Structure and function of peroxisomal ABC transporters and regulation of cellular lipid metabolism

atp结合盒（ABC）转运蛋白是一个膜结合蛋白的超家族，其结构和功能从真细菌到哺乳动物一直高度保守，并催化多种底物的atp依赖跨膜易位，包括抗肿瘤药物、氨基酸、肽、蛋白质、糖、低聚糖和几种脂类。迄今为止，在哺乳动物过氧化物酶体中已鉴定出四种过氧化物酶体ABC转运蛋白；70-kDa过氧化物酶体膜蛋白（PMP70）、肾上腺白质营养不良蛋白（ALDP）、ALOP相关蛋白（ALDRP）和PMP70相关蛋白（P70R）。PMP70是过氧化物酶体膜的主要成分之一，在过氧化物酶体增殖和诱导过氧化物酶体脂肪酸β -氧化酶的同时，可通过施用降血脂药物显著诱导。为了研究PMP70在过氧化物酶体生物发生和功能中的作用，我们将大鼠PMP70 cDNA转染CHO细胞，建立了稳定表达PMP70的细胞株。与对照细胞相比，转基因细胞中PM P70的含量增加了约5倍。亚细胞分离研究表明，过表达的PMP70在过氧化物酶体中富集。免疫荧光和免疫电镜证实了这种过氧化物酶体的定位。在转染细胞中，过氧化物酶体上的免疫金颗粒数量明显增加，但在转染细胞和对照细胞中，过氧化物酶体的大小和数量基本相同。棕榈酸的β -氧化在转基因中增加了约2-3倍，而木质素酸的氧化则减少了约30-40%。当从两种细胞系制备的完整过氧化物酶体与棕榈酰辅酶a孵育时，ATP刺激氧化，但转染细胞的刺激程度高于对照细胞。此外，我们建立了三个稳定表达突变体PM P70的CHO细胞系（EAA-样或Walker A基序错义突变体，以及c端包含部分Walker B基序的缺失突变体）。在这些细胞中，棕榈酸的13-氧化明显降低。这些结果表明，PM P70参与了长链酰基辅酶a跨过氧化物酶体膜的代谢运输，PM P70的增加与过氧化物酶体的增殖无关。少

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 … More 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