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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 encodes an integral peroxisomal membrane  protein. (A) A postnuclear supernatant of HepG2 cells was fractionated by density gradient centrifugation. Equal proportions of  each fraction were assayed for the marker enzymes catalase, a  peroxisomal enzyme, SDH, a mitochondrial marker, and NCR,  an ER-associated enzyme. Equal proportions of each fraction  were also assayed for levels of PEX16 by immunoblot (bottom).  These antibodies also detect an unknown protein of slightly  higher mobility in fraction 10. (B) PEX16 is present in high pH  carbonate-washed membranes. A light mitochondrial fraction  from HepG2 cells was separated into a hypotonic extraction supernatant (lane 1); a high salt extraction supernatant (lane 2); a  high pH carbonate supernatant (lane 3); and a pellet of carbonate-washed membranes (lane 4). Equal proportions of each fraction were separated by SDS-PAGE and assayed for levels of  PEX16 by Western blot. (C) Protease protection experiments indicate that PEX16 spans the membrane twice with its NH2 and  COOH termini exposed to the cytoplasm. A light mitochondrial  fraction was prepared from HepG2 cells and incubated with no  trypsin (lane 1), 15 μg trypsin (lane 2), 30 μg trypsin (lane 3), and  60 μg trypsin (lane 4). PEX16 is reduced to a protease-resistant  form of 15 kD, the size of the two transmembrane domains and  the intermembrane loop. Digestion in the presence of detergent  (1% Triton X-100) did not yield the 15-kD species (data not  shown).
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Figure 6: PEX16 encodes an integral peroxisomal membrane protein. (A) A postnuclear supernatant of HepG2 cells was fractionated by density gradient centrifugation. Equal proportions of each fraction were assayed for the marker enzymes catalase, a peroxisomal enzyme, SDH, a mitochondrial marker, and NCR, an ER-associated enzyme. Equal proportions of each fraction were also assayed for levels of PEX16 by immunoblot (bottom). These antibodies also detect an unknown protein of slightly higher mobility in fraction 10. (B) PEX16 is present in high pH carbonate-washed membranes. A light mitochondrial fraction from HepG2 cells was separated into a hypotonic extraction supernatant (lane 1); a high salt extraction supernatant (lane 2); a high pH carbonate supernatant (lane 3); and a pellet of carbonate-washed membranes (lane 4). Equal proportions of each fraction were separated by SDS-PAGE and assayed for levels of PEX16 by Western blot. (C) Protease protection experiments indicate that PEX16 spans the membrane twice with its NH2 and COOH termini exposed to the cytoplasm. A light mitochondrial fraction was prepared from HepG2 cells and incubated with no trypsin (lane 1), 15 μg trypsin (lane 2), 30 μg trypsin (lane 3), and 60 μg trypsin (lane 4). PEX16 is reduced to a protease-resistant form of 15 kD, the size of the two transmembrane domains and the intermembrane loop. Digestion in the presence of detergent (1% Triton X-100) did not yield the 15-kD species (data not shown).

Mentions: While the above results demonstrated that PEX16 is required for peroxisome biogenesis they did not reveal how PEX16 might perform this role. Since the physical location of proteins can provide useful insight into their function we first determined the subcellular distribution of PEX16. Antibodies to PEX16 were generated and recognized a polypeptide of 38 kD, the predicted molecular mass of PEX16, in human cell extracts (data not shown). These antibodies were used to localize PEX16 in subcellular fractionation experiments. A postnuclear supernatant was prepared from HepG2 cells and fractionated by Nycodenz density gradient centrifugation. Fractions were assayed for marker enzymes of peroxisomes, mitochondria, and microsomes, as well as PEX16 (Fig. 6 A). PEX16 behaved as a typical PMP, localizing only to the high-density, catalase-containing peroxisomal fractions (rupture of peroxisomes during homogenization and centrifugation leads to the presence of peroxisomal matrix proteins at the top of the gradient, demonstrated here by the presence of catalase activity in the low density fractions).


Peroxisome synthesis in the absence of preexisting peroxisomes.

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

PEX16 encodes an integral peroxisomal membrane  protein. (A) A postnuclear supernatant of HepG2 cells was fractionated by density gradient centrifugation. Equal proportions of  each fraction were assayed for the marker enzymes catalase, a  peroxisomal enzyme, SDH, a mitochondrial marker, and NCR,  an ER-associated enzyme. Equal proportions of each fraction  were also assayed for levels of PEX16 by immunoblot (bottom).  These antibodies also detect an unknown protein of slightly  higher mobility in fraction 10. (B) PEX16 is present in high pH  carbonate-washed membranes. A light mitochondrial fraction  from HepG2 cells was separated into a hypotonic extraction supernatant (lane 1); a high salt extraction supernatant (lane 2); a  high pH carbonate supernatant (lane 3); and a pellet of carbonate-washed membranes (lane 4). Equal proportions of each fraction were separated by SDS-PAGE and assayed for levels of  PEX16 by Western blot. (C) Protease protection experiments indicate that PEX16 spans the membrane twice with its NH2 and  COOH termini exposed to the cytoplasm. A light mitochondrial  fraction was prepared from HepG2 cells and incubated with no  trypsin (lane 1), 15 μg trypsin (lane 2), 30 μg trypsin (lane 3), and  60 μg trypsin (lane 4). PEX16 is reduced to a protease-resistant  form of 15 kD, the size of the two transmembrane domains and  the intermembrane loop. Digestion in the presence of detergent  (1% Triton X-100) did not yield the 15-kD species (data not  shown).
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Figure 6: PEX16 encodes an integral peroxisomal membrane protein. (A) A postnuclear supernatant of HepG2 cells was fractionated by density gradient centrifugation. Equal proportions of each fraction were assayed for the marker enzymes catalase, a peroxisomal enzyme, SDH, a mitochondrial marker, and NCR, an ER-associated enzyme. Equal proportions of each fraction were also assayed for levels of PEX16 by immunoblot (bottom). These antibodies also detect an unknown protein of slightly higher mobility in fraction 10. (B) PEX16 is present in high pH carbonate-washed membranes. A light mitochondrial fraction from HepG2 cells was separated into a hypotonic extraction supernatant (lane 1); a high salt extraction supernatant (lane 2); a high pH carbonate supernatant (lane 3); and a pellet of carbonate-washed membranes (lane 4). Equal proportions of each fraction were separated by SDS-PAGE and assayed for levels of PEX16 by Western blot. (C) Protease protection experiments indicate that PEX16 spans the membrane twice with its NH2 and COOH termini exposed to the cytoplasm. A light mitochondrial fraction was prepared from HepG2 cells and incubated with no trypsin (lane 1), 15 μg trypsin (lane 2), 30 μg trypsin (lane 3), and 60 μg trypsin (lane 4). PEX16 is reduced to a protease-resistant form of 15 kD, the size of the two transmembrane domains and the intermembrane loop. Digestion in the presence of detergent (1% Triton X-100) did not yield the 15-kD species (data not shown).
Mentions: While the above results demonstrated that PEX16 is required for peroxisome biogenesis they did not reveal how PEX16 might perform this role. Since the physical location of proteins can provide useful insight into their function we first determined the subcellular distribution of PEX16. Antibodies to PEX16 were generated and recognized a polypeptide of 38 kD, the predicted molecular mass of PEX16, in human cell extracts (data not shown). These antibodies were used to localize PEX16 in subcellular fractionation experiments. A postnuclear supernatant was prepared from HepG2 cells and fractionated by Nycodenz density gradient centrifugation. Fractions were assayed for marker enzymes of peroxisomes, mitochondria, and microsomes, as well as PEX16 (Fig. 6 A). PEX16 behaved as a typical PMP, localizing only to the high-density, catalase-containing peroxisomal fractions (rupture of peroxisomes during homogenization and centrifugation leads to the presence of peroxisomal matrix proteins at the top of the gradient, demonstrated here by the presence of catalase activity in the low density fractions).

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