A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3*
A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3*
复制标题
DOI：
10.1074/jbc.m114.588566
复制
发表时间：
2014-12
期刊：
影响因子：
--
通讯作者：
Marianne Kluth;J. Stindt;C. Dröge;Doris Linnemann;R. Kubitz;L. Schmitt
中科院分区：
文献类型：
--
作者：
Marianne Kluth;J. Stindt;C. Dröge;Doris Linnemann;R. Kubitz;L. Schmitt
背景：MDR3延长的X环突变导致遗传性肝胆汁淤积症。结果：野生型MDR3有PC诱导的ATPase活性，而Q1174E突变体无刺激作用。结论：ABC信号基序之前的谷氨酰胺将TMD内的底物结合传递到NBD的扩展X环。意义：这项研究为ATP水解酶与底物转运之间的传递接口提供了证据。人多药耐药蛋白3(MDR3/ABCB4)属于广泛存在的三磷酸腺苷结合盒(ABC)转运蛋白家族，位于肝细胞的小管膜上。在那里，它使磷脂酰胆碱(PC)家族的磷脂从内到外的小叶。在这里，我们报告了野生型MDR3和Q1174E突变的特征，这是以前在一例进行性家族性肝内胆汁淤积症3型(PFIC-3)患者中发现的。我们在巴斯德毕赤酵母中表达了MDR3的不同变异体，通过串联亲和层析纯化了这些蛋白，并测定了在磷脂存在或不存在的情况下MDR3特异的ATPase活性。肝PC或1，2-dioleoyl-sn-glycero-3-phosphatidylethanolamine脂对野生型MDR3的ATPase活性有2倍的刺激作用。此外，MDR3与硫醇反应的荧光团的交联阻止了ATP的水解，并且没有表现出PC刺激。类似地，磷脂酰乙醇胺、磷脂酰丝氨酸和鞘磷脂不能诱导野生型MDR3 ATPase活性的增加。磷酸类似物氟化铍和氟化铝完全抑制MDR3的ATPase活性，而原钒酸则完全抑制PC刺激的MDR3的ATPase活性。Q1174E突变位于ABC特征基序亮氨酸附近的核苷酸结合域，并延伸了在ABC出口子中发现的X环。我们对Q1174E突变体的数据显示了基础的ATPase活性，但PC脂类不能刺激ATPase活性，突出了扩展的X环在核苷酸结合域和跨膜域的串扰中的作用。
Background: A mutation of the extended X loop of MDR3 caused hereditary liver cholestasis. Results: Wild type MDR3 exhibited PC-induced ATPase activity, but the Q1174E mutant displayed no stimulation. Conclusion: The glutamine preceding the ABC signature motif communicates substrate binding within the TMD to the extended X loop of the NBD. Significance: This study provides evidence for a transmission interface coupling ATP hydrolysis to substrate transport. The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain.