Vacca F , Gomes AS , Murashita K , Cinquetti R , Roseti C , Barca A , Rønnestad I , Verri T , Bossi E .

247978 Regional Research Fund West project SalmoFeedPlus, 267626 Research Council of Norway projects LeuSense, 262096 GUTASTE, 311627 Gut2Brain 2020

	Figure 2. Phylogenetic relationship of fish and mammalian PepT2 based on predicted protein sequencesThe (unrooted) phylogenetic tree was constructed based on deduced PepT2 amino acid sequences using the maximum likelihood method, 1000 bootstrap replicates, and Jones‐Taylor‐Thornton + G matrix‐based model in MEGA X. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Protein Acc. Nos. for GenBank or Ensembl databases are provided next to species common name. [Colour figure can be viewed at wileyonlinelibrary.com]
	Figure 3. Synteny analysis of slc15a2 genomic region in teleostsFrom top, slc15a2a and slc15a2b genes from Atlantic salmon (Salmo salar), slc15a2 from Northern pike (Esox lucius) and zebrafish (Danio rerio). The chromosome (Chr) number is indicated below each species name. The central pentagons in dark orange indicate the slc15a2 genes. For each slc15a2 gene, 10 flanking genes upstream and downstream are represented by different coloured pentagons. Each colour identifies sets of orthologous genes based on the degree of conservation between species and between the chromosomes within species and white pentagons represent non‐identified genes. The pentagons point in the direction of transcription and only protein‐coding genes are indicated. [Colour figure can be viewed at wileyonlinelibrary.com]
	Figure 4. mRNA tissue distribution of slc15a2a and slc15a2b in head kidney and kidney (A) and head tissues and along the gastrointestinal tract of the Atlantic salmon (B) using quantitative RT‐PCRResults are shown as target slc15a2 copy number per ng of total RNA normalized using β‐actin copy number per ng of total RNA. The line in the boxplot indicates the median and boxes the 1st to 3rd quartiles, whiskers mark variation outside 1st and 3rd quartiles and dots the outliers (n = 8 for all tissues, except for HK, K, GL, OC, AMG, PMG and AHG where n = 7). HK, head kidney; K, kidney; BR, brain; GL, gills; OC, olfactory cavity; TG, tongue; ES, oesophagus; AST, anterior stomach; PST, posterior stomach; PC, pyloric caeca; AMG, anterior midgut; MG, midgut; PMG, posterior midgut; AHG, anterior hindgut; PHG, posterior hindgut. [Colour figure can be viewed at wileyonlinelibrary.com]
	Figure 5. Transport of Gly‐Gln in Xenopus oocytes expressing Atlantic salmon PepT2a (A, B) and PepT2b (C, D)In A and C, representative traces (1 mmol l−1 Gly‐Gln in NaCl solution), the dashed line represents the baseline (conventionally fixed at the value in NaCl solution at pH 7.6). B and D, current/voltage relationships (the values are the means (SD) of the current normalized to the mean value of the current at −140 mV and pH 7.6); insets, currents at pH 6.5 in the presence of 98 mmol l−1 NaCl or tetraethylammonium chloride. Data are means (SD) from 4–24 oocytes from 1–5 batches.
	Figure 6. Box plots of the transport current values recorded as reported in Fig. 5, for PepT2a (left) and PepT2b (right) at −120 mV (A) and at −60 mV (B)Dots indicate the single oocyte transport current value. In the top part of each figure the statistical comparison between different pH conditions for each transporter is shown. In the bottom, the statistical comparison between transporters at the same pH (two‐sample t test or Mann‐Whitney's U test; * P < 0.05, ** P < 0.01 and *** P < 0.001) is shown. Samples for box plots are the same as for Fig. 5. The detailed statistical values are reported in the statistical summary document.
	Figure 7. Current vs. substrate concentration (I/S) relationships at the indicated voltages for Atlantic salmon PepT2a in A, B and C, and for PepT2b in D, E and F The mean value of the currents at each concentration (from 3 μmol l−1 to 3 mmol l−1 for PepT2a and from 3 μmol l−1 to 300 μmol l−1 for PepT2b) are plotted at the indicated voltage and pH. Data are means (SD) from 10–19 oocytes, obtained from 2–3 batches. The full series of the mean values (SD) for each concentration, voltage and pH are reported in the statistical summary document. The raw data are available at the link given in the data availability statement.
	Figure 8. Dose–response analysis: I max, K 0.5 and transport efficiency of Atlantic salmon PepT2a in A, B and C, and PepT2b in D, E and F The current values evaluated in the presence of increasing concentrations of Gly‐Gln under each tested voltage (reported in Fig. 7 as means (SD)) were subsequently fitted with eqn (1) to obtain the relative maximal current (I max) in A and D, the K 0.5 in B and E, i.e. the substrate concentration that elicits half of the maximal current (I max), and the transport efficiency in C and F, evaluated as the ratio I max/K 0.5 under each membrane potential and pH condition. Data are here reported as means (SE) result from Origin fitting. All fit values are reported in the statistical summary document. The raw data are also available at the link indicated in the data availability statement.
	Figure 9. Representative traces of currents elicited by voltage steps protocol applied to Xenopus oocytes expressing the Atlantic salmon PepT2 transporters, PepT2a (top) and PepT2b (bottom)The voltage pulses were in the range −140 to +20 mV. At all tested pH, the presence of saturating concentrations of Gly‐Gln (1 mmol l−1 for PepT2a and 0.3 mmol l‐1 for PepT2b) eliminates the transporter transient component (I PSS) and produces large inwardly directed steady‐state currents that raise increasing the proton chemical gradient and in hyperpolarization.
	Figure 10. The time constant of decay (τ) and normalized displaced charge (Q/Q max) for Atlantic salmon PepT2 transportersPepT2a in A and B and PepT2b in C and D. τ/V in A and C and (Q/Q max)/V in B and D. The sample sizes for τ/V and Q/V for each condition were between 14 and 22 oocytes, from 2–3 batches. The τ/V values are the means (SD). The Q/V values are the means normalized against the maximal moveable charge Q max values obtained by fitting using eqn (3). The unitary charge level was set to the saturation value at positive potential. In B and D, the horizontal arrow intersects each curve at half completion of charge movement corresponding to V 0.5 given by the Boltzmann eqn (3).
	Figure 11. The unidirectional rate constantsGraphical representation of a two‐state system (left). Inward (open symbols) and outward (solid symbols) rate constants of the intramembrane charge movement of Atlantic salmon PepT2 transporters, PepT2a in A, B and C, and PepT2b in D, E and F. The unidirectional rates, plotted as a function of membrane potentials, were calculated from τ/V (and (Q/Q max)/V (in Fig. 10) at the different pH. The rate (1/τ) of the two transporters are plotted as stars. [Colour figure can be viewed at wileyonlinelibrary.com]
	Figure 12. Representation of two kinetic models for PepT2 transporterEach configuration represents a state, from I to IV (outside) and from I’ to IV’ (inside). The presteady‐state currents arise from the redistribution of the negatively charged transporter between states I’, I and II (blue boxes). The binding of the first external proton to state I accelerates the inward rate constant, while the binding of a second proton to state II leads to state III, effectively slowing down the outward rate. Transitions between I’, I and II are voltage dependent. S represents the binding of the substrate. Modified from Chen et al. (1999) and Sala‐Rabanal et al. (2008). [Colour figure can be viewed at wileyonlinelibrary.com]

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