SUMMARY Amino acids play a pivotal role in every living organism as they function as building blocks of proteins, signaling molecules, metabolic precursor, etc. Due to their multiple roles in the body, amino acids availability must be tightly controlled and relies on their transport across cellular membrane via amino acid transporters. In the kidney proximal tubule and in the small intestine, the overall directional (re)absorptive transport of amino acids is mainly driven by accumulative transporters located in the apical membrane of epithelial cells. The best characterized luminal transporter for neutral amino acids is B0AT1 (SLC6A19). Expression and function of B0AT1 in the kidney have been shown to depend on the associated protein collectrin/TMEM27 (Danilczyk et al., 2006). To better understand the cellular and molecular mechanism underlying this interaction, we generated MDCK cells inducibly overexpressing B0AT1 and/or TMEM27. Our data showed that TMEM27 increases B0AT1 function by upregulating its surface expression. Interestingly, we found that the expression of the amino acid transporter and its accessory protein strongly decreased following subsequent cell culture passages. We tested the impact of cell culture medium amino acid concentrations on the transporter and found that high amino acid levels inhibit B0AT1 protein abundance. In contrast, the expression of a control transgene remained stable. To test whether this loss was due to inappropriately high amino acid uptake, freshly transduced MDCK cell lines were cultivated either with physiological amounts of amino acids or with the high concentration found in standard cell culture media. Expression of exogenous transporters was unaffected by physiological amino acid concentration in the media. Interestingly, mycoplasma infection resulted in a significant increase in transgene expression and correlated with the rapid metabolism of L-arginine. However, L-arginine metabolites were shown to play no role in transgene expression. In contrast, activation of the GCN2 pathway revealed by an increase in eIF2α phosphorylation may trigger transgene derepression. In addition to the transgene regulation, we found that mycoplasma-induced arginine depletion dramatically affected MDCK epithelia, resulting in a decrease in cell number and trans-epithelial electrical resistance corresponding in a delay in tight junction formation. Taken together, high extracellular amino acid concentration provided by cell culture media appears to inhibit the constitutive expression of concentrative amino acid transporters whereas L-arginine depletion by mycoplasma induces the expression of transgenes via stimulation of the GCN2 pathway.