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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 expression restores peroxisome synthesis in the  CG9 cell line, PBD061. (A) The deduced amino acid sequence of  human PEX16. Amino acid positions are noted on the left and  the two predicted transmembrane domains are underlined.  PBD061 cells were transfected with the plasmids pcDNA3 (B  and D), and pcDNA3-PEX16 (C and E). 3 d later the cells were  processed for indirect immunofluorescence using rabbit antibodies specific for PMP70 (B and C), and sheep antibodies specific  for catalase (D and E), followed by fluorescein-labeled goat anti– rabbit and Texas red–labeled goat anti–sheep secondary antibodies. Note the colocalization of PMP70 and catalase in the rescued  cell (C and E). Bar, 10 μm.
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Figure 4: PEX16 expression restores peroxisome synthesis in the CG9 cell line, PBD061. (A) The deduced amino acid sequence of human PEX16. Amino acid positions are noted on the left and the two predicted transmembrane domains are underlined. PBD061 cells were transfected with the plasmids pcDNA3 (B and D), and pcDNA3-PEX16 (C and E). 3 d later the cells were processed for indirect immunofluorescence using rabbit antibodies specific for PMP70 (B and C), and sheep antibodies specific for catalase (D and E), followed by fluorescein-labeled goat anti– rabbit and Texas red–labeled goat anti–sheep secondary antibodies. Note the colocalization of PMP70 and catalase in the rescued cell (C and E). Bar, 10 μm.

Mentions: The prevailing model of peroxisome biogenesis holds that peroxisomes are formed by division of preexisting peroxisomes. Thus, it was an open question as to whether peroxisome synthesis could be restored in a cell line such as PBD061; and the identification of the gene defective in PBD061 cells would likely have a significant impact on our understanding of peroxisome biogenesis. We employed the homology probing strategy that has been used previously to identify human homologues of other yeast PEX genes (Dodt et al., 1996). The database of expressed sequence tags was searched for mammalian homologues of the Y. lipolytica PEX16 gene and a single human homologue was identified (Fig. 4 A). This human PEX16 cDNA was inserted into a mammalian expression vector and the resulting plasmid, pcDNA3-PEX16, was transfected into PBD061 cells. 3 d after transfection the cells were processed for indirect immunofluorescence using antibodies specific for the peroxisome membrane marker, PMP70, and a peroxisomal matrix protein, catalase. Expression of PEX16 rescued both PMP and matrix protein import in PBD061 cells, demonstrating that peroxisomes can form in the absence of preexisting peroxisomes (Fig. 4). No rescue was observed in cells transfected with the vector alone.


Peroxisome synthesis in the absence of preexisting peroxisomes.

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

PEX16 expression restores peroxisome synthesis in the  CG9 cell line, PBD061. (A) The deduced amino acid sequence of  human PEX16. Amino acid positions are noted on the left and  the two predicted transmembrane domains are underlined.  PBD061 cells were transfected with the plasmids pcDNA3 (B  and D), and pcDNA3-PEX16 (C and E). 3 d later the cells were  processed for indirect immunofluorescence using rabbit antibodies specific for PMP70 (B and C), and sheep antibodies specific  for catalase (D and E), followed by fluorescein-labeled goat anti– rabbit and Texas red–labeled goat anti–sheep secondary antibodies. Note the colocalization of PMP70 and catalase in the rescued  cell (C and E). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 4: PEX16 expression restores peroxisome synthesis in the CG9 cell line, PBD061. (A) The deduced amino acid sequence of human PEX16. Amino acid positions are noted on the left and the two predicted transmembrane domains are underlined. PBD061 cells were transfected with the plasmids pcDNA3 (B and D), and pcDNA3-PEX16 (C and E). 3 d later the cells were processed for indirect immunofluorescence using rabbit antibodies specific for PMP70 (B and C), and sheep antibodies specific for catalase (D and E), followed by fluorescein-labeled goat anti– rabbit and Texas red–labeled goat anti–sheep secondary antibodies. Note the colocalization of PMP70 and catalase in the rescued cell (C and E). Bar, 10 μm.
Mentions: The prevailing model of peroxisome biogenesis holds that peroxisomes are formed by division of preexisting peroxisomes. Thus, it was an open question as to whether peroxisome synthesis could be restored in a cell line such as PBD061; and the identification of the gene defective in PBD061 cells would likely have a significant impact on our understanding of peroxisome biogenesis. We employed the homology probing strategy that has been used previously to identify human homologues of other yeast PEX genes (Dodt et al., 1996). The database of expressed sequence tags was searched for mammalian homologues of the Y. lipolytica PEX16 gene and a single human homologue was identified (Fig. 4 A). This human PEX16 cDNA was inserted into a mammalian expression vector and the resulting plasmid, pcDNA3-PEX16, was transfected into PBD061 cells. 3 d after transfection the cells were processed for indirect immunofluorescence using antibodies specific for the peroxisome membrane marker, PMP70, and a peroxisomal matrix protein, catalase. Expression of PEX16 rescued both PMP and matrix protein import in PBD061 cells, demonstrating that peroxisomes can form in the absence of preexisting peroxisomes (Fig. 4). No rescue was observed in cells transfected with the vector alone.

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