Peroxisomes Are Formed from Complex Membrane Structures inPEX6-deficient CHO Cells upon Genetic Complementation

Abstract

Pex6p belongs to the AAA family of ATPases. Its CHO mutant, ZP92, lacks normal peroxisomes but contains peroxisomal membrane remnants, so called peroxisomal ghosts, which are detected with anti-70-kDa peroxisomal membrane protein (PMP70) antibody. No peroxisomal matrix proteins were detected inside the ghosts, but exogenously expressed green fluorescent protein (GFP) fused to peroxisome targeting signal-1 (PTS-1) accumulated in the areas adjacent to the ghosts. Electron microscopic examination revealed that PMP70-positive ghosts in ZP92 were complex membrane structures, rather than peroxisomes with reduced matrix protein import ability. In a typical case, a set of one central spherical body and two layers of double-membraned loops were observed, with endoplasmic reticulum present alongside the outer loop. In the early stage of complementation by PEX6 cDNA, catalase and acyl-CoA oxidase accumulated in the lumen of the double-membraned loops. Biochemical analysis revealed that almost all the peroxisomal ghosts were converted into peroxisomes upon complementation. Our results indicate that 1) Peroxisomal ghosts are complex membrane structures; and 2) The complex membrane structures become import competent and are converted into peroxisomes upon complementation with PEX6.

Kidney-specific Overexpression of Sirt1 Protects against Acute Kidney Injury by Retaining Peroxisome Function

“…The sections were incubated overnight in the 1/1000 diluted primary antibody, an anti-PMP70 antibody. After being rinsed with phosphate-buffered saline, the sections were incubated for 30 min on a drop of protein A-gold (15 nm) prepared as described previously (21). The sections were briefly contrasted with uranyl acetate and lead citrate before examination.…”

Deficiency of a Lipid Droplet Protein, Perilipin 5, Suppresses Myocardial Lipid Accumulation, Thereby Preventing Type 1 Diabetes-Induced Heart Malfunction

“…For preembedding immunoelectron microscopy, the heart was perfused and fixed with 4% formaldehyde-0.025% glutaraldehyde in PBS, and 6-m-thick frozen sections were prepared. Sections were blocked and permeabilized with 1% BSA-1% saponin in PB for 1 h. Sections were incubated with a primary antibody against Plin2 (ab108323; Abcam) overnight at 4°C, washed with 0.005% saponin in PB, and incubated with a secondary antibody, Alexa Fluor 594 FluoroNanogold-conjugated anti-rabbit IgG (Nanoprobes), for 2 h. After samples were washed, sections were fixed with 1% glutaraldehyde in PB for 10 min, and silver enhancement was performed as described previously (30). Samples were then postfixed with 1% reduced osmium tetroxide, and ultrathin sections were prepared, followed by staining with 4% uranyl acetate and lead stain solution (Sigma).…”

Identification of a new complementation group of the peroxisome biogenesis disorders andPEX14 as the mutated gene

“…pGFPSKL has been described previously [Hashiguchi et al, 2002] and pPTS2-GFP, which encodes GFP, with N-terminal 32 amino acid residues of PT as peroxisome targeting signal 2. pECFPNLS was made by insertion of oligonucleotides (5 0 -CCCCCCAAGAAGAAGCGCAAGGTGCG-3 0 and 5 0 -AGCTC-CCCCCAAGAAGAAGCGCAAGGTGCGAATT-3 0 ) encoding the nuclear localization signal of SV40 T antigen into the SacI/ EcoRI sites of pECFP-C1 (Clontech, www.clontech.com). Fulllength HsPEX14 cDNA expression plasmid, named pUcD2-PEX14, was constructed into pUcD2SRalphaMCS [Tsukamoto et al, 1995] with the two partial human cDNA clones (I.M.A.G.E.…”