(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).

(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.

(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).

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.

(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.

(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.