<i>Hs</i>Pex11pβ is an integral membrane protein with two transmembrane spans flanking a protease-protected region.

<p>(<b>A</b>) COS-7 cells were transfected with Myc-tagged Pex11pα, Pex11pβ, or Pex11pγ and subjected to carbonate extraction (Carb.) at pH 11.5 or were mock treated (Con). Equal amounts of protein (P, membrane fraction; S, carbonate extract) were separated by SDS-PAGE on 12.5% acrylamide gels and subjected to immunoblotting with anti-Myc antibodies. PMP70 and Pex19p served as controls for integral and peripheral proteins, respectively. (<b>B</b>) Schematic view of potential results of a proteinase K (PK) digest depending on the number and location of putative transmembrane spans within Pex11pβ (see also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053424#pone.0053424.s002" target="_blank">Fig. S2</a></b>). AB, epitope recognized by anti-Pex11pβ. (<b>C</b>) COS-7 cells were transfected with YFP-Pex11pβ or mock transfected (UT). 48 h after transfection, peroxisome-enriched fractions were prepared. Equal amounts of protein were digested with proteinase K in the presence or absence of Triton X-100 (TX-100). Controls were left untreated. Samples were separated by 12.5% SDS-PAGE and immunoblotted using anti-Pex11pβ. As a loading control, the membrane was re-incubated with anti-GFP after membrane stripping. Asterisks indicate the YFP-Pex11pβ band before and after digest. Note that the nonspecific band (approx. 60 kDa) is no longer recognized after repeated use of the Pex11pβ antibody (see <b>D</b>). (<b>D</b>) As an alternative to (<b>C</b>), peroxisome fractions were ruptured by sonication prior to proteinase K digest and immunoblotted as described. As a loading control, the membrane was re-incubated with anti-GFP. Successful membrane rupture was verified by incubation with anti-AOX, a peroxisomal matrix marker.</p

A glycine-rich internal region specific for human Pex11pβ is dispensable for peroxisome elongation and division.

<p>COS-7 cells expressing Myc-Pex11pβ (<b>A, C</b>) and Myc-Pex11pβΔGly (<b>B, D</b>) were processed for immunofluorescence microscopy after 12 and 48 h using anti-Myc (<b>A–D</b>). (<b>E</b>) Quantitative evaluation of peroxisome morphology over time. Data are from 3 independent experiments and are presented as means ± S.D. Bars, 20 µm.</p

Mutations of N-terminal cysteines within Pex11pβ do not affect peroxisome membrane elongation.

<p>COS-7 cells were transfected with Pex11pβ-Myc (<b>A–C</b>), Pex11pβ-Myc^C18S-C25S (<b>D–F</b>) and Pex11pβ-Myc^{C18S-C25S-C85S} (<b>G–I</b>), and were processed for immunofluorescence microscopy 24 h after transfection using anti-Myc (<b>A, D, G</b>) and anti-Pex14p (<b>B, E, H</b>) antibodies. (<b>J</b>) Quantitative evaluation of peroxisome morphology. Data are from 3 independent experiments and are presented as means ± S.D. Bars, 20 µm.</p

ΔN40-Pex11pβ-Myc shows altered membrane distribution within tubular peroxisomal accumulations (TPAs) and fails to induce them when co-expressed with YFP-Pex11pβ.

<p>COS-7 cells were co-transfected with Pex11pβ-YFP/Pex11pβΔN40-Myc (<b>A–C</b>), YFP-Pex11pβ/Pex11pβ-Myc (<b>D–F</b>), or YFP-Pex11pβ/Pex11pβΔN40-Myc (<b>G–I</b>) and processed as described below. Note that Pex11pβ-YFP localizes to tubular membranes (<b>A, C</b>), whereas Pex11pβΔN40-Myc distributes over both tubular and spherical membrane domains (<b>B, C</b>). Note that N-terminally tagged YFP-Pex11pβ only induces TPAs when co-expressed with Pex11pβ-Myc (<b>D–F</b>), but not with Pex11pβΔN40-Myc (<b>G–I</b>). (<b>J</b>) Quantitative evaluation of TPA formation in cells expressing Pex11pβ-YFP (a strong inducer of TPAs), YFP-Pex11pβ or co-expressing YFP-Pex11pβ/Pex11pβ-Myc or YFP-Pex11pβ/Pex11pβΔN40-Myc. Cells were fixed after 24 h, stained for immunofluorescence with anti-Myc antibodies and analyzed. Data are from 3–4 independent experiments and are presented as means ± S.D. (*p<0.01). Bars, 20 µm.</p

Permeabilization of the peroxisome membrane is required for epitope recognition of the Pex11pβ antibody.

<p>COS-7 cells were transfected with Pex11pβ-Myc (<b>A–I</b>) or YFP-Pex11pβ (<b>J–R</b>) and fixed after 24 hours. Cell membranes were permeabilized with 0.2% Triton X-100 (TX-100) (<b>A–C, J–L</b>), 25 µg/ml digitonin (<b>D–F, M–O</b>) or methanol (MetOH) (<b>G–I, P–R</b>) prior to immunostaining with anti-Myc (<b>A, D, G</b>) and anti-Pex11pβ (<b>middle column)</b> antibodies. Note that Pex11pβ-Myc is liberated from peroxisomal membranes after postfixation TX-100 treatment (<b>A–C</b>), while YFP-Pex11pβ is retained (<b>J–L</b>). Bars, 20 µm.</p

An intact Helix 2 within the first 40 N-terminal aa of Pex11pβ influences dimer formation.

<p>COS-7 cells expressing Pex11pβ-Myc (WT) (<b>A, B</b>), Pex11pβΔN40-Myc (<b>B</b>), or Pex11pβ-Myc^A21P (<b>B</b>) were fixed with 4% para-formaldehyde 24 h after transfection and subjected to postfixation Triton X-100 (TX) or digitonin (Dig) extraction. Equal amounts of protein from supernatants (S) (TX-extracts), remaining cell pellets (P) and untreated lysates (L) were separated by 10% SDS-PAGE and immunoblotted using anti-Myc. Note that Pex11pβ-Myc is extracted by postfixation Triton X-100 treatment but not by digitonin (<b>A</b>). (<b>C</b>) <b>Crosslinking of Pex11pβ-Myc with DSP.</b> COS-7 cells expressing Pex11pβ-Myc were cross-linked with DSP and either lysed with 1% Triton X-100 or 1% digitonin. Equal protein amounts of the lysates were separated by reducing and non-reducing (non-red.) SDS-PAGE and immunoblotted using anti-Myc. Arrowheads highlight monomeric and dimeric forms of Pex11pβ-Myc. (<b>D</b>) <b>Migration of Pex11pβ in native sucrose gradients</b>. COS-7 cells expressing Pex11pβ-Myc were either lysed in buffer containing 1% Triton X-100 (after cross-linking with DSP) (TX, CL) or in buffer containing 1% digitonin (without cross-linking) (Dig). Cell lysates were applied on top of each gradient (*), separated by sucrose density gradient ultracentrifugation (10–47%) into 12 fractions and analyzed by immunoblotting using anti-Myc. A gradient with a molecular mass marker was run in parallel for size calibration; correspondent masses are indicated at the bottom. Note the difference in the molecular mass of Pex11pβ complexes indicating different oligomerization states depending on the detergent used.</p

An intact Helix 2 within the first 40 N-terminal aa of Pex11pβ is required to elongate the peroxisomal membrane.

<p>COS-7 cells were transfected with Pex11pβ-Myc (<b>A–C</b>), the N-terminal deletions Pex11pβΔN40-Myc (<b>D–F</b>), Pex11pβΔN60-Myc, Pex11pβΔN70-Myc and the Helix 2-breaking mutant Pex11pβ-Myc^A21P. Cells were processed for immunofluorescence microscopy after 24 h using anti-Myc (<b>A, D</b>) and anti-PMP70 (<b>B, E</b>) antibodies. (<b>G</b>) Quantitative evaluation of peroxisome morphology. Data are from 3–4 independent experiments and are presented as means ± S.D. (*p<0.01). Bars, 20 µm.</p

Phospho-mimicking mutants of Pex11pβ do not influence peroxisomal elongation and division.

<p>COS-7 cells expressing Pex11pβ-Myc, Pex11pβ-Myc^S11A, Pex11pβ-Myc^S11D, Pex11pβ-Myc^S38A and Pex11pβ-Myc^S38D were processed for immunofluorescence using anti-Myc and anti-Pex14p antibodies (<b>Suppl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053424#pone.0053424.s004" target="_blank">Fig. S4</a></b>) and peroxisome morphology was quantified. Data are from 3 independent experiments and are presented as means ± S.D.</p