the indicated antibodies. (B) Representative confocal micrographs (single Z plane near the center of the cell) of fixed, permeabilized HEK293T cells of the indicated genotypes expressing FLAG-Pex26-HA (introduced via lentiviral transduction and expressed under control of the UbC promoter) and stained with anti-PMP70, anti-Pex14, anti-FLAG and anti-calatase antibodies and DAPI as indicated. Endogenous Pex14 and exogenous FLAG-Pex26-HA colocalized with the peroxisomal marker PMP70 in Pex5 WT and KO cells, whereas catalase colocalized with PMP70 in Pex5 WT cells but became diffusely cytosolic in PEX5 KO cells. (C) HEK293T of the indicated PEX5 genotype expressing endogenous Pex14 and exogenous FLAG-Pex26-HA (introduced via lentiviral transduction and expressed under control of the UbC promoter) were subjected to gentle lysis. Following centrifugation of lysates, membranes were resuspended in buffer with or without Triton X-100 and treated with proteinase K (doses: 0 and 200 ng/μL) for 40 minutes on ice. Following proteinase K quenching, samples were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with the indicated antibodies. Arrows, full length proteins; arrowheads, protected fragments; asterisk, partial cleavage products.

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: Expressing, Transduction, SDS Page, Staining, Marker, Lysis, Centrifugation