Fairweather SJ , Bröer A , O'Mara ML , Bröer S .

	Figure 1. Isolation of intestinal brush-border protein complexesMurine intestinal BBMVs were prepared using MgCl2 precipitation and centrifugation. (A) BBMVs (50 μg/sample) at 1 mg/ml were solubilized in detergent buffer as indicated and mixed with Coomassie Brilliant Blue G-250 loading dye before separation. Samples were separated by Blue native PAGE. Following semi-dry transfer on to a PVDF membrane, individual proteins were detected by immunoblot analysis. The blot was stripped and re-probed for the proteins indicated, to allow a direct comparison of band positions. Protein complexes and individual proteins are numbered 1–7 (discussed in the main text). (B) Murine BBMVs were solubilized in co-immunoprecipitation buffer, followed by incubation with Protein A and primary antibodies against either B0AT1 or APN. After overnight incubation of BBMV samples, the cleared lysate and intestinal homogenate were separated by SDS/PAGE. Following semi-dry transfer on to a nitrocellulose membrane, the blots were pre-stripped using stripping buffer and blocked overnight to reduce background signal. Individual proteins were then detected as indicated above the blot and visualized using immunoblot analysis. Molecular masses are indicated to the left-hand side in kDa.
	Figure 2. Co-localization of B0AT1 and APNHEK-293 cells in eight-well microscope slide dishes were transfected with plasmid DNA (constructs used are indicated on the left-hand side) when cells had achieved >90% confluency. The eGFP fluorescence was visualized with a Leica SP5 confocal system and processed with LAS AF software. Indicated above the panels is whether or not cells were co-transfected with the pcDNA mammalian expression vector encoding the trafficking protein collectrin. The white scale bars indicate 25 μm. All images were taken at ×63 magnification.
	Figure 3. Effect of APN on B0AT1 transport activityOocytes were injected with 10 ng of B0AT1 cRNA, 15 ng of APN cRNA or 10 ng of ACE2 cRNA. (A) Uptake of 100 μM [14C]leucine was measured over 30 min on day 5 post-injection. Oocyte endogenous [14C]leucine uptake has been subtracted from all conditions. Each bar represents the means±S.D. (n=8–10, e=3). Note that there is a break in the ordinate axis between 50 and 150 pmol/30 min per oocyte. (B) Oocytes were voltage-clamped at −50 mV and subsequently superfused with 10 mM leucine or Leu-Ser-Lys-Leu tetrapeptide. Substrate-induced Na+ currents were recorded on day 5 post-injection for all oocytes, except for those co-injected with B0AT1 and collectrin, which were recorded on day 3 or 4 post-injection. Each tracing is a typical example of currents observed in all oocytes injected with the cRNA indicated. (C) Oocyte substrate-induced Na+ currents were recorded as described in (B) and superfused with 10 mM leucine. (D) Concurrent uptake and electrophysiological experiments were conducted under the same conditions as in (A) and (B), with uptake experiments using 100 μM [14C]leucine and electrophysiology 10 mM unlabelled leucine. Uptake and currents under control conditions was set to 1. Each data point represents the mean±S.D. (n=6–10, e=3).
	Figure 4. Changes in B0AT1 transporter kinetics by APN and ACE2Oocytes were injected with combinations of the following amounts of cRNA: B0AT1, 10 ng; APN, 15 ng; ACE2, 10 ng; and collectrin, 2 ng. Leucine-induced Na+ currents were recorded on day 5 post-injection, except those co-injected with B0AT1 and ACE2, which were recorded on day 3 or 4 post-injection. (A) Oocytes were voltage-clamped at −50 mV and subsequently superfused with serial concentrations of leucine (0.1–10 mM) in descending and then ascending order. B0AT1-expressing oocytes were not superfused with 0.1 mM leucine due to a low signal to noise ratio elicited at this concentration. An Eadie–Hofstee linear regression plot of the data points is shown. Each data point represents the mean±S.D. (n=6–8, e=3). (B) Uptake of 100 μM [14C]leucine was measured over 15 min on days 1–6 post-injection. Oocyte endogenous [14C]leucine uptake has been subtracted. Each data point represents the mean±S.D. (n=8–12, e=3). (C) A total of 15 oocytes per sample were incubated in 0.5 mg/ml sulfo-NHS-LC-biotin on day 5 post-injection before being lysed and treated with streptavadin-coated agarose beads. Samples were separated by SDS/PAGE, blotted, detected and visualized for B0AT1 using immunoblot analysis. (D) A total of 15 oocytes per sample were prepared as in (C). Following SDS/PAGE, proteins were detected and visualized using immunoblot analysis for APN. Membranes were prepared for detection of subsequent proteins by stripping, re-blocking and re-probing of the protein indicated. Molecular masses are indicated to the left-hand side in kDa. (E) Oocytes were voltage-clamped at −50 mV and subsequently superfused with 20 mM of the amino acid (AA) indicated. All substrate-induced currents were normalized to a 20 mM leucine current (ILeu) to account for transporter de-sensitization. Each bar represents the mean±S.D. (n=6–8, e=3).
	Figure 5. Mechanism of APN-mediated increase in substrate affinity of B0AT1Oocytes were injected with 10 ng of B0AT1 cRNA, 15 ng of APN cRNA and 2 ng of collectrin cRNA. (A) Oocytes were voltage clamped at −50 mV and subsequently superfused with either 200 μM leucine or 1 mM Leu-Ser-Lys-Leu tetrapeptide. Leucine-induced sodium currents were recorded 4–6 days post-injection. All Na+ currents were normalized to the sodium currents elicited by 200 μM leucine (ILeu) at 100%. Each bar represents the mean±S.D. (n=6–8, e=3). (B) Oocytes were recorded as indicated in (A) and superfused with either 200 μM leucine or 1 mM leucine tripeptide. All Na+ currents were normalized to the Na+ currents elicited by 200 μM leucine (ILeu) at 100%. Each bar represents the mean±S.D. (n=18).
	Figure 6. Mechanism of APN-mediated increase in substrate affinity of B0AT1Oocytes were injected with either 10 ng of B0AT1 cRNA or 15 ng of APN cRNA. Uptake of [14C]leucine was measured over 30 min on day 5 post-injection. Bestatin (70 μM) was pre-incubated for 1 min before leucine was added. Oocyte endogenous leucine transport has been subtracted in all experimental conditions. Uptake was normalized to that measured in oocytes expressing B0AT1 alone in the presence of 100 μM leucine. Each bar represents the mean±S.E.M. of data combined from four experiments (n=33–38).
	Figure 7. Homology model of mouse APNThe homology model of mouse APN was generated using HHpred Homology detection and structure prediction tool (Max-Planck Institute for Developmental Biology; http://toolkit.tuebingen.mpg.de/hhpred) and visualized using PyMOL v9.9 (DeLano Scientific; http://www.pymol.org). Depicted are E. coli LAP (PDB code 3B34) (A) and murine APN (B) with phenylalanine bound to their active site. The top panels display a lateral three-dimensional view of the murine and E. coli structures. Domains are shaded as follows: magenta (domain I for E. coli, domain IV for mouse); green (domain II for E. coli, domain V for mouse); cyan (domain III for E. coli, domain VI for mouse); and yellow (domain IV for E. coli, domain VII for mouse). Domains I–III from mouse APN are not depicted (residues 1 to 89) as no homologous sequence exists in E. coli for these. The HEXXH……….BXLXE and GXMEN consensus sequences for zinc and amino acid binding are shaded red and orange respectively. The zinc ion is shaded blue-grey. The active sites of E. coli LAP and murine APN are depicted in panels (C) and (D) respectively. The zinc ion is shaded blue-grey, and the bound phenylalanine is depicted as a space-filled model, with oxygen and nitrogen atoms coloured red and blue respectively. All residues predicted to bind and interact with the substrate are shown and labelled.
	Figure 8. Sequence alignments of aminopeptidase family membersPotential homologues of mouse APN (P97449) were identified by a NCBI blastp search. Peptide sequences were aligned using COBALT (http://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?CMD=Web). Highly conserved regions are boxed. In the consensus sequence row, X is any amino acid and B is bulky side chain neutral amino acids. The full-length aligned sequence from E. coli LAP (not shown) was used as the basis for the homology modelling of mouse APN. The three conserved residues in the binding domains of murine APN selected for site-directed mutagenesis are indicated with a vertical line above them, a fourth mutated residue, Tyr476, is not shown. AAP1, alanine/arginine aminopeptidase 1; LTA4, leukotriene A-4 hydrolase.
	Figure 9. Mechanism of APN-mediated increase in substrate affinity of B0AT1Oocytes were injected with either 10 ng of B0AT1 cRNA, 15 ng of APN cRNA or 2 ng of collectrin cRNA. (A) Oocytes were voltage clamped at −50 mV and subsequently superfused with either 10 mM leucine or 10 mM Leu-Ser-Lys-Leu tetrapeptide. Each tracing is a typical example of currents observed in all oocytes injected with the cRNA indicated. Leucine-induced sodium currents were recorded on day 4 and 5 post-injection for all oocytes. (B) Oocytes were recorded as indicated in (A). Each data point indicates an APN mutant co-expressed with B0AT1, or B0AT1 co-expressed with collectrin. All peptide-induced sodium currents were normalized to the corresponding leucine-induced Na+ current (IPep/ILeu). Each data point represents the mean±S.D. for both peptide-induced sodium currents and apparent Km values. The trend line was fitted using linear regression (r=−0.84, P=0.035 and n=74). (C) A total of 15 oocytes/sample were incubated in 0.5 mg/ml sulfo-NHS-LC-biotin on day 5 post-injection before being lysed and treated with streptavadin-coated agarose beads. Samples were separated by SDS/PAGE. Subsequently B0AT1 was detected by immunoblotting. Molecular masses are indicated to the left-hand side in kDa.

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