Essential amino acid transporter Lat4 (Slc43a2) is required for mouse development

Amino acid (AA) uniporter Lat4 (Slc43a2) mediates facilitated diffusion of branched-chain AAs, methionine and phenylalanine, although its physiological role and subcellular localization are not known. We report that Slc43a2 knockout mice were born at expected Mendelian frequency but displayed an ∼10% intrauterine growth retardation and low amniotic fluid AAs, suggesting defective transplacental transport. Postnatal growth was strongly reduced, with premature death occurring within 9 days such that further investigations were made within 3 days of birth. Lat4 immunofluorescence showed a strong basolateral signal in the small intestine, kidney proximal tubule and thick ascending limb epithelial cells of wild-type but not Slc43a2 null littermates and no signal in liver and skeletal muscle. Experiments using Xenopus laevis oocytes demonstrated that Lat4 functioned as a symmetrical low affinity uniporter with a K0.5 of ∼5 mm for both in- and efflux. Plasma AA concentration was decreased in Slc43a2 null pups, in particular that of non-essential AAs alanine, serine, histidine and proline. Together with an increased level of plasma long chain acylcarnitines and a strong alteration of liver gene expression, this indicates malnutrition. Attempts to rescue pups by decreasing the litter size or by nutrients injected i.p. did not succeed. Radioactively labelled leucine but not lysine given per os accumulated in the small intestine of Slc43a2null pups, suggesting the defective transcellular transport of Lat4 substrates. In summary, Lat4 is a symmetrical uniporter for neutral essential AAs localizing at the basolateral side of (re)absorbing epithelia and is necessary for early nutrition and development. Key points Lat4 (Slc43a2) transports branched-chain amino acids, phenylalanine and methionine, and is expressed in kidney tubule and small intestine epithelial cells. Using a new knockout model as a negative control, it is shown that Lat4 is expressed at the basolateral side of small intestine enterocytes and kidney epithelial cells of the proximal tubule, thick ascending limb and distal convoluted tubule. In the Xenopus oocyte expression system, Lat4 is shown to function as a uniporter with symmetric intracellular and extracellular apparent affinities for phenylalanine. Mice lacking Lat4 display a slight intrauterine growth restriction, postnatal malnutrition and early death, presumably as a result of defective amino acid (re)absorption. These results demonstrate the crucial role that the uniporter Lat4 plays for amino acid transport across cellular barriers and mouse development. Introduction Dietary protein intake is an essential and tightly regulated process that needs to meet physiological body requirements, especially during highly anabolic periods (Poncet & Taylor, 2013). The enzyme-mediated hydrolysis of dietary proteins in the gastrointestinal lumen produces oligopeptides and individual amino acids (AAs) that are absorbed by moving sequentially across apical and basolateral cell membranes of small intestine enterocytes (Nassl et al. 2011). The zwitterionic nature of neutral AAs prevents the direct crossing of the epithelial lipid bilayer by simple diffusion and imposes the presence of membrane-spanning transporter proteins (Braun et al. 2011). Severe malabsorption and aminoaciduria syndromes have been linked to defects of specific AA transporters, substantiating the essential role that transepithelial transport plays for body AA homeostasis (Verrey et al. 2009; Broer & Palacin, 2011). The luminal step of neutral AA absorption and reabsorption at the level of the small intestine and kidney, respectively, is largely mediated by the apical transporter B0AT1 (SLC6A19). This luminal symporter uses the driving force exerted by the Na+ gradient and the membrane potential on the co-transported Na+ ion for the concentrative import of its neutral AA substrates (secondary active transport) (Rudnick et al. 2014). Once inside enterocytes or kidney proximal tubule cells, neutral AAs can be effluxed across the basolateral membrane by cooperating AA exchangers and uniporters. Indeed, all neutral AAs can be effluxed by the major basolateral neutral AA anti-porter (obligatory exchanger) LAT2–4F2hc (SLC7A8-SLC3A2). It is, however, important to realize that, for each exported AA, this anti-porter imports another AA. Thus, this anti-porter does not perform a net transmembrane AA efflux (Meier et al. 2002). Unexpectedly, no genetic diseases involving defects of LAT2–4F2hc have been described to date, whereas mutations in the other basolateral anti-porter y+LAT1–4F2hc (SLC7A7-SLC3A2), which exchanges preferentially intracellular cationic AAs against neutral AAs and Na+, cause lysinuric protein intolerance, an autosomal recessive disease (Borsani et al. 1999). The respective physiological roles of both exchangers were also investigated using knockout (KO) mice. The lack of LAT2 resulted in a mild phenotype with a minor aminoaciduria (Braun et al. 2011), whereas the lack of y+LAT1 caused intrauterine growth restriction and resulted in premature death for ∼90% of newborn Slc7a7−/− homozygous mice (Sperandeo et al. 2007). To achieve a net efflux of all different AAs from small intestine enterocytes and proximal kidney tubule epithelial cells, transporters are required to mediate the net efflux of some AAs that can then recycle into the cells via the above mentioned anti-porters and thereby drive their efflux activity (Verrey et al. 2009). The T-type aromatic AA uniporter TAT1 (SLC16A10) represents the best characterized such transporter expressed at the basolateral membrane (Ramadan et al. 2006; Ramadan et al. 2007). Using the Xenopus laevis oocyte expression system, it was shown to mediate facilitated diffusion of the essential aromatic AAs phenylalanine, tryptophan and tyrosine with symmetric apparent affinities (i.e. Km ≈ 30 mm for phenylalanine) and thereby drive the efflux of other LAT2–4F2hc substrates such as the non-essential AA glutamine (Ramadan et al. 2006; Ramadan et al. 2007). Recently, we characterized the physiological role of TAT1 using Slc16a10 deficient mice. Interestingly, these mice presented a mild phenotype with increased plasma, kidney and muscle aromatic AAs, independently of dietary protein quantity, combined with a major aromatic aminoaciduria under a high protein diet (Mariotta et al. 2012). The lack of neurological symptoms and important metabolic dysfunctions, despite the defective intestinal absorption of aromatic AAs such as tyrosine, which is the metabolic precursors of the hormone thyroxine and the neurotransmitter dopamine, suggested a possible compensatory mechanism by (an)other basolateral uniporter(s). We proposed that the uniporter LAT4 (SLC43A2) could play this role. LAT4 belongs to the SLC43 family that includes two additional members: LAT3 (SLC43A1), which is a uniporter for large essential neutral AAs and has been shown to be upregulated in liver and skeletal muscle upon starvation and displays only a low mRNA expression level in the small intestine and kidney (Babu et al. 2003; Fukuhara et al. 2007), and EEG1 (SLC43A3), the function of which remains unknown. The latter gene product was shown to be prominently expressed in human liver and heart and, based on immunohistochemistry, to be expressed at the basolateral membrane of human kidney proximal convoluted tubule (Bodoy et al. 2005). LAT4 shows only ∼30% identity with EEG1 and has been shown by Northern blot analysis of human RNA to be highly expressed in placenta, peripheral blood leucocytes and kidney and, additionally, by Northern blot analysis of mouse RNA, to be expressed in the small intestine and brain (Bodoy et al. 2005). Microarray data (www.biogps.org) confirm this expression pattern and additionally show substantial expression in macrophages, microglia and osteoclasts. Based on in situ hybridization, Lat4 was suggested to be expressed in the kidney distal tubule and collecting duct and the images also appeared to be compatible with expression in the proximal tubule. In the small intestine, the signal was localized more to crypt cells (Bodoy et al. 2005). Lat4 mRNA was also detected in cell lines isolated from the mouse kidney proximal tubule, in agreement with the mRNA expression in isolated proximal tubules reported by Cheval et al. (2011). Furthermore, kinetic analysis of phenylalanine uptake in these cells suggested the involvement of Lat4, supporting the possibility that this uniporter plays a role in proximal tubule AA reabsorption. The preferential substrates of LAT4 comprise only essential AAs, specifically the branched-chain ones (leucine, isoleucine, valine) and additionally phenylalanine and methionine (Bodoy et al. 2005) To test the physiological role of Lat4 in whole-body AA homeostasis and epithelial transport, we generated a global Slc43a2–/– KO mouse and we now report the analysis of its phenotype.