Limits...
Peroxisome synthesis in the absence of preexisting peroxisomes.

South ST, Gould SJ - J. Cell Biol. (1999)

Bottom Line: We also identified human PEX16, a novel integral peroxisomal membrane protein, and found that PBD061 had inactivating mutations in the PEX16 gene.These results demonstrate that peroxisomes do not necessarily arise from division of preexisting peroxisomes.We propose that peroxisomes may form by either of two pathways: one that involves PEX11-mediated division of preexisting peroxisomes, and another that involves PEX16-mediated formation of peroxisomes in the absence of preexisting peroxisomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
Zellweger syndrome and related diseases are caused by defective import of peroxisomal matrix proteins. In all previously reported Zellweger syndrome cell lines the defect could be assigned to the matrix protein import pathway since peroxisome membranes were present, and import of integral peroxisomal membrane proteins was normal. However, we report here a Zellweger syndrome patient (PBD061) with an unusual cellular phenotype, an inability to import peroxisomal membrane proteins. We also identified human PEX16, a novel integral peroxisomal membrane protein, and found that PBD061 had inactivating mutations in the PEX16 gene. Previous studies have suggested that peroxisomes arise from preexisting peroxisomes but we find that expression of PEX16 restores the formation of new peroxisomes in PBD061 cells. Peroxisome synthesis and peroxisomal membrane protein import could be detected within 2-3 h of PEX16 injection and was followed by matrix protein import. These results demonstrate that peroxisomes do not necessarily arise from division of preexisting peroxisomes. We propose that peroxisomes may form by either of two pathways: one that involves PEX11-mediated division of preexisting peroxisomes, and another that involves PEX16-mediated formation of peroxisomes in the absence of preexisting peroxisomes.

Show MeSH

Related in: MedlinePlus

Rescue of peroxisome synthesis in PBD061  cells occurs in a multistep  process. PBD061 cells were  injected with pcDNA3- PEX16 and processed for indirect immunofluorescence  3 h later using rabbit antibodies specific for P70R (A); and  sheep antibodies specific for  catalase (B); followed by fluorescein-labeled goat anti– rabbit and Texas red–labeled  goat anti–sheep secondary  antibodies. Note that many  of the peroxisomes appeared  to be undergoing elongation,  the first step in PEX11β-mediated peroxisome proliferation (Schrader et al., 1998).  Cells processed for 22 h (C  and D) and 72 h (E and F) after introduction of pcDNA3- PEX16 were also processed  for indirect immunofluorescence using rabbit antibodies specific for P70R (C and  E) and sheep antibodies specific for catalase (D and F),  followed by fluorescein-labeled  goat anti–rabbit and Texas  red–labeled goat anti–sheep  secondary antibodies. (G and  H) PBD061 cells were preincubated for 30 min with 10  μg/ml BFA, injected with  pcDNA3-PEX16, and incubated in 10 μg/ml BFA for  30 h. These cells were processed for immunofluorescence using rabbit antibodies  specific for P70R (G); and  sheep antibodies specific for  catalase (H); followed by fluorescein-labeled goat anti– rabbit and Texas red–labeled  goat anti–sheep secondary  antibodies. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132891&req=5

Figure 9: Rescue of peroxisome synthesis in PBD061 cells occurs in a multistep process. PBD061 cells were injected with pcDNA3- PEX16 and processed for indirect immunofluorescence 3 h later using rabbit antibodies specific for P70R (A); and sheep antibodies specific for catalase (B); followed by fluorescein-labeled goat anti– rabbit and Texas red–labeled goat anti–sheep secondary antibodies. Note that many of the peroxisomes appeared to be undergoing elongation, the first step in PEX11β-mediated peroxisome proliferation (Schrader et al., 1998). Cells processed for 22 h (C and D) and 72 h (E and F) after introduction of pcDNA3- PEX16 were also processed for indirect immunofluorescence using rabbit antibodies specific for P70R (C and E) and sheep antibodies specific for catalase (D and F), followed by fluorescein-labeled goat anti–rabbit and Texas red–labeled goat anti–sheep secondary antibodies. (G and H) PBD061 cells were preincubated for 30 min with 10 μg/ml BFA, injected with pcDNA3-PEX16, and incubated in 10 μg/ml BFA for 30 h. These cells were processed for immunofluorescence using rabbit antibodies specific for P70R (G); and sheep antibodies specific for catalase (H); followed by fluorescein-labeled goat anti– rabbit and Texas red–labeled goat anti–sheep secondary antibodies. Bar, 10 μm.

Mentions: The restoration of peroxisome synthesis in PBD061 cells demonstrated that peroxisomes do not necessarily arise by division of preexisting peroxisomes. To characterize the process by which this occurs we followed the appearance of peroxisomes at various times after injection of PBD061 cells with the PEX16 expression vector. At each time point, cells were processed for indirect immunofluorescence using antibodies specific for the peroxisomal membrane marker, PMP70, and the peroxisomal matrix marker, catalase. We were unable to detect any peroxisomal structures 30 min, 1, or 2 h after injection. However, by 3 h after injection we started to observe P70R-containing structures that could be detected over and above background staining (Fig. 9, A and B). These structures generally did not contain the matrix protein catalase, and were much reduced in number as compared to normal cells or fully rescued PBD061 cells. In addition, peroxisomes detected at early time points often appeared to form elongated structures similar to those that form at early times in the PEX11β-mediated proliferation of human peroxisomes.


Peroxisome synthesis in the absence of preexisting peroxisomes.

South ST, Gould SJ - J. Cell Biol. (1999)

Rescue of peroxisome synthesis in PBD061  cells occurs in a multistep  process. PBD061 cells were  injected with pcDNA3- PEX16 and processed for indirect immunofluorescence  3 h later using rabbit antibodies specific for P70R (A); and  sheep antibodies specific for  catalase (B); followed by fluorescein-labeled goat anti– rabbit and Texas red–labeled  goat anti–sheep secondary  antibodies. Note that many  of the peroxisomes appeared  to be undergoing elongation,  the first step in PEX11β-mediated peroxisome proliferation (Schrader et al., 1998).  Cells processed for 22 h (C  and D) and 72 h (E and F) after introduction of pcDNA3- PEX16 were also processed  for indirect immunofluorescence using rabbit antibodies specific for P70R (C and  E) and sheep antibodies specific for catalase (D and F),  followed by fluorescein-labeled  goat anti–rabbit and Texas  red–labeled goat anti–sheep  secondary antibodies. (G and  H) PBD061 cells were preincubated for 30 min with 10  μg/ml BFA, injected with  pcDNA3-PEX16, and incubated in 10 μg/ml BFA for  30 h. These cells were processed for immunofluorescence using rabbit antibodies  specific for P70R (G); and  sheep antibodies specific for  catalase (H); followed by fluorescein-labeled goat anti– rabbit and Texas red–labeled  goat anti–sheep secondary  antibodies. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132891&req=5

Figure 9: Rescue of peroxisome synthesis in PBD061 cells occurs in a multistep process. PBD061 cells were injected with pcDNA3- PEX16 and processed for indirect immunofluorescence 3 h later using rabbit antibodies specific for P70R (A); and sheep antibodies specific for catalase (B); followed by fluorescein-labeled goat anti– rabbit and Texas red–labeled goat anti–sheep secondary antibodies. Note that many of the peroxisomes appeared to be undergoing elongation, the first step in PEX11β-mediated peroxisome proliferation (Schrader et al., 1998). Cells processed for 22 h (C and D) and 72 h (E and F) after introduction of pcDNA3- PEX16 were also processed for indirect immunofluorescence using rabbit antibodies specific for P70R (C and E) and sheep antibodies specific for catalase (D and F), followed by fluorescein-labeled goat anti–rabbit and Texas red–labeled goat anti–sheep secondary antibodies. (G and H) PBD061 cells were preincubated for 30 min with 10 μg/ml BFA, injected with pcDNA3-PEX16, and incubated in 10 μg/ml BFA for 30 h. These cells were processed for immunofluorescence using rabbit antibodies specific for P70R (G); and sheep antibodies specific for catalase (H); followed by fluorescein-labeled goat anti– rabbit and Texas red–labeled goat anti–sheep secondary antibodies. Bar, 10 μm.
Mentions: The restoration of peroxisome synthesis in PBD061 cells demonstrated that peroxisomes do not necessarily arise by division of preexisting peroxisomes. To characterize the process by which this occurs we followed the appearance of peroxisomes at various times after injection of PBD061 cells with the PEX16 expression vector. At each time point, cells were processed for indirect immunofluorescence using antibodies specific for the peroxisomal membrane marker, PMP70, and the peroxisomal matrix marker, catalase. We were unable to detect any peroxisomal structures 30 min, 1, or 2 h after injection. However, by 3 h after injection we started to observe P70R-containing structures that could be detected over and above background staining (Fig. 9, A and B). These structures generally did not contain the matrix protein catalase, and were much reduced in number as compared to normal cells or fully rescued PBD061 cells. In addition, peroxisomes detected at early time points often appeared to form elongated structures similar to those that form at early times in the PEX11β-mediated proliferation of human peroxisomes.

Bottom Line: We also identified human PEX16, a novel integral peroxisomal membrane protein, and found that PBD061 had inactivating mutations in the PEX16 gene.These results demonstrate that peroxisomes do not necessarily arise from division of preexisting peroxisomes.We propose that peroxisomes may form by either of two pathways: one that involves PEX11-mediated division of preexisting peroxisomes, and another that involves PEX16-mediated formation of peroxisomes in the absence of preexisting peroxisomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
Zellweger syndrome and related diseases are caused by defective import of peroxisomal matrix proteins. In all previously reported Zellweger syndrome cell lines the defect could be assigned to the matrix protein import pathway since peroxisome membranes were present, and import of integral peroxisomal membrane proteins was normal. However, we report here a Zellweger syndrome patient (PBD061) with an unusual cellular phenotype, an inability to import peroxisomal membrane proteins. We also identified human PEX16, a novel integral peroxisomal membrane protein, and found that PBD061 had inactivating mutations in the PEX16 gene. Previous studies have suggested that peroxisomes arise from preexisting peroxisomes but we find that expression of PEX16 restores the formation of new peroxisomes in PBD061 cells. Peroxisome synthesis and peroxisomal membrane protein import could be detected within 2-3 h of PEX16 injection and was followed by matrix protein import. These results demonstrate that peroxisomes do not necessarily arise from division of preexisting peroxisomes. We propose that peroxisomes may form by either of two pathways: one that involves PEX11-mediated division of preexisting peroxisomes, and another that involves PEX16-mediated formation of peroxisomes in the absence of preexisting peroxisomes.

Show MeSH
Related in: MedlinePlus