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Research Article
Pex11mediates peroxisomal proliferation by promoting deformation of the lipid membrane
Yumi Yoshida, Hajime Niwa, Masanori Honsho, Akinori Itoyama, Yukio Fujiki
Biology Open 2015 4: 710-721; doi: 10.1242/bio.201410801
Yumi Yoshida
1Department of Biology, Faculty of Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
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Hajime Niwa
1Department of Biology, Faculty of Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
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Masanori Honsho
1Department of Biology, Faculty of Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
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Akinori Itoyama
2Graduate School of Systems Life Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
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Yukio Fujiki
1Department of Biology, Faculty of Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
2Graduate School of Systems Life Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
32, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
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  • For correspondence: yfujiki@kyudai.jp
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  • Fig. 1.
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    Fig. 1. Pex11pβ forms Pex11pβ-enriched membrane subregions on the peroxisomal membrane.

    (A) HeLa cells were transfected with a plasmid encoding human Pex11pβ for 6–24 h and immunostained with antibodies against Pex11pβ (green) and Pex14p (red). Panels are arranged according to the stage of peroxisomal proliferation. Arrowheads indicate regions enriched in Pex11pβ compared with Pex14p. Scale bars: 10 µm and 5 µm (enlarged). (B) HeLa cells stably expressing EGFP-SKL (a–e) and control HeLa cells (g–k) were treated with a control siRNA (a,g) or DLP1 siRNA (b–e,h–k) for 72 h. Cells were subjected to immunostaining using the anti-Pex11pβ antibody (a–e) or double-stained with the anti-Pex11pβ antibody and an anti-Pex14p antibody (g–k). Magnified view of DLP1 siRNA-treated cells (c–e,i–k), and pixel intensity by line scanning along the elongated peroxisome (arrow) were plotted (f,l). Clear peaks of Pex11pβ were highlighted by arrowheads. Scale bars: 10 µm and 5 µm (enlarged). (C) HeLa cells were treated with a control siRNA (a) or DLP1 siRNA (b) for 72 h, and stained the DLP1 siRNA-treated cells with antibodies to Pex14p and PMP70 in magnified views (c–e). Line scanning of elongated peroxisomes was shown in (f). Scale bars: 10 µm and 5 µm (magnified view).

  • Fig. 2.
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    Fig. 2. Reconstitution of recombinant Pex11pβ into proteo-liposomes.

    (A) Coomassie Brilliant Blue-stained SDS-PAGE gel of recombinant His-Pex11pβ protein. (B) Schematic model of incorporation into proteo-liposomes. Purified Pex11pβ was snap-frozen in the presence or absence of liposomes and the mixture was then thawed and diluted in Hepes-KOH buffer. Proteo-liposomes were collected by centrifugation, and incorporation of Pex11pβ was evaluated by western blotting using the anti-Pex11pβ antibody. (C) Pex11pβ was reconstituted into rhodamine-labeled proteo-liposomes, which were observed under confocal microscopy (b,c). Liposomes were also snap-frozen with the buffer used in the purification of Pex11pβ, as a control (a). Confocal micrographs were also taken under short-time exposure and enlarged (inset). Scale bars: 10 µm and 5 µm (inset).

  • Fig. 3.
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    Fig. 3. The effect of the N-terminal amphipathic helix of Pex11pβ on the morphology of reconstituted liposomes.

    (A) Molecular structure and helical wheel representation of the region comprising the amphipathic helix encompassing the amino acid residues at positions 53–70 of Hs-Pex11pβ. The helical wheels are presented by looking down into the helical axis and taking Ser55 as the first amino acid residue with the largest circle, the second residue with the second “largest”, followed by those with smaller and smaller sizes. Amino acids are colored according to the physicochemical properties of the side chains (hydrophobic – yellow; polar, positively charged – blue; polar, negatively charged – pink; polar, uncharged – green). The hydrophobic surface was mutated to glutamate (indicated in blue) and proline (indicated in red). (B) Myc-tagged Pex11p proteins were overexpressed in HeLa cells and immunostained with antibodies to Myc (green) and Pex14p (red), respectively (a–f). Peroxisomal fission was observed under confocal microscopy. Scale bars: 10 µm and 5 µm (inset). (C) Confocal micrographs of control liposomes (a) and proteo-liposomes which were reconstituted with Pex11pβ-P (b), Pex11pb-φ (c), Pex11pα (d), Pex11pγ (e), or Pex14p (f). Scale bar: 10 µm. Incorporation of recombinant protein into liposomes was evaluated by western blotting (g). (D) Sizes of liposomes were quantitated, and the size distribution is represented as histograms. The rightmost bar (*) indicates total percentage of over 0.3 µm2. At least 1000 particles from randomly selected fields were counted.

  • Fig. 4.
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    Fig. 4. Localization of Pex11p protein in reconstituted proteo-liposomes.

    (A) EGFP-tagged Pex11pβ protein was introduced into rhodamine-labeled liposomes, which were observed under confocal microscopy (a–f). Photos were enlarged in panels d–f. Arrowheads indicate clusters of Pex11pβ on the proteo-liposomes. Image stacks were collected along the z-axis and rendered as maximum intensity projections using Zen 2011 software (Carlzeiss) (g–i). Scale bars: 5 µm and 2 µm (enlarged). (B) Rhodamine-labeled liposomes were reconstituted with EGFP-fused mutants, Pex11pβ-P (a–e) and Pex11pβ-φ (f–j). Close-up views of the proteo-liposomes for Pex11pβ-P (c–e) and Pex11pβ-φ (h–j). Scale bars: 10 µm and 1 µm (close-up view).

  • Fig. 5.
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    Fig. 5. Membrane continuity of Pex11pβ-containing proteo-liposomes.

    A small region (white circle) of Pex11pβ-reconstituted proteo-liposomes was repeatedly photobleached. Images were taken before and after 30 rounds of photobleaching. In the control experiment, an adjacent region of proteo-liposomes was photobleached. The fluorescence intensity of the liposomes was measured and normalized to the intensity prior to photobleaching. Data represent the means±SD. Scale bar: 2 µm.

  • Fig. 6.
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    Fig. 6. Morphology of Pex11pβ-reconstituted proteo-liposomes.

    (A–C) Negative staining electron micrographs of Pex11pβ-reconstituted proteo-liposomes. (A) Control liposomes (a) and Pex11pβ-reconstituted proteo-liposomes (b–d) were negatively stained with 2% uranyl acetate. Pex11pβ-reconstituted liposomes are magnified in panels c and d. Scale bars: 1 µm (a,b) and 0.5 µm (c,d). (B) Negative staining of proteo-liposomes reconstituted with Pex11pβ-P (a), Pex11pβ-Φ (b), Pex11pα (c), or Pex11pγ (d). Scale bar: 1 µm. (C) Reconstituted proteo-liposomes were subjected to immune labeling, and stained with uranyl acetate. Pex11pβ- (a) or Pex11pβ-P- (b) reconstituted proteo-liposomes were sequentially incubated with the anti-Pex11pβ antibody and an anti-rabbit IgG conjugated to 10 nm gold particles. The anti-Pex11pβ antibody and gold-labeled anti-rabbit IgG did not react with the control liposomes (c). Scale bar: 0.5 µm. Arrowheads indicate the gold particles (see text). (D) The reconstituted proteo-liposomes were subjected to electron microscopy. Control-treated liposomes (left) and Pex11pβ-reconstituted liposomes (right) were pelleted by ultracentrifugation and prepared for embedding and ultra-thin sectioning as described in the Materials and Methods. Note that the constriction of the liposomal membrane was observed in Pex11pβ-reconstituted liposomes (arrowheads). Scale bar: 0.2 µm.

  • Fig. 7.
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    Fig. 7. Knockdown of PEX11 in HeLa cells.

    (A) HeLa cells were treated for 96 h with a control siRNA (a) or siRNA for PEX11α (b), PEX11β (c), or PEX11γ (d). Cells were then immunostained with an anti-Pex14p antibody. Scale bars: 10 µm and 5 µm (inset). The efficiency of knockdown was evaluated by RT-PCR (e, upper panel) and western blotting (e, lower panel). PEX11α and PEX11γ mRNA levels were assessed by RT-PCR using total RNA. Actin was used as a loading control. Pex11pβ protein levels were verified by immunoblot analysis using the antibody to Pex11pβ. Peroxisomal marker proteins, Pex3p and Pex14p, were also assessed. Tubulin was used as a loading control. Peroxisomal abundance per cell (f), average peroxisomal size (g), and peroxisomal area per cell (h) in each siRNA-treated cell type were determined as described in the Materials and Methods. At least 20 cells were randomly selected and analyzed at each time point. Data represent the means±SD. *p<0.05. (B) HeLa cells were treated with a control siRNA or siRNA for PEX5 for 48 h and immunostained with antibodies to Pex14p (a,c) and catalase (b,d). Scale bar: 10 µm. Cells were lysed and analyzed by SDS-PAGE and immunoblotting using the antibodies indicated on the right in the panel (e). ‘p’ and ‘m’ in the panel of ADAPS indicates the larger ADAPS precursor harboring PTS2 and its mature form, respectively. (C) HeLa cells were transfected for 96 h with siRNAs each for DLP1 (a) in combination with the indicated genes (b–e). Cells were then subjected to immunostaining with an anti-Pex14p antibody. Scale bars: 10 µm and 5 µm (inset). The average lengths of peroxisomes of the cells were measured and plotted in (f). Cells were lysed and analyzed by SDS-PAGE and immunoblotting using the antibodies indicated on the left in the panel (g). Data represent the means±SD. *p<0.05.

  • Fig. 8.
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    Fig. 8. A schematic model for the Pex11pβ function in peroxisomal fission.

    Schematic drawing of peroxisomes during fission process as maturation was represented in red. The whole process consists of three steps, maturation, constriction/elongation, and fission. Pex11pβ is highlighted in green only in the constriction/elongation step, where it locates at the constriction sites. Note that the Pex11pβ forms the active oligomer at the stage to deform peroxisomal membrane.

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Keywords

  • Peroxisomes
  • Pex11p
  • Liposomes
  • Membrane deformation
  • Amphiphathic helix

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Research Article
Pex11mediates peroxisomal proliferation by promoting deformation of the lipid membrane
Yumi Yoshida, Hajime Niwa, Masanori Honsho, Akinori Itoyama, Yukio Fujiki
Biology Open 2015 4: 710-721; doi: 10.1242/bio.201410801
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Research Article
Pex11mediates peroxisomal proliferation by promoting deformation of the lipid membrane
Yumi Yoshida, Hajime Niwa, Masanori Honsho, Akinori Itoyama, Yukio Fujiki
Biology Open 2015 4: 710-721; doi: 10.1242/bio.201410801

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