Formation of Peroxisomes from Peroxisomal Ghosts in a Peroxisome-deficient Mammalian Cell Mutant upon Complementation by Protein Microinjection

Abstract

Most mammalian cell strains genetically deficient in peroxisome biogenesis have abnormal membrane structures called ghosts, containing integral peroxisomal membrane protein, PMP70, but lacking the peroxisomal matrix proteins. Upon genetic complementation, these mutants regain the ability of peroxisome biogenesis. It is postulated that, in this process, the ghosts act as the precursors of peroxisomes, but there has been no evidence to support this. In the present study, we investigated this issue by protein microinjection to a mutant Chinese hamster ovary cell line defective of PEX5, encoding a peroxisome-targeting signal receptor. When recombinant Pex5p and green fluorescent protein (GFP) carrying a peroxisome-targeting signal were co-injected into the mutant cells, the GFP fluorescence gathered over time to particulate structures where PMP70 was co-localized. This process was dependent on both Pex5p and the targeting signal, and, most importantly, occurred even in the presence of cycloheximide, a protein synthesis inhibitor. These findings suggest that the ghosts act as acceptors of matrix proteins in the peroxisome recovery process at least in the PEX5 mutant, and support the view that peroxisomes can grow by incorporating newly synthesized matrix proteins.

The Newly Identified Human Nuclear Protein NXP-2 Possesses Three Distinct Domains, the Nuclear Matrix-binding, RNA-binding, and Coiled-coil Domains

“…Plasmids and Constructs-The GFP-tagged expression vector used in this study was the phGFP(105) series, encoding a GFP mutant that has increased brightness (30). phGFP-NXP-2-(25-939) was obtained by introducing a fragment excised from KIAA0136 with SpeI and DraI into the blunted EcoRI site of the phGFP(105)-C1 vector.…”

Active involvement of micro-lipid droplets and lipid-droplet-associated proteins in hormone-stimulated lipolysis in adipocytes

“…Mouse perilipin cDNA was obtained from adipocyte total RNA by reverse transcription (RT)-PCR. GFP-fusion constructs were prepared using phGFP(105), a GFP mutant that has an increased brightness (Yamasaki et al, 1999). From a lentiviral expression vector, CSIIhMTIIA(DGRE)-MCS-IRES2-Venus (Fumoto et al, 2007), the IRES2-Venus region was removed using appropriate restriction sites, and GFP(105)-coding sequence was inserted into the multicloning site, yielding CSII-MT-GFP.…”

Evaluation of the role of the Endoplasmic Reticulum-Golgi transit in the biogenesis of peroxisomal membrane proteins in wild type and peroxisome biogenesis mutant CHO cells

“…In some experiments, transfected cells were incubated in the presence of 1 μg/ml CHX (Yamasaki et al, 1999), for 6 h after transfection. Then, BFA was added to the medium (10 μg/ml final concentration) (Fujiwara et al, 1988) and the cells were incubated in the presence of both CHX and BFA for 1 h. Finally, cells were further incubated in the presence of BFA (alone) for an additional 6 h. At the end of this treatment, the cells were fixed with 4% paraformaldehyde and analyzed directly under the fluorescence microscope or processed for indirect immunofluorescence.…”