Human BSEP and MDR3 expression in <i>Pichia pastoris.</i>

<p><b>A</b> 5 µg of membranes derived from <i>P. pastoris</i> cells carrying the empty expression plasmid pSGP18 (neg ctrl), BSEP or BSEP-GFP were subjected to SDS-PAGE and immunoblotting (lanes from left to right). The negative control (left lane) did not react with the monoclonal antibody (F-6), while BSEP (middle lane) and BSEP-GFP (right lane) could be detected by the same antibody. <b>B</b> Identical samples were probed with a monoclonal GFP antibody. The negative control (left lane) as well as BSEP (middle lane) showed no signal with anti-GFP antibody, while BSEP-GFP could be detected (right lane). <b>C</b> In case of MDR3 the negative control (left lane) showed no signal with the monoclonal antibody C219; MDR3 (middle lane) as well as MDR3-GFP (right lane) could be detected with the monoclonal antibody C219. <b>D</b> Identical MDR3 samples were probed with a monoclonal GFP antibody. The negative control (left lane) as well as MDR3 (middle lane) showed no signal with anti-GFP antibody, while MDR3-GFP could be detected (right lane). The position of the molecular weight markers are shown on the left.</p

Characterization of purified human MDR3 in Fos-choline-16.

<p><b>A</b> Coomassie Brilliant Blue-stained SDS-PAGE and immunoblot using an anti-MDR3 antibody of purified MDR3 wild-type and the MDR3 EQ/EQ-mutant via TAP. Molecular weight markers are shown on the left. <b>B</b> Normalized ATPase activity of MDR3 wild-type (black) and of an ATPase deficient mutant (E558Q E1207Q, white) in FC-16 without and with different phospholipids. The ATPase activity of three independent MDR3 purifications was determined ± SD (n = 3).</p

Purification and nucleotide binding of human BSEP.

<p><b>A</b> Coomassie Brilliant Blue-stained SDS-PAGE of purified BSEP solubilized in Fos-choline-16 or in β-DDM and Cymal5, which were exchanged after solubilization. Molecular weight markers are indicated on the left. <b>B</b> Purified BSEP in all three detergents was incubated with ATP-agarose and bound protein was eluted in SDS sample buffer and examined with immunoblotting with a monoclonal antibody (F-6). BSEP signals could be detected in β-DDM and Cymal5, but not in Fos-Choline-16, indicating only binding to ATP in maltosides.</p

Solubilization screen of human BSEP and MDR3 using Dot Blot.

<p>Solubilization screen of <i>P. pastoris</i> membranes containing BSEP (<b>A</b>), BSEP-GFP (<b>B</b>) or MDR3 (<b>C</b>) with over 100 different detergents were analysed regarding the solubilization efficiacy. The solubilized protein was spotted onto a nitrocellulose membrane and examined via dot blotting with BSEP or MDR3 specific monoclonal antibodies (F-6 and C219). Compared areas are marked with a red box. All dot blots were performed in duplicate.</p

Fluorescence microscopy of BSEP-GFP and MDR3-GFP expressing <i>Pichia pastoris</i> cells.

<p><i>P. pastoris</i> cells expressing GFP, BSEP-GFP or MDR3-GFP were harvested 48 h after induction and examined for GFP fluorescence (upper row) by confocal LSM. BSEP-GFP as well as MDR-GFP was located in the plasma membrane of <i>P. pastoris</i> cells in contrast to soluble GFP, which was homogenously distributed within the cell. Bottom row: merge of the GFP fluorescence and the Differential Interference Contrast (DIC) scans.</p

Detergent screening utilizing FSEC.

<p>FSEC analysis of BSEP-GFP (<b>A</b>) in five representative detergents and MDR3-GFP in five detergents (<b>B</b>). The arrows indicate the estimated elution position of the void volume and free GFP. Additional FSEC profiles are summarized in the supplementary material.</p

PCR oligonucleotides used in this study.

<p>PCR oligonucleotides used in this study.</p

Functional Impact of a Single Mutation within the Transmembrane Domain of the Multidrug ABC Transporter Pdr5

The pleiotropic drug resistance network in budding yeast presents a first line of defense against xenobiotics, which is formed by primary and secondary active membrane transporters. Among these transporters, the ABC transporter Pdr5 is a key component, because it confers resistance against a broad spectrum of such cytotoxic agents. Furthermore, it represents a model system for homologous transporters from pathogenic fungi and has been intensively studied in the past. In addition to other mutational studies, the S1360F mutation of Pdr5 was found to modulate substrate specificity and resistance. Notably, in the S1360F background, the resistance against the immunosuppressant FK506 is drastically increased. We present a detailed analysis of this mutation that is located in the predicted cytosolic part of transmembrane helix 11. Our data demonstrate that kinetic and thermodynamic parameters of the S1360F mutant are similar to those of the wild-type protein, except for FK506-inhibited ATPase activity and the degree of competitive inhibition. In summary, our results indicate that the S1360F mutation within the transmembrane domain interferes drastically with the ability of the nucleotide-binding domains to hydrolyze ATP by interfering with interdomain crosstalk

Analysis of the Bile Salt Export Pump (<i>ABCB11</i>) Interactome Employing Complementary Approaches

<div><p>The bile salt export pump (BSEP, <i>ABCB11</i>) plays an essential role in the formation of bile. In hepatocytes, BSEP is localized within the apical (canalicular) membrane and a deficiency of canalicular BSEP function is associated with severe forms of cholestasis. Regulation of correct trafficking to the canalicular membrane and of activity is essential to ensure BSEP functionality and thus normal bile flow. However, little is known about the identity of interaction partners regulating function and localization of BSEP. In our study, interaction partners of BSEP were identified in a complementary approach: Firstly, BSEP interaction partners were co-immunoprecipitated from human liver samples and identified by mass spectrometry (MS). Secondly, a membrane yeast two-hybrid (MYTH) assay was used to determine protein interaction partners using a human liver cDNA library. A selection of interaction partners identified both by MYTH and MS were verified by <i>in vitro</i> interaction studies using purified proteins. By these complementary approaches, a set of ten novel BSEP interaction partners was identified. With the exception of radixin, all other interaction partners were integral or membrane-associated proteins including proteins of the early secretory pathway and the bile acyl-CoA synthetase, the second to last, ER-associated enzyme of bile salt synthesis.</p></div

Proteins identified as interaction partners of BSEP in MYTH and co-IP / MS/MS screens.

<p>Proteins identified as interaction partners of BSEP in MYTH and co-IP / MS/MS screens.</p