Abstract
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.
Highlights
A mutation of the extended X loop of MDR3 caused hereditary liver cholestasis
Expression and Purification of the Human ATP-binding cassette (ABC) Transporter MDR3 in P. pastoris—Previously, we described the expression of wild type MDR3 and the ATP hydrolysis-deficient mutant (E558Q/E1207Q, later called the EQ/EQ mutant) in the methylotrophic yeast P. pastoris
The purity of wild type MDR3, EQ/EQ, and Q1174E mutants was analyzed by Coomassie Brilliant Blue-stained SDS gels, and MDR3 was identified by immunoblot analysis using the C219 antibody, which is specific for the human ABC transporters MDR1 and MDR3 (Fig. 1A, left and middle panels)
Summary
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. 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. The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the family of ATP-binding cassette (ABC) transporters and is highly expressed in the canalicular membrane of hepatocytes. We analyzed the ATPase activity of the MDR3 Q1174E mutant in vitro, which was identified in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3) and described previously in Kubitz et al [19] This mutation is located in the extended X loop of NBD2 (TRVGDKGTQ). We suggest that glutamine 1174 is involved in the cross-talk of NBD and TMD
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have