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

PBD061 cells have an inactivating mutation in PEX16.  Direct sequence analysis of a PEX16 fragment from (A) PBD061  and (B) an unaffected individual showing the CGA to TGA nonsense mutation in PBD061 genomic DNA. The homogeneous nature of the PBD061 sequence indicates that this patient is homozygous for the R176ter mutation. The effect of this mutation  was assayed by transfecting PBD061 cells with pcDNA3-PEX16/ R176ter (C and D) a plasmid designed to express the mutated  form of the cDNA; and pcDNA3-PEX16 (E and F). 2 d after  transfection the cells were processed for indirect immunofluorescence using rabbit antibodies specific for PMP70 (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. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132891&req=5

Figure 5: PBD061 cells have an inactivating mutation in PEX16. Direct sequence analysis of a PEX16 fragment from (A) PBD061 and (B) an unaffected individual showing the CGA to TGA nonsense mutation in PBD061 genomic DNA. The homogeneous nature of the PBD061 sequence indicates that this patient is homozygous for the R176ter mutation. The effect of this mutation was assayed by transfecting PBD061 cells with pcDNA3-PEX16/ R176ter (C and D) a plasmid designed to express the mutated form of the cDNA; and pcDNA3-PEX16 (E and F). 2 d after transfection the cells were processed for indirect immunofluorescence using rabbit antibodies specific for PMP70 (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. Bar, 10 μm.

Mentions: The functional complementation experiments described above indicated that this patient would have mutations in the PEX16 gene. The PEX16 cDNA was amplified from control and PBD061 RNAs by RT-PCR, and direct sequence analysis revealed the presence of a nonsense mutation in the PEX16/PBD061 cDNA population (data not shown). This mutation, a C-to-T transition at a CpG dinucleotide, converts the arginine 176 codon to a nonsense codon (R176ter), and appeared to represent the only form of PEX16 mRNA present in PBD061 cells. To determine whether this mutation was present at the genomic DNA level we amplified an appropriate fragment of the PEX16 gene from control and PBD061 genomic DNAs. Direct sequence analysis showed only the mutant form of the gene in PBD061 (Fig. 5, A and B), indicating that this patient is homozygous for the R176ter mutation.


Peroxisome synthesis in the absence of preexisting peroxisomes.

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

PBD061 cells have an inactivating mutation in PEX16.  Direct sequence analysis of a PEX16 fragment from (A) PBD061  and (B) an unaffected individual showing the CGA to TGA nonsense mutation in PBD061 genomic DNA. The homogeneous nature of the PBD061 sequence indicates that this patient is homozygous for the R176ter mutation. The effect of this mutation  was assayed by transfecting PBD061 cells with pcDNA3-PEX16/ R176ter (C and D) a plasmid designed to express the mutated  form of the cDNA; and pcDNA3-PEX16 (E and F). 2 d after  transfection the cells were processed for indirect immunofluorescence using rabbit antibodies specific for PMP70 (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. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: PBD061 cells have an inactivating mutation in PEX16. Direct sequence analysis of a PEX16 fragment from (A) PBD061 and (B) an unaffected individual showing the CGA to TGA nonsense mutation in PBD061 genomic DNA. The homogeneous nature of the PBD061 sequence indicates that this patient is homozygous for the R176ter mutation. The effect of this mutation was assayed by transfecting PBD061 cells with pcDNA3-PEX16/ R176ter (C and D) a plasmid designed to express the mutated form of the cDNA; and pcDNA3-PEX16 (E and F). 2 d after transfection the cells were processed for indirect immunofluorescence using rabbit antibodies specific for PMP70 (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. Bar, 10 μm.
Mentions: The functional complementation experiments described above indicated that this patient would have mutations in the PEX16 gene. The PEX16 cDNA was amplified from control and PBD061 RNAs by RT-PCR, and direct sequence analysis revealed the presence of a nonsense mutation in the PEX16/PBD061 cDNA population (data not shown). This mutation, a C-to-T transition at a CpG dinucleotide, converts the arginine 176 codon to a nonsense codon (R176ter), and appeared to represent the only form of PEX16 mRNA present in PBD061 cells. To determine whether this mutation was present at the genomic DNA level we amplified an appropriate fragment of the PEX16 gene from control and PBD061 genomic DNAs. Direct sequence analysis showed only the mutant form of the gene in PBD061 (Fig. 5, A and B), indicating that this patient is homozygous for the R176ter mutation.

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