Okamoto Takumi, Kawaguchi Kosuke, Watanabe Shiro, Agustina Rina, Ikejima Toshiki, Ikeda Keisuke, Nakano Minoru, Morita Masashi, Imanaka Tsuneo
Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.
Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.
Biochem Biophys Res Commun. 2018 Feb 19;496(4):1122-1127. doi: 10.1016/j.bbrc.2018.01.153. Epub 2018 Feb 3.
In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1‒3 are located on peroxisomal membrane and play an important role in the transportation of various fatty acid-CoA derivatives, including very long chain fatty acid-CoA, into peroxisomes. ABCD4 is located on lysosomal membrane and is suggested to be involved in the transport of vitamin B from lysosomes to the cytosol. However, the precise transport mechanism by which these ABC transporters facilitate the import or export of substrate has yet to be well elucidated. In this study, the overexpression of human ABCD1‒4 in the methylotrophic yeast Pichia pastoris and a purification procedure were developed. The detergent-solubilized proteins were reconstituted into liposomes. ABCD1‒4 displayed stable ATPase activity, which was inhibited by AlF. Furthermore, ABCD1‒4 were found to possess an equal levels of acyl-CoA thioesterase activity. Proteoliposomes is expected to be an aid in the further biochemical characterization of ABCD transporters.
在哺乳动物中，已鉴定出属于D亚家族的四种ATP结合盒（ABC）蛋白。ABCD1-3位于过氧化物酶体膜上，在包括极长链脂肪酸辅酶A在内的各种脂肪酸辅酶A衍生物转运到过氧化物酶体的过程中发挥重要作用。ABCD4位于溶酶体膜上，据推测参与维生素B从溶酶体到细胞质的转运。然而，这些ABC转运蛋白促进底物进出的确切转运机制尚未得到充分阐明。在本研究中，开发了在甲基营养型酵母毕赤酵母中过表达人ABCD1-4的方法以及纯化程序。将去污剂溶解的蛋白重构成脂质体。ABCD1-4表现出稳定的ATP酶活性，该活性受到AlF的抑制。此外，发现ABCD1-4具有同等水平的酰基辅酶A硫酯酶活性。蛋白脂质体有望有助于对ABCD转运蛋白进行进一步的生化特性分析。
