Peroxisomes function together with mitochondria in a number of essential biochemical pathways, from bile acid synthesis to fatty acid oxidation1. Peroxisomes grow and divide from pre-existing organelles2, but can also emerge de novo in the cell3. The physiological regulation of de novo peroxisome biogenesis remains unclear, and it is thought that peroxisomes emerge from the endoplasmic reticulum in both mammalian and yeast cells4. However, in contrast to the yeast system5,6,7,8, a number of integral peroxisomal membrane proteins are imported into mitochondria in mammalian cells in the absence of peroxisomes, including Pex3, Pex12, Pex13, Pex14, Pex26, PMP34 and ALDP9,10,11,12,13,14,15. Overall, the mitochondrial localization of peroxisomal membrane proteins in mammalian cells has largely been considered a mis-targeting artefact in which de novo biogenesis occurs exclusively from endoplasmic reticulum-targeted peroxins16. Here, in following the generation of new peroxisomes within human patient fibroblasts lacking peroxisomes, we show that the essential import receptors Pex3 and Pex14 target mitochondria, where they are selectively released into vesicular pre-peroxisomal structures. Maturation of pre-peroxisomes containing Pex3 and Pex14 requires fusion with endoplasmic reticulum-derived vesicles carrying Pex16, thereby providing full import competence. These findings demonstrate the hybrid nature of newly born peroxisomes, expanding their functional links to mitochondria.
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This work was supported by CIHR grant MOP#133549 and a Canada Research Chair to H.M.M. A.S. was supported by a JSPS Postdoctoral Fellowship for Research Abroad, S.M. through a Quebec FRQS Studentship, and J.P. by a CIHR Postdoctoral Fellowship (MFE-140925). The authors thank P. Kim for critical reagents and advice, and M. Nguyen for help with import assays.
The authors declare no competing financial interests.
Extended data figures and tables
a, Levels of PEX19, PEX3 and PEX16 mRNA in each cell line were quantified by qRT–PCR. Data show mean ± s.e.m. of each mRNA normalized to control cells from three biological independent experiments. b, Levels of PMP70 and Pex14 protein were determined by immunoblotting analysis using the indicated antibodies. c, Control, Pex3mut and Pex16mut cells were subjected to immunfluorescence using the indicated antibodies. Representatives of captured images: Ctrl (16), Pex3mut (51), Pex16mut (98) shown. Scale bars, 10 μm. d, Subcellular fractionation of control and mutant cells. Isolation of whole-cell extracts (WCE), a 2.3K pellet enriched for mitochondria, a 23K pellet containing ER and peroxisomes, and a 100K pellet as a light membrane fraction showed clear separation of resident markers of these organelles. We analysed 7.5 μg of each fraction isolated from cells by immunoblotting using the indicated antibodies as organelle markers. 2.3K, 23K and 100K: precipitates after centrifugation at indicated speeds. Cyt: supernatants after centrifugation at 100,000g. e, f, The 23K fraction isolated from control cells or 2.3K fraction isolated from mutant cells were further separated into soluble (S) and membrane (P) fractions by alkaline carbonate extraction (e) or digested with trypsin at indicated concentrations (f). After the treatments, samples were analysed by immunoblotting using the indicated antibodies.
Extended Data Figure 2 Pex3–YFP is inserted into mitochondrial outer membrane and assembles into mitochondria-derived pre-peroxisomal structures.
a, Left, 23K fraction enriched for peroxisomes, isolated from control cells infected with Ad-Pex3–YFP for 1 day, was separated into soluble and membrane fractions by alkaline carbonate extraction. Internal controls for the efficiency of extraction are shown using antibodies detecting endogenous mitochondrial proteins. Right, protease sensitivity of each protein. Isolated peroxisomes were digested by trypsin at indicated concentrations in the absence or presence of 1% tritonX-100 to ensure full protease accessibility as control (right). After the treatments, samples were analysed by immunoblotting using the indicated antibodies. b, Pex3, but not Pex16, was imported efficiently into the mitochondrial outer membrane in vitro. In vitro translated Pex3 and Pex16 were incubated with mitochondria isolated from mouse heart34,37. Following the reaction, mitochondria were isolated by centrifugation (P), and the supernatants collected (S). The initial total reaction (T) was also loaded for comparison. Fractions were analysed by immunoblotting using the indicated antibodies. Arrowheads indicate Pex-myc-His. Single and double asterisks indicate nonspecific signals from mitochondria and rabbit reticulocyte lysate, respectively. c, Post-import fractionation of Pex3–YFP. Left, following the import reaction, membrane proteins were extracted using sodium carbonate and pelleted, with soluble proteins remaining in the supernatant. Right, protease protection experiment with increasing concentrations of trypsin and samples analysed by immunblotting, along with the indicated internal controls. Pex3–YFP is shown to be trypsin sensitive and alkali resistant, confirming its carbonate-resistant import into the mitochondrial outer membrane in vitro. d, Co-translational import of Pex3 into microsomes in vitro. Synthesized RNA was incubated with rabbit reticulocyte lysate and canine pancreas microsomes (cpm)35. Supernatants and pellets after centrifugation at 100,000g were analysed by immunoblotting using the indicated antibodies. In contrast to a previous study18, but consistent with the primary mitochondrial localization of Pex3–YFP in Pex3mut fibroblasts, Pex3 was very weakly detected in the microsome fraction following the import reaction. Calnexin (CNX) reveals the efficient isolation of microsomal membranes in the pellet fraction. e, Captured images from live cell imaging of Pex3mut cells infected with Ad-Pex3–YFP for 1 day and stained with MitoTracker deep red (sVideo4). Arrowheads indicate Pex3–YFP-enriched structure budding from mitochondrion and lacking MitoTracker, distinguishing the release of Pex3–YFP-positive structure from normal mitochondrial fission. Representative of 47 movies is shown. Scale bar: 2 μm. f, Z series images of Pex3mut cells infected with Ad-Pex3–YFP were acquired (21 sections, 0.1 μm per section). Left, stacked image. Numbers in right panels indicate Z sections of inset in left (all sections shown in Supplementary Video 6). Representative of 60 captured cells is presented. Scale bar: left, 10 μm; right, 2 μm.
a, PMP70 is rapidly incorporated into pre-peroxisomes. Pex3mut cells infected with Ad-Pex3–YFP for 1 day were subjected to immunofluorescence using the indicated antibodies. Representatives of 50 captured cells are presented. Arrowheads, Pex3–YFP- and Pex14-positive but PMP70-negative pre-peroxisomes, defined as stage I. Circles, all positive structures. Scale bars: 2 μm. b, Pex3mut cells infected with Ad-GFP or Ad-Pex3–YFP for 1 or 2 days were fractionated. Each fraction was loaded with 7.5 μg protein, and cellular levels of Pex3–YFP and PMP70 increased throughout the rescue. PMP70 is known to be degraded when peroxisomes are absent, and stabilized upon the generation of import-competent organelles. Quantification of Pex3–YFP, Pex14 and PMP70 in each fraction at each time point was normalized to the total expression in whole cell extracts (WCE). The graphs represent means from three independent experiments. Two days after infection, Pex3–YFP was enriched in the 23K peroxisomal fraction by more than twofold, and PMP70 levels were stabilized in the newly generated peroxisomes. Endogenous Pex14 was found in the mitochondrial fraction (2.3K) of both GFP and Pex3–YFP infected cells, and shifted into the peroxisomal fraction after 2 days of Pex3–YFP expression.
Extended Data Figure 4 N-terminal YFP-tagged Pex3 fails to import into mitochondria and cannot rescue peroxisomal biogenesis in Pex3mut cells.
a, b, Control (a) or Pex3mut cells (b) transfected with Pex3–YFP or YFP–Pex3 were subjected to immunofluorescence using the indicated antibodies. Representatives of captured cells: Ctrl + Pex3–YFP (12), Ctrl + YFP–Pex3 (27), Pex3mut + Pex3–YFP (27), Pex3mut + YFP–Pex3 (62) are presented. YFP–Pex3 inefficiently targets peroxisomes in control cells and mitochondria in Pex3mut cells. Scale bars: low magnification, 10 μm; high magnification, 5 μm. c, Representatives of counted cells from 1 day after transfection are shown. The graph represents percentage of cells at each stage of de novo synthesis. Error bars show means from three biological replicates. Pex3–YFP: day 1 (52, 51, 51), day 2 (51, 53, 51); YFP–Pex3: day 1 (50, 50, 52), day 2 (50, 50, 51) cells were counted in each experiment. Scale bars: 20 μm
a, Control, Pex3mut, and Pex16mut cells infected with Ad-Pex3–YFP for 1 day were subjected to immunofluorescence using the indicated antibodies. Representative of counted cells are presented. Quantification of the Pex3–YFP localization was performed from fixed confocal images. Error bars represent the mean from three biological independent experiments. Cells counted in each experiment: control (59, 81, 79), Pex3mut (69, 62, 73), Pex16mut (40, 45, 51). Scale bars: 10 μm. b, Control cells infected with varying amounts of Ad-GFP or Pex3–YFP were analysed by immunoblotting using indicated antibodies. c, Captured images from live cell imaging (Supplementary Video 7). Control cells were monitored for 2 days after infection with Ad-Pex3–YFP. Times indicate hours after infection. At 20 h, Pex3–YFP is exclusively peroxisomal within the cell (asterisk). The number of peroxisomes then steady declines between 20 and 32 h. Pex3–YFP expression is very low between 32 and 35 h, and slowly rises as it is imported into mitochondria, evident by 38–41 h. Representative of 30 movies is shown. Scale bar: 20 μm.
Extended Data Figure 6 Pex3-mediated peroxisomal biogenesis is blocked upon addition of MG132 or nocodazole.
a, Ad-Pex3–YFP did not colocalize with lysosomal markers within Pex3mut cells. Representative of 171 captured cells shown. Scale bars: low magnification, 10 μm; high magnification, 5 μm. b, Representative images of PMP70 and catalase staining shown in Fig. 3c. Scale bars: low magnification, 10 μm; high magnification, 5 μm. c, Ten hours after infection with Ad-Pex3–YFP, Pex3mut cells were incubated with 0.02% DMSO, 1.5 μM nocodazole or 5 μM Y-27632 for 14 h. Stages of de novo synthesis were classified and quantified using confocal imaging. The graphs represent mean from three biological replicates. Cells counted in each experiment: DMSO (50, 56, 51), Noc (50, 54, 51), Y-27632 (53, 51, 53), Noc + Y-27632 (51, 54, 51).
Extended Data Figure 7 Pex3 is rapidly turned over from both mitochondrial and peroxisomal locations.
a, b, Control and Pex3mut cells infected with Ad-Pex3–YFP or not infected for 1 day were chased with 20 μg ml−1 cycloheximide (CHX) for indicated times. Protein levels were analysed by immunoblotting using the indicated antibodies and quantified by densitometric analysis. Line graphs and plots of different shapes represent mean and each value from three independent experiments. Pex14 levels were very stable over the 5-h period, but Pex3–YFP was rapidly depleted between 1 and 5 h. c, One day after infection, cells were incubated with 20 μg ml−1 CHX and 2 μM MG132 for 5 h. Protein levels were analysed by immunoblotting using the indicated antibodies. Asterisks, nonspecific bands. The data show that loss of Pex3–YFP upon cyclohexamide treatment is through proteasome-dependent degradation from both the peroxisomal location (in control human fibroblasts) and from mitochondria (in Pex3mut cells). Note that total expression of Pex3–YFP in Pex3mut cells is significantly lower than in control fibroblasts. d, Pex3mut cells infected with Ad-Pex3–YFP for 1 day were treated under the indicated conditions and subjected to immunoprecipitation using anti-FP antibody. Input and immunoprecipitates were analysed by immunoblotting using the indicated antibodies. GFP, Ad-GFP; Pex3–YFP, Ad-Pex3–YFP; MG132, 500 nM for 14 h. The immunoprecipitation shown in the last lane reveals the accumulation of ubiquitin-conjugated forms of Pex3–YFP in the presence of MG132 (Pex3–YFPUb), indicating that a ubiquitin E3 ligase is required for the steady-state turnover of Pex3.
Extended Data Figure 8 Pex3–YFP pre-peroxisomes are released from mitochondria by distinct machinery from mitochondrial fission and mitochondrial vesicular transport to peroxisomes.
a, b, Pex3mut cells were silenced with siRNAs targeting Drp1, Vps35 or Pex19 for 3 days. Drp1 is a fission GTPase that is essential for mitochondrial and peroxisomal membranes22. Mitochondrial vesicle formation occurs in a manner independent of Drp1 (ref. 38), and this experiment was to test whether exit of Pex3–YFP from mitochondria in vesicular profiles requires this GTPase. Vps35 is a subunit of the retromer complex, which plays a critical role in the steady-state transport of mitochondrial vesicles to peroxisomes23. Pex19 is the cytosolic chaperone that binds and delivers peroxisomal membrane proteins to Pex3–Pex16 for insertion9,17. This experiment was to test whether Pex19 was required for Pex3 import into mitochondria. Knockdown efficiency was determined by immunoblotting using the indicated antibodies (a) or qRT–PCR (b). c, Pex3mut cells silenced (as indicated) for 3 days were infected with Ad-Pex3–YFP. One or two days after infection, cells were subjected to immunofluorescence using the indicated antibodies. Left panels, representatives of counted cells at day 2. The graph represents the percentage of cells at each stage. Error bars show mean from three biologically independent experiments. Cells counted in each experiment: control (N): day 1 (55, 86, 55), day 2 (90, 90, 70); Drp1: day 1 (56, 63, 65), day 2 (103, 103, 87); Vps35: day 1 (59, 81, 77), day 2 (83, 83, 88); Pex19: day 1 (39, 66, 68), day 2 (61, 61, 59). Although Drp1 was essential for peroxisomal division, its loss did not alter peroxisome biogenesis or import competence. Loss of Vps35 appeared to enhance the rate of peroxisomal biogenesis, but Vps35 was clearly not required for Pex3–YFP-mediated rescue. Loss of Pex19 did not alter Pex3–YFP insertion into mitochondria or the generation of stage I pre-peroxisomes. These peroxisomes did not mature, suggesting that the roughly 60% loss of Pex19 was functionally sufficient to completely block peroxisomal protein import. Scale bars: 20 μm.
Extended Data Figure 9 Ad-Pex16–YFP restores peroxisomal biogenesis in human fibroblasts lacking Pex16 from a patient with Zellweger.
a, Pex16mut cells infected with Ad-Pex16–YFP for 1 day were subjected to immunofluorescence using anti-Tom20 (mitochondria) and anti-KDEL (ER) antibodies. A representative of 34 captured cells is presented. Scale bar: 10 μm. The ER is primarily sheet-like rather than reticular within Pex16mut cells, and Pex16–YFP primarily follows that pattern, which is completely distinct from the mitochondrial staining. b, Each fraction isolated from Pex16mut cells infected with Ad-GFP or Ad-Pex16–YFP for 1 or 4 days was analysed by immunoblotting. c, Pex16-positive vesicular profiles localize to mitochondrial tubules where they acquire endogenous Pex14. Z series images of Pex16mut cells infected with Ad-Pex16–YFP and immunostained with anti-Pex14 and anti-Tom20 (32 sections, 0.1 μm per section). The numbers represent images at indicated Z (all sections shown in Supplementary Video 8). Representatives of 31 captured cells are presented. Scale bar: 2 μm. d, Pex16–YFP transition to stage I is unaffected by nocodazole. Ten hours after infection with Ad-Pex16–YFP, Pex16mut cells were incubated with 0.02% DMSO or 1.5 μM nocodazole for 14 h. Representatives of counted cells are presented. Stages of de novo synthesis (left) or localization of Pex16–YFP (right) were determined under the confocal microscope. Error bars show mean from three biologically independent experiments. Cells counted in each experiment: DMSO (56, 51, 52), Noc (54, 53, 51). Representative images of Pex16–YFP are shown. Scale bars: 20 μm.
Extended Data Figure 10 Peroxisome assembly requires both mitochondrial and ER-derived pre-peroxisomes.
a, One day after transfection with Pex16–mRFP, Pex3mut cells were infected with Ad-Pex3–YFP for further 1 day. Cells were subjected to immunofluorescence using anti-Pex14 and anti-PMP70 antibodies. Insets in left images are shown in right panels. Representatives of 32 captured cells are presented. Scale bars: low magnification, 20 μm; high magnification, 5 μm. b. Pex3mut cells were transfected with mRFP or Pex16–mRFP for 24 h before infection with Ad-Pex3–YFP. Cells were subjected to immunofluorescence 1 or 2 days after Pex3–YFP infection. The graph represents the percentage of cells at each stage of de novo synthesis as defined in Fig. 1a. The graph represents mean from three biological replicates. Cells counted in each experiment: mRFP: day 1 (58, 49, 68), day 2 (47, 73, 61); Pex16–mRFP: day 1 (37, 52, 55), day 2 (54, 50, 50). c, Pex16mut cells expressing Pex3–YFP and Pex3mut cells expressing Pex16–mRFP were co-plated in a glass-bottom cell culture dish. A representative of 17 images captured before addition of PEG is presented. Scale bar: 20 μm. d, Pex16mut cells expressing Pex3–YFP and Pex3mut cells expressing Pex16–mRFP were fused by PEG. Captured images are from live-cell imaging 3 h after PEG-mediated whole-cell fusion (Supplementary Video 10). Circles highlight Pex16 vesicle structures that fuse with Pex3 vesicle structures. Scale bar: 4 μm. e, Model of peroxisomal biogenesis in mammalian system. Pex16-containing pre-peroxisomes emerge from the ER. They then fuse with Pex3–Pex14 pre-peroxisomes at the mitochondrial surface, thereby generating an import-competent, functional peroxisome that proliferates through growth and division.
This file contains the uncropped blots. (PDF 4310 kb)
Pex3mut cells infected with Ad-Pex3-YFP were subjected to long-term live cell imaging with Viva View microscope. Cells were chased 25 h to 45 h after the infection every 1 h. Following a cell division event Pex3-YFP exits mitochondria in only one of the two daughter cells, illustrating the stochastic nature of peroxisomal biogenesis. Captured images are shown in Fig. 2c. (AVI 1393 kb)
Pex3mut cells infected with Ad-Pex3-YFP for 24 h were observed with spinning disk confocal microscope. Images were acquired every second. Captured images are shown in Fig. 2d (AVI 197 kb)
Pex3mut cells infected with Ad-Pex3-YFP (yellow) for 24 h were stained with 100 nM MitoTracker deep red (magenta). Images were acquired every second with spinning disk confocal microscope. Captured images of sVideo4 are shown in extended Fig. 2f. (AVI 542 kb)
Pex3mut cells infected with Ad-Pex3-YFP (yellow) for 24 h were stained with 100 nM MitoTracker deep red (magenta). Images were acquired every second with spinning disk confocal microscope. Captured images of sVideo4 are shown in extended Fig. 2f. (AVI 360 kb)
Bottom to top z series images of Pex3mut cells infected with Ad-Pex3-YFP (yellow) for 1 day and immunostained with anti-Tom20 (magenta) antibody (21 sections, 0.1 μm / section). Section 10, 14, 18, 22 and 26 are shown in extended Fig. 2g (AVI 214 kb)
Serial section revealing the incorporation of endogenous Pex14 within Pex3-YFP containing vesicular profiles emerging from mitochondria
Bottom to top z series images of Pex3mut cells infected with Ad-Pex3-YFP (yellow) for 1 day and immunostained with anti-Pex14 (magenta) and -Tom20 (cyan) antibodies (36 sections, 0.1 μm / section). Section 10, 14, 18 22 and 26 are shown in Fig. 2e. (AVI 679 kb)
Peroxisomal Pex3-YFP is imported into mitochondria following pexophagy in wild type human fibroblast cells
Control cells infected with Ad-Pex3-YFP were subjected to long-term live cell imaging with Viva View microscope. Cells were chased 24 h to 56 h after the infection every 1 h. Captured images are shown in extended Fig. 5c. (AVI 441 kb)
Serial sections of ER derived Pex16-YFP that colocalize with mitochondrial Pex14 in vesicular profiles that segregate from Tom20
Bottom to top z series images of Pex16mut cells infected with Ad-Pex16-YFP (yellow) for 1 day and immunostained with anti-Pex14 (magenta) and -Tom20 (cyan) antibodies (32 sections, 0.1 μm / section). Section 10, 14, 18 22 and 26 are shown in extended Fig. 9c (AVI 546 kb)
ER derived Pex16-mRFP fused with mitochondrial Pex3-YFP enriched domains are released together from the mitochondria
Pex3mut cells expressing Pex16-mRFP and Pex16mut cells expressing Pex3-YFP for 1 day were fused by PEG. 3 h after, cells were observed with spinning disk confocal microscope. Captured images are shown in Fig. 4d (AVI 9 kb)
Another example of PEG assay. Captured images are shown in extended Fig. 10d. (AVI 18 kb)
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Sugiura, A., Mattie, S., Prudent, J. et al. Newly born peroxisomes are a hybrid of mitochondrial and ER-derived pre-peroxisomes. Nature 542, 251–254 (2017). https://doi.org/10.1038/nature21375
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