PEX11 promotes peroxisome division independently of peroxisome metabolism

Abstract

The PEX11 peroxisomal membrane proteins are the only factors known to promote peroxisome division in multiple species. It has been proposed that PEX11 proteins have a direct role in peroxisomal fatty acid oxidation, and that they only affect peroxisome abundance indirectly. Here we show that PEX11 proteins are unique in their ability to promote peroxisome division, and that PEX11 overexpression promotes peroxisome division in the absence of peroxisomal metabolic activity. We also observed that mouse cells lacking PEX11beta display reduced peroxisome abundance, even in the absence of peroxisomal metabolic substrates, and that PEX11beta(-/-) mice are partially deficient in two distinct peroxisomal metabolic pathways, ether lipid synthesis and very long chain fatty acid oxidation. Based on these and other observations, we propose that PEX11 proteins act directly in peroxisome division, and that their loss has indirect effects on peroxisome metabolism.

Figure 1.

PEX11β-mediated peroxisomal proliferation is a multistep process. Wild-type human skin fibroblasts GM5756 were microinjected with pcDNA3-PEX11βmyc and processed at 1.5 h (A and B), 4.5 h (C and D) and 48 h (E and F) after injection for double indirect immunofluorescence with antibodies to the myc epitope (A, C, and E) and PEX14 (B, D, and F). Note that elongated peroxisomes appear 4.5 h after microinjection. Bar, 20 μm.

Figure 2.

Overexpression of PEX11β increases peroxisome abundance in wild-type human skin fibroblasts. (A) Peroxisome abundance in GM5756 cells, GM5756 cells overexpressing PMP34myc, and GM5756 cells overexpressing PEX11βmyc. (B–E) Representative cells transfected with pcDNA3-PMP34myc (B and C) and pcDNA3-PEX11βmyc (D and E). GM5756 cells were transfected with pcDNA3-PEX11βmyc or pcDNA3-PMP34myc. 2 d after transfection, cells were processed for indirect immunofluorescence with antibodies to the myc epitope (B and D) and PEX14 (C and E). Samples were examined under a confocal fluorescence microscope and the number of peroxisomes present in at least 30 randomly selected cells was counted. Results in A are presented as the average peroxisome abundance ± one standard deviation. Bar, 20 μm.

Figure 3.

Overexpression of PEX11β increases peroxisome abundance in PEX5-deficient cell line PBD005. (A) Peroxisome abundance in PBD005 cells, PBD005 cells overexpressing PMP34myc, and PBD005 cells overexpressing PEX11βmyc. (B–E) Representative cells transfected with pcDNA3-PMP34myc (B and C) and pcDNA3-PEX11βmyc (D and E). PBD005 cells were transfected with pcDNA3-PEX11βmyc or pcDNA3-PMP34myc. 2 d after transfection, the cells were processed for indirect immunofluorescence with antibodies to the myc epitope (B and D) and PEX14 (C and E). Samples were examined under a confocal fluorescence microscope and the number of peroxisomes present in at least 30 randomly selected cells was counted. Results in A are presented as the average peroxisome abundance ± one standard deviation. Bar, 20 μm.

Figure 4.

Overexpression of PEX11 in S. cerevisiae increases peroxisome abundance in lipid-free medium. Various S. cerevisiae strains were cultured by different conditions and the peroxisome number was counted under fluorescence microscopy in 120 cells for each sample. Figures shown here are the frequency distribution of cells with different peroxisome number. (A and B) BY4733 cells transformed with pPGK1-GFP/PTS1 and pRS425/GAL1 were precultured in S minimal medium with 2% glucose to midlog phase (A), shifted to minimal medium containing 0.2% oleic acid and 0.02% Tween 40, cultured for 17 h (B). (C and D) XLY1 cells carrying pRS425/GAL1 were precultured in minimal S medium with 2% glucose to midlog phase (C), shifted to minimal medium containing 1% galactose and cultured for 17 h (B). (E–G) Galactose induced XLY1 cells transformed with pRS425/GAL1-PEX13 (E), pRS425/GAL1-YPR128C (F), and pRS425/GAL1-PEX11 (G). (H and I) Galactose induced XYL2 cells transformed with pRS425/GAL1-PEX13 (H) and pRS425/GAL1-PEX11 (I). Note that profile shifts in XYL1 and XYL2 cells overexpressing PEX11 were comparable to that of oleic acid induced wild-type cells.

Figure 5.

Loss of mouse PEX11β reduces peroxisome abundance in serum-free medium. (A) Peroxisome abundance in wild-type and PEX11β^{− / −} mouse fibroblasts. Solid bars represent peroxisome abundance in fibroblasts cultured in normal conditions, and open bars represent peroxisome abundance in fibroblasts cultured in serum-free medium for 24 h. (B–E) Representative wild-type (B and C) and PEX11β^{− / −} mouse fibroblasts (D and E) cultured in serum-free medium. Wild-type and PEX11β^{− / −} mouse were cultured 24 h in serum-free medium and then processed for indirect immunofluorescence with antibodies to the peroxisomal membrane protein PEX14 (B and D) and matrix marker enzyme catalase (C and E). Samples were examined under a confocal fluorescence microscope and the number of peroxisomes present in 60 randomly selected cells was counted. Results in A are presented as the average peroxisome abundance ± one standard deviation. Bar, 10 μm.