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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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The NH2 and COOH termini of PEX16 are exposed to  the cytoplasm. Normal human fibroblasts were transfected with  pcDNA3-mycPEX16 (A–D) or pcDNA3-PEX16myc (E–H). 2 d  after transfection cells were fixed and processed for indirect  immunofluorescence under standard permeabilization conditions  (A, B, E, and F) or under differential permeabilization conditions (C, D, G, and H). Cells were labeled with mouse mAbs specific for the myc epitope tag (A, C, E, and G) and with sheep antibodies specific for catalase (B, D, F, and H), followed by  Texas red–labeled goat anti–mouse and fluorescein-labeled goat  anti–sheep secondary antibodies. Bar, 10 μm.
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Figure 7: The NH2 and COOH termini of PEX16 are exposed to the cytoplasm. Normal human fibroblasts were transfected with pcDNA3-mycPEX16 (A–D) or pcDNA3-PEX16myc (E–H). 2 d after transfection cells were fixed and processed for indirect immunofluorescence under standard permeabilization conditions (A, B, E, and F) or under differential permeabilization conditions (C, D, G, and H). Cells were labeled with mouse mAbs specific for the myc epitope tag (A, C, E, and G) and with sheep antibodies specific for catalase (B, D, F, and H), followed by Texas red–labeled goat anti–mouse and fluorescein-labeled goat anti–sheep secondary antibodies. Bar, 10 μm.

Mentions: To test PEX16 orientation by an independent method we generated tagged versions of the PEX16 cDNA expression vectors: one designed to express the c-myc tag at the NH2 terminus of PEX16 (pcDNA3-mycPEX16), and one designed to express the c-myc tag at its COOH terminus (pcDNA3-PEX16myc). Normal human fibroblasts were transfected with these plasmids. After 2 d of incubation, the cells were processed for immunofluorescence using antibodies specific for the myc epitope tag and for catalase, a peroxisomal matrix protein (Fig. 7). The mycPEX16 and PEX16myc proteins both colocalized with catalase, confirming that PEX16 is a peroxisomal protein. Furthermore, the mycPEX16 and PEX16myc proteins were detected under differential permeabilization conditions that preclude the detection of intraperoxisomal antigens, confirming that PEX16 is a peroxisomal membrane protein with its NH2 and COOH termini extending into the cytoplasm. Surprisingly, overexpression of mycPEX16 or PEX16myc never induced peroxisome proliferation, suggesting that there may be a functional distinction between factors that are required for peroxisome membrane synthesis and those that are involved in peroxisome proliferation, such as PEX11α and PEX11β (Schrader et al., 1998). In control experiments, the mycPEX16 and PEX16myc expression vectors were able to rescue peroxisome synthesis in PBD061 cells, confirming that these localization studies were performed with functional forms of PEX16 (data not shown).


Peroxisome synthesis in the absence of preexisting peroxisomes.

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

The NH2 and COOH termini of PEX16 are exposed to  the cytoplasm. Normal human fibroblasts were transfected with  pcDNA3-mycPEX16 (A–D) or pcDNA3-PEX16myc (E–H). 2 d  after transfection cells were fixed and processed for indirect  immunofluorescence under standard permeabilization conditions  (A, B, E, and F) or under differential permeabilization conditions (C, D, G, and H). Cells were labeled with mouse mAbs specific for the myc epitope tag (A, C, E, and G) and with sheep antibodies specific for catalase (B, D, F, and H), followed by  Texas red–labeled goat anti–mouse and fluorescein-labeled goat  anti–sheep secondary antibodies. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 7: The NH2 and COOH termini of PEX16 are exposed to the cytoplasm. Normal human fibroblasts were transfected with pcDNA3-mycPEX16 (A–D) or pcDNA3-PEX16myc (E–H). 2 d after transfection cells were fixed and processed for indirect immunofluorescence under standard permeabilization conditions (A, B, E, and F) or under differential permeabilization conditions (C, D, G, and H). Cells were labeled with mouse mAbs specific for the myc epitope tag (A, C, E, and G) and with sheep antibodies specific for catalase (B, D, F, and H), followed by Texas red–labeled goat anti–mouse and fluorescein-labeled goat anti–sheep secondary antibodies. Bar, 10 μm.
Mentions: To test PEX16 orientation by an independent method we generated tagged versions of the PEX16 cDNA expression vectors: one designed to express the c-myc tag at the NH2 terminus of PEX16 (pcDNA3-mycPEX16), and one designed to express the c-myc tag at its COOH terminus (pcDNA3-PEX16myc). Normal human fibroblasts were transfected with these plasmids. After 2 d of incubation, the cells were processed for immunofluorescence using antibodies specific for the myc epitope tag and for catalase, a peroxisomal matrix protein (Fig. 7). The mycPEX16 and PEX16myc proteins both colocalized with catalase, confirming that PEX16 is a peroxisomal protein. Furthermore, the mycPEX16 and PEX16myc proteins were detected under differential permeabilization conditions that preclude the detection of intraperoxisomal antigens, confirming that PEX16 is a peroxisomal membrane protein with its NH2 and COOH termini extending into the cytoplasm. Surprisingly, overexpression of mycPEX16 or PEX16myc never induced peroxisome proliferation, suggesting that there may be a functional distinction between factors that are required for peroxisome membrane synthesis and those that are involved in peroxisome proliferation, such as PEX11α and PEX11β (Schrader et al., 1998). In control experiments, the mycPEX16 and PEX16myc expression vectors were able to rescue peroxisome synthesis in PBD061 cells, confirming that these localization studies were performed with functional forms of PEX16 (data not shown).

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