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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.

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PEX16 is rapidly targeted to peroxisomes in a PEX1-independent and BFA-independent manner. The PEX1-deficient  cell line, PBD009, was transfected with pcDNA3-PEX16myc (A  and B), and processed for indirect immunofluorescence 2 d later  using mouse mAbs specific for the myc epitope tag (A), and rabbit antibodies specific for PMP70 (B), followed by fluorescein-  labeled goat anti–mouse and Texas red–labeled goat anti–rabbit  secondary antibodies. The normal human fibroblast cell line,  5756T, was injected with pcDNA3-PEX16myc and processed for  indirect immunofluorescence 2 h later using mouse mAbs specific for the myc epitope tag (C) and rabbit antibodies specific for  P70R (D), followed by fluorescein-labeled goat anti–mouse and  Texas red–labeled goat anti–rabbit secondary antibodies. (E and  F) 5756T cells that had been pretreated for 30 min with 10 μg/ml  BFA were injected with pcDNA3-PEX16myc, incubated for 2 h  in 10 μg/ml BFA, and processed for immunofluorescence using  mouse mAbs specific for the myc epitope tag (E) and rabbit antibodies specific for P70R (F), followed by fluorescein-labeled goat  anti–mouse and Texas red–labeled goat anti–rabbit secondary  antibodies. Bar, 10 μm.
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Figure 8: PEX16 is rapidly targeted to peroxisomes in a PEX1-independent and BFA-independent manner. The PEX1-deficient cell line, PBD009, was transfected with pcDNA3-PEX16myc (A and B), and processed for indirect immunofluorescence 2 d later using mouse mAbs specific for the myc epitope tag (A), and rabbit antibodies specific for PMP70 (B), followed by fluorescein- labeled goat anti–mouse and Texas red–labeled goat anti–rabbit secondary antibodies. The normal human fibroblast cell line, 5756T, was injected with pcDNA3-PEX16myc and processed for indirect immunofluorescence 2 h later using mouse mAbs specific for the myc epitope tag (C) and rabbit antibodies specific for P70R (D), followed by fluorescein-labeled goat anti–mouse and Texas red–labeled goat anti–rabbit secondary antibodies. (E and F) 5756T cells that had been pretreated for 30 min with 10 μg/ml BFA were injected with pcDNA3-PEX16myc, incubated for 2 h in 10 μg/ml BFA, and processed for immunofluorescence using mouse mAbs specific for the myc epitope tag (E) and rabbit antibodies specific for P70R (F), followed by fluorescein-labeled goat anti–mouse and Texas red–labeled goat anti–rabbit secondary antibodies. Bar, 10 μm.

Mentions: We first examined the distribution of PEX16myc in cells that lack PEX1. PBD009 cells have inactivating mutations on both PEX1 alleles, lack PEX1 mRNA and protein (Reuber et al., 1997), and would be expected to accumulate PEX16myc in the ER. In contrast to the expected results, we found that PEX16myc colocalized with the peroxisomal membrane marker, P70R, in PEX1-deficient PBD009 cells (Fig. 8, A and B). Next, we replicated the pulsed expression experiments. However, because of the need to detect PEX16myc at the very earliest time points during its expression, we had to deliver the PEX16myc expression vector by nuclear microinjection rather than electroporation. The normal human fibroblast line 5756T was injected with pcDNA3-PEX16myc and then processed for indirect immunofluorescence at 0.5, 1, 2, and 3 h after injection. The first time point that we detected PEX16myc expression was 2 h after injection, at which point all of the PEX16myc appeared to colocalize with the peroxisomal membrane marker, P70R (Fig. 8, C and D). Examination of multiple cells at the 2- and 3-h time points showed a similar peroxisomal distribution of PEX16myc. The fact that human PEX16 lacks a consensus sequence for N-linked oligosaccharide addition, Asn-Xaa-Ser/Thr, made it impossible to follow N-linked glycosylation as a test of whether PEX16 entered the ER before its delivery to peroxisomes.


Peroxisome synthesis in the absence of preexisting peroxisomes.

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

PEX16 is rapidly targeted to peroxisomes in a PEX1-independent and BFA-independent manner. The PEX1-deficient  cell line, PBD009, was transfected with pcDNA3-PEX16myc (A  and B), and processed for indirect immunofluorescence 2 d later  using mouse mAbs specific for the myc epitope tag (A), and rabbit antibodies specific for PMP70 (B), followed by fluorescein-  labeled goat anti–mouse and Texas red–labeled goat anti–rabbit  secondary antibodies. The normal human fibroblast cell line,  5756T, was injected with pcDNA3-PEX16myc and processed for  indirect immunofluorescence 2 h later using mouse mAbs specific for the myc epitope tag (C) and rabbit antibodies specific for  P70R (D), followed by fluorescein-labeled goat anti–mouse and  Texas red–labeled goat anti–rabbit secondary antibodies. (E and  F) 5756T cells that had been pretreated for 30 min with 10 μg/ml  BFA were injected with pcDNA3-PEX16myc, incubated for 2 h  in 10 μg/ml BFA, and processed for immunofluorescence using  mouse mAbs specific for the myc epitope tag (E) and rabbit antibodies specific for P70R (F), followed by fluorescein-labeled goat  anti–mouse and Texas red–labeled goat anti–rabbit secondary  antibodies. Bar, 10 μm.
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Figure 8: PEX16 is rapidly targeted to peroxisomes in a PEX1-independent and BFA-independent manner. The PEX1-deficient cell line, PBD009, was transfected with pcDNA3-PEX16myc (A and B), and processed for indirect immunofluorescence 2 d later using mouse mAbs specific for the myc epitope tag (A), and rabbit antibodies specific for PMP70 (B), followed by fluorescein- labeled goat anti–mouse and Texas red–labeled goat anti–rabbit secondary antibodies. The normal human fibroblast cell line, 5756T, was injected with pcDNA3-PEX16myc and processed for indirect immunofluorescence 2 h later using mouse mAbs specific for the myc epitope tag (C) and rabbit antibodies specific for P70R (D), followed by fluorescein-labeled goat anti–mouse and Texas red–labeled goat anti–rabbit secondary antibodies. (E and F) 5756T cells that had been pretreated for 30 min with 10 μg/ml BFA were injected with pcDNA3-PEX16myc, incubated for 2 h in 10 μg/ml BFA, and processed for immunofluorescence using mouse mAbs specific for the myc epitope tag (E) and rabbit antibodies specific for P70R (F), followed by fluorescein-labeled goat anti–mouse and Texas red–labeled goat anti–rabbit secondary antibodies. Bar, 10 μm.
Mentions: We first examined the distribution of PEX16myc in cells that lack PEX1. PBD009 cells have inactivating mutations on both PEX1 alleles, lack PEX1 mRNA and protein (Reuber et al., 1997), and would be expected to accumulate PEX16myc in the ER. In contrast to the expected results, we found that PEX16myc colocalized with the peroxisomal membrane marker, P70R, in PEX1-deficient PBD009 cells (Fig. 8, A and B). Next, we replicated the pulsed expression experiments. However, because of the need to detect PEX16myc at the very earliest time points during its expression, we had to deliver the PEX16myc expression vector by nuclear microinjection rather than electroporation. The normal human fibroblast line 5756T was injected with pcDNA3-PEX16myc and then processed for indirect immunofluorescence at 0.5, 1, 2, and 3 h after injection. The first time point that we detected PEX16myc expression was 2 h after injection, at which point all of the PEX16myc appeared to colocalize with the peroxisomal membrane marker, P70R (Fig. 8, C and D). Examination of multiple cells at the 2- and 3-h time points showed a similar peroxisomal distribution of PEX16myc. The fact that human PEX16 lacks a consensus sequence for N-linked oligosaccharide addition, Asn-Xaa-Ser/Thr, made it impossible to follow N-linked glycosylation as a test of whether PEX16 entered the ER before its delivery to 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