Characterization of the 70-kDa peroxisomal membrane protein, an ATP binding cassette transporter.
The 70-kDa peroxisomal membrane protein (PMP70) is one of the major components of rat liver peroxisomal membranes and belongs to a superfamily of proteins known as ATP binding cassette transporters. PMP70 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 Chinese hamster ovary cells and established cell lines stably expressing PMP70. The content of PMP70 in the transfectants increased about 5-fold when compared with the control cells. A subcellular fractionation study showed that overexpressed PMP70 was enriched in peroxisomes. This peroxisomal localization was confirmed by immunofluorescence and immunoelectron microscopy. The number of immuno-gold particles corresponding to PMP70 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 prepared from 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. Furthermore, we established three Chinese hamster ovary cell lines stably expressing mutant PMP70. In these cells, beta-oxidation of palmitic acid decreased markedly. These results suggest that PMP70 is involved in metabolic transport of long chain acyl-CoA across peroxisomal membranes and that increase of PMP70 is not associated with proliferation of peroxisomes.
Imanaka
T
T
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. imanaka@ms.toyama-mpu.ac.jp
Aihara
K
K
Takano
T
T
Yamashita
A
A
Sato
R
R
Suzuki
Y
Y
Yokota
S
S
Osumi
T
T
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008830:Microbodies / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D019308:Palmitic Acid / Q000378:metabolism; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; C489272:Abcd3 protein, rat; D017931:DNA Primers; D008565:Membrane Proteins; D019308:Palmitic Acid