Abstract
The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.
Highlights
ATP-binding cassette (ABC) transporters constitute one of the largest families of membrane transport proteins present in all three kingdoms of life
Except for the transport of different cyclic nucleotides, glucuronide and glutathione conjugates through MRPs (MRP 1–6, note that MRP1 is detected only in fetal hepatocytes) [8] and the transport of endo- and xenobiotics by MDR1 (P-gp) [9] and ABCG2 [10], one of the main function of ABC transporters in the liver is the formation of bile depending on the ABC transporters bile salt export pump (BSEP) (ABCB11), multidrug resistance protein 3 (MDR3) (ABCB4) and ABCG5/8 [11]
Chloupkovaet al. tested 25 human ABC transporters for expression in P. pastoris [34], but BSEP and MDR3 were not included in this study, while for example MRP2, another human liver ABC transporter, could not be expressed
Summary
ATP-binding cassette (ABC) transporters constitute one of the largest families of membrane transport proteins present in all three kingdoms of life. The bile salt export pump (BSEP) is the main bile salt transporter in humans and is localized in the apical membrane of hepatocytes [14] It is a 1321 amino acid large, glycosylated full-size ABC transporter and mediates the ATP-dependent bile flow by transporting monovalent bile salts like taurine and glycine conjugates of primary and secondary bile salts (e.g. tauro- and glycocholate or taurodeoxycholate) into the canaliculus [15]. New successful forms of therapy include treatment with chemical chaperones like 4-phenylbutyrate for misfolded BSEP mutants [27] Because of their high clinical interest, MDR3 and especially BSEP have been characterized extensively in cell culture as well as animal models [22,28,29,30,31,32]. The purified protein could be isolated in a functional state as judged by substrateinduced ATPase activity of MDR3 and ATP binding in the case of BSEP
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