Key points Amino acid absorption requires luminal uptake into and subsequent basolateral efflux out of epithelial cells, with the latter step being critical to regulate the intracellular concentration of the amino acids. The basolateral essential neutral amino acid uniporter LAT4 (SLC43A2) has been suggested to drive the net efflux of non‐essential and cationic amino acids via parallel amino acid antiporters by recycling some of their substrates; its deletion has been shown to cause defective postnatal growth and death in mice. Here we test the regulatory function of LAT4 phosphorylation sites by mimicking their phosphorylated and dephosphorylated states in Xenopus laevis oocytes and show that dephosphorylation of S274 and phosphorylation of S297 increase LAT4 membrane localization and function. Using new phosphorylation site‐specific antibodies, we observe changes in LAT4 phosphorylation in mouse small intestine that correspond to its upregulation at the expected feeding time. These results strongly suggest that LAT4 phosphorylation participates in the regulation of transepithelial amino acid absorption. Abstract The essential amino acid uniporters LAT4 and TAT1 are located at the basolateral side of intestinal and kidney epithelial cells and their transport function has been suggested to control the transepithelial (re)absorption of neutral and possibly also cationic amino acids. Uniporter LAT4 selectively transports the branched chain amino acids leucine, isoleucine and valine, and additionally methionine and phenylalanine. Its deletion leads to a postnatal growth failure and early death in mice. Since LAT4 has been reported to be phosphorylated in vivo, we hypothesized that phosphorylation regulates its function. Using Xenopus laevis oocytes, we tested the impact of LAT4 phosphorylation at Ser274 and Ser297 by expressing mutant constructs mimicking phosphorylated and dephosphorylated states. We then investigated the in vivo regulation of LAT4 in mouse small intestine using new phosphorylation site‐specific antibodies and a time‐restricted diet. In Xenopus oocytes, mimicking non‐phosphorylation of Ser274 led to an increase in affinity and apparent surface membrane localization of LAT4, stimulating its transport activity, while the same mutation of Ser297 decreased LAT4's apparent surface expression and transport rate. In wild‐type mice, LAT4 phosphorylation on Ser274 was uniform at the beginning of the inactive phase (ZT0). In contrast, at the beginning of the active phase (ZT12), corresponding to the anticipated feeding time, Ser274 phosphorylation was decreased and restricted to relatively large patches of cells, while Ser297 phosphorylation was increased. We conclude that phosphorylation of small intestinal LAT4 is under food‐entrained circadian control, leading presumably to an upregulation of LAT4 function at the anticipated feeding time.

Determining the contributions of different transporter species to overall cellular transport is fundamental for understanding the physiological regulation of solutes. We calculated the relative activities of Solute Carrier (SLC) transporters using the Michaelis-Menten equation and global fitting to estimate the normalized maximum transport rate for each transporter (Vmax). Data input were the normalized measured uptake of the essential neutral amino acid (AA) L-leucine (Leu) from concentration-dependence assays performed using Xenopus laevis oocytes. Our methodology was verified by calculating Leu and L-phenylalanine (Phe) data in the presence of competitive substrates and/or inhibitors. Among 9 potentially expressed endogenous X. laevis oocyte Leu transporter species, activities of only the uniporters SLC43A2/LAT4 (and/or SLC43A1/LAT3) and the sodium symporter SLC6A19/B0AT1 were required to account for total uptake. Furthermore, Leu and Phe uptake by heterologously expressed human SLC6A14/ATB0,+ and SLC43A2/LAT4 was accurately calculated. This versatile systems biology approach is useful for analyses where the kinetics of each active protein species can be represented by the Hill equation. Furthermore, its applicable even in the absence of protein expression data. It could potentially be applied, for example, to quantify drug transporter activities in target cells to improve specificity.

Basolateral efflux is a crucial step for amino acid (AA) (re)absorption across small intestine and kidney proximal tubule epithelia mediated by various transporters. There are uniporters that mediate the facilitated diffusion of essential AAs, as does for instance aromatic AA transporter TAT1 (Slc16a10) and also antiporters such as LAT2‐4F2hc (SLC7A8‐SLC3A2) that exchanges neutral AAs. To test the hypothesis that the recycling of aromatic AAs via TAT1 allows the vectorial efflux of other AAs via obligatory exchanger LAT2‐4F2hc, LAT2‐/‐ TAT1‐/‐ double knockout (dKO) mice were generated. These mice have a reduced body weight (~‐17% at 3 months) but no other overt phenotypic alteration. Under normal protein diet, they excrete in the urine, compared to LAT2‐/‐ and TAT1‐/‐ knockout mice, higher amounts of aromatic AAs and of some other AAs that are not substrates of TAT1. The amino aciduria was further increased under high protein diet and involved all proteogenic AAs but some charged ones. Screening the mRNA levels of ~20 AA transporters in the kidney suggested a trend of compensatory changes as the level of the transcript of 5 basolateral AA transporters was increased in dKO mice under high protein diet. Preliminary transport experiments with small intestine gut sacs confirm the alteration of transepithelial transport. These observations support the hypothesis that the basolateral efflux of LAT2 substrates depends on the recycling of aromatic AAs via TAT1.Supported by Swiss National Science Foundation.