Pieri M , Gan C , Bailey P , Meredith D .

082051/Z/07/Z Wellcome Trust

	Fig. 1. (A) Surface expression quantification of all TMD tyrosine to phenylalanine mutants using luminometry against the FLAG epitope incorporated in each mutant. Wild type rPepT1-FLAG (wt-PepT1) surface expression was used as a positive control, and non-injected oocyte values were subtracted from all data as a negative control. Results are normalised to wild-type and expressed as mean ± SEM, n = 3 separate experiments, >20 oocytes per experiment. (B) Uptake of 0.4 μM [3H]-d-Phe-l-Gln into Xenopus oocytes expressing wild-type rPepT1 (wt-Pept1) or the individual tyrosine mutants. Non-injected control oocyte values have been subtracted from the data. Each data point is expressed as the mean ± SEM, *p < 0.05, n ≥ 3 separate experiments. The wild-type rPepT1 uptake corresponds to 342 ± 37 fmol peptide/(oocyte h). (C) Uptake of 0.4 μM [3H]-d-Phe-l-Gln into Xenopus oocytes expressing wild-type rPepT1 (wt-PepT1) or individual tyrosine mutants corrected for surface expression. Non-injected control oocyte values have been subtracted from the data. Note that adjustment has been made to normalize uptake to the level of protein expression for each mutant in order to express the functional effect of each mutation. Each data point is the mean ± SEM, *p < 0.05, n ≥ 3 separate experiments.
	Fig. 2. Comparison of pH dependence of [3H]-d-Phe-l-Gln uptake by the wild-type rPepT1 (wt) and individual tyrosine mutants. Uptake of [3H]-d-Phe-l-Gln by oocytes was measured at extracellular pH 5.5 and pH 7.4 after one hour incubation. Values represent mean ± SEM for three separate oocyte preparations and are normalized to the uptake at pHout 5.5 for each construct, *p < 0.01, n = 3, Student's t-test compared to pHout 7.4.
	Fig. 3. Graphs show the affinity constants (Ki, mM) for the Y12F-, Y56F-, Y92F- and Y345F-rPepT1 mutants for both pHout 5.5 (A) and 7.4 (B). The uptake of 0.4 μM [3H]-d-Phe-l-Gln into oocytes was measured in the presence of increasing concentrations of the non-radioactive (cold) Gly-l-Gln dipeptide. Each Ki was calculated from the best fit of the data to Michaelis–Menten kinetics for binding to a single site (Deves and Boyd, 1989), *p < 0.05, n = 3, Student's t-test compared to wild-type.
	Fig. 4. (A) Chemical structures of Phe-Tyr-NH2 and its parent compound Phe-Tyr. (B and C) Ki determination of carboxyl-terminal amidated Phe-Tyr-NH2 and the parent compound Phe-Tyr by wild-type-PepT1 (B) and Y56F-rPepT1 mutant (C) at pHout 5.5. Uptake of 0.4 μM [3H]-d-Phe-l-Gln into oocytes was measured in the presence of increasing concentrations of Phe-Tyr or Phe-Tyr-NH2 at pHout 5.5. Lines represent best fit of the data to Michaelis–Menten kinetics for binding to a single site (Deves and Boyd, 1989). Each data point is the mean ± SEM of 5 oocytes, and the graphs are representative of n = 4 oocyte preparations.
	Fig. 5. (A) Uptake of [3H]-d-Phe-l-Gln in Xenopus oocytes expressing wild-type rPepT1 (wt-PepT1), the single T58F or the double Y56F/T58Y-rPepT1 mutants. *p < 0.01 versus wild-type uptake at pHout 5.5 (black bars) and #p < 0.01 versus wild-type uptake at pHout 7.4 (grey bars), Student's t-test, n = 3. (B) pH dependence of [3H]-d-Phe-l-Gln uptake normalised to the uptake of pHout 5.5 for each construct. pH dependence of uptake is seen only in the wild-type rPepT1 (*p < 0.01, Student's t-test, n = 3).
	Fig. 6. (A) Uptake of [3H]-d-Phe-l-Gln in Xenopus oocytes expressing wild-type rPepT1 (wt-PepT1), Y91F-, Y91Q- or Y91R-PepT1. *p < 0.05 versus wild-type uptake at pHout 5.5 (black bars) and #p < 0.05 versus wild-type uptake at pHout 7.4 (grey bars), Student's t-test, n = 3). (B) pH dependence of [3H]-d-Phe-l-Gln uptake normalised to the uptake of pHout 5.5 for each construct. pH dependence of uptake is maintained only in the Y91F-rPepT1 mutant (*p < 0.01, Student's t-test, n = 3). (C and D) Ki determination of Gly-l-Gln against 0.4 μM [3H]-d-Phe-l-Gln uptake by wild-type rPepT1 (wt-PepT1) and the Y91F-, Y91Q- and Y91R-rPepT1 mutants at pHout 5.5 (C) and 7.4 (D). Lines represent best fit of the data to Michaelis–Menten kinetics for binding to a single site (Deves and Boyd, 1989). Each data point is the mean ± SEM of 5 oocytes, and the graphs are representative of n ≥ 2 oocyte preparations.
	Fig. 7. (A) Uptake of [3H]-d-Phe-l-Gln in Xenopus oocytes expressing wild-type rPepT1 (wt-PepT1), Y167F-, Y167Q- or Y167R-rPepT1 mutants at pHout 5.5 and 7.4. Uptake is reduced by >90% as compared to the wild-type at both pHout 5.5 (black bars, *p < 0.001, Student's t-test, n > 3) and pHout 7.4 (grey bars, #p < 0.001, Student's t-test, n > 3). (B) pH dependence of [3H]-d-Phe-l-Gln uptake normalised to the uptake of pHout 5.5 for each construct. pH dependence of uptake is maintained in all Y167 mutants (*p < 0.05, Student's t-test, n > 3).
	Fig. 8. Cartoon representation of rPepT1 to show the putative position of the transmembrane tyrosines (adapted from Fei et al., 1994).

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