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.
Highlights
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
When green fluorescent protein (GFP) attached with peroxisomal targeting signal-1 (PTS-1) was microinjected into the mutant cells together with recombinant Pex5p, the fluorescence gathered to particulate structures, being colocalized with PMP70, even in the presence of cycloheximide, a protein synthesis inhibitor
We observed that the above concentration of cycloheximide suppressed the incorporation of radioactive methionine into trichloroacetic acid-precipitable materials by 98% during 5 h of incubation, in both wild type Chinese hamster ovary (CHO) and ZP102. These findings indicate that cycloheximide of the concentration used in the present study was fully effective in inhibiting de novo protein synthesis, and most importantly, de novo formation of peroxisomes or ghosts
Summary
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. We employed a CHO cell mutant [14] deficient in PEX5, a gene encoding the peroxisomal targeting signal-1 (PTS-1) receptor [15,16,17]. When green fluorescent protein (GFP) attached with PTS-1 was microinjected into the mutant cells together with recombinant Pex5p, the fluorescence gathered to particulate structures, being colocalized with PMP70, even in the presence of cycloheximide, a protein synthesis inhibitor.
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