|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1998 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1997 : ¥1,700,000 (Direct Cost : ¥1,700,000)
ATP-binding cassette (ABC) transporters are a superfamily of membrane-bound proteins whose structure and function have been highly conserved from eubacteria to mammals, and catalyze the ATP-dependent transmembrane translocation of a wide variety of substrates, including anti -tumor drugs, amino acids, peptides, proteins, sugars, oligosaccharides, and several kind of lipids. To date, four peroxisomal ABC transporters have been identified in mammalian peroxisomes ; 70-kDa peroxisomal membrane protein (PMP70), adrenoleukodystrophy protein (ALDP), ALOP related protein (ALDRP) and PM P70 related protein (P70R). PMP70 is one of the major components of peroxisomal membranes and is markedly induced by administration of hypolipidemic agents in parallel with peroxisome proliferation and induction of peroxisomal fatty acid beta-oxidation enzymes.
To characterize the role of PMP70 in biogenesis and function of peroxisomes, we transfected the cDNA of rat PMP70 into CHO cells and established cell lin
es stably expressing PM P70. The content of PM P70 in the transfectants increased about 5 fold when compared with the control cells. Subcellular fractionation study showed that over expressed PMP70 was enriched in peroxisomes. This peroxisomal localization was confirmed by Immuno fluorescence and immuno electron microscopy. The number of immuno - gold particles on peroxisomes increased markedly in the transfectants, but the size and the number of peroxisomes were essentially the same in both the transfectants and the control cells. beta-Oxidation of palmitic acid increased about 2-3 fold in the transfectants, whereas the oxidation of lignoceric acid decreased about 30-40%. When intact peroxisomes preparedfrom both the cell lines were incubated with palmitoyl-CoA, oxidation was stimulated with ATP, but the degree of the stimulation was higher in the transfectants than in the control cells. Further, we established three CHO cell lines stably expressing mutant PM P70 (missense mutants in EAA- like or Walker A motifs, and a deletion mutant in C-terminal segment including a part of Walker B motif). ln these cells, 13-oxidation of palmitic acid decreased markedly. These results suggest that PM P70 is involved in metabolic transport of long-chain acyl-CoA across peroxisomal membranes and increase of PM P70 is not associated with proliferation of peroxisomes. Less