The CG9 cell line lacks detectable peroxisomes. Human fibroblasts from representatives of nine different complementation groups of the PBDs were processed for indirect immunofluorescence using rabbit antibodies specific for PMP70, and Texas red–labeled goat anti–rabbit IgG secondary antibodies. Numerous peroxisomal structures were detected in (A) the PEX1-deficient CG1 cell line, PBD009; (B) the PEX5-deficient CG2 cell line, PBD005; (C) the PEX12-deficient CG3 cell line, PBD097; (D) the PEX6-deficient CG4 cell line, PBD105; (E) the PEX10-deficient CG7 cell line, PBD100; (F) the CG8 cell line, PBD109; (G) the PEX2-deficient CG10 cell line, PBD094; and (H) the CG13 cell line, PBD222. In contrast, (I) the CG9 cell line, PBD061, lacks detectable peroxisomes. Bar, 10 μm.

The PMP import defect of CG9 cells applies to numerous PMPs. The CG9 cell line, PBD061 (A–C); and the CG10 cell line, PBD094 (D–F), were transfected with vectors designed to express PMP70 myc (A and D), PEX12myc (B and E), and PMP32myc (C and F). 2 d after transfection the cells were processed for indirect immunofluorescence using antibodies specific for the myc tag. The peroxisomal nature of the punctate structures in PBD094 cells was confirmed by double label experiments with peroxisomal membrane markers (data not shown). Bar, 10 μm.

Different fates for different PMPs in PBD061 cells. Total cellular protein was extracted from the CG9 cell line, PBD061, and the CG10 cell line, PBD094. Equal amounts of each lysate (by protein) were separated by SDS-PAGE, and blotted with antibodies specific for the PMPs PMP70 (A), P70R (B); and PEX14 (C). In addition, total cellular protein was extracted from PBD061 and PBD094 cells that had been transfected with the PEX12myc expression vector. Equal amounts of each lysate (by protein) were separated by SDS-PAGE, and blotted with antibodies specific for the myc epotope tag (D).

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.

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.

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).

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.

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.

Rescue of peroxisome synthesis in PBD061 cells occurs in a multistep process. PBD061 cells were injected with pcDNA3- PEX16 and processed for indirect immunofluorescence 3 h later using rabbit antibodies specific for P70R (A); and sheep antibodies specific for catalase (B); followed by fluorescein-labeled goat anti– rabbit and Texas red–labeled goat anti–sheep secondary antibodies. Note that many of the peroxisomes appeared to be undergoing elongation, the first step in PEX11β-mediated peroxisome proliferation (Schrader et al., 1998). Cells processed for 22 h (C and D) and 72 h (E and F) after introduction of pcDNA3- PEX16 were also processed for indirect immunofluorescence using rabbit antibodies specific for P70R (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. (G and H) PBD061 cells were preincubated for 30 min with 10 μg/ml BFA, injected with pcDNA3-PEX16, and incubated in 10 μg/ml BFA for 30 h. These cells were processed for immunofluorescence using rabbit antibodies specific for P70R (G); and sheep antibodies specific for catalase (H); followed by fluorescein-labeled goat anti– rabbit and Texas red–labeled goat anti–sheep secondary antibodies. Bar, 10 μm.

Import of PEX16myc can precede P70R import in peroxisomes of rescued PBD061 cells. PBD061 cells were injected with pcDNA3-PEX16myc and processed for indirect immunofluorescence 3 h later using mouse mAbs specific for the myc epitope tag (A and C) and rabbit antibodies specific for P70R (B and D), followed by fluorescein-labeled goat anti– mouse and Texas red–labeled goat anti–sheep secondary antibodies. Bar, 10 μm.

A model of peroxisome biogenesis in the absence (top) and presence (bottom) of preexisting peroxisomes. During rescue of PBD061 cells, PEX16 creates nascent peroxisomes, possibly from a preperoxisomal vesicle. These nascent PEX16-containing peroxisomes then import additional PMPs. The import of PEX11 proteins allows these structures to proliferate by fission or by budding, and the import of other peroxins leads to formation of a functional matrix protein import apparatus. Under normal conditions, peroxisomes would form primarily from preexisting peroxisomes in a PEX11-mediated process. Although the targeting of PEX16 to preperoxisomal structures may be enhanced in the absence of peroxisomal membranes, such a process may also occur under normal conditions.