SLC36A4 (hPAT4) Is a High Affinity Amino Acid Transporter When Expressed in Xenopus laevis Oocytes

Abstract

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [ 3 H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 M. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 M). Other amino acids that inhibited [ 3 H]proline were isoleucine (K i 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K i >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K i 1.09 mM), whereas ␥-aminobutyric acid (GABA), ␤-alanine, L-Dopa, D-serine, and ␦-aminolevulinic acid were without effect on [ 3 H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.

Functional Characterization of 5-Oxoproline Transport via SLC16A1/MCT1

“…These results indicate that the transport process is proton/ 5-oxoproline cotransport. We then examined the transport of 5-oxoproline under the conditions of normal (2 mM) and high (50 mM) extracellular potassium concentrations as the latter condition acts to depolarize the membrane potential (19). SLC16A1-mediated 5-oxoproline uptake was not changed by a high potassium concentration condition.…”

Dennd3 Functions as a Guanine Nucleotide Exchange Factor for Small GTPase Rab12 in Mouse Embryonic Fibroblasts

“…3 and 6A), presumably a protein involved in the amino acid sensing mechanism modifies the Dennd3 GEF function that activates Rab12, thereby leading to a decrease in the PAT4 protein level and the intracellular amino acid concentration to the basal level. Extensive research will be necessary to determine whether treatment of MEF cells with an excess of amino acids, specifically, proline, which has been found to be a high affinity substrate of PAT4 (25), will alter posttranslational modification of Dennd3 and Rab12-GEF activity. Despite our unexpected discovery that Dennd3 is involved in the Akt-mTORC1 pathway (Fig.…”

“…Because the effect of Dennd3 overexpression on mTORC1 activity was canceled out by exposure of the Dennd3-overexpressing cells to proline (Fig. 6, D and E), which has been reported to be a high affinity substrate of PAT4 in Xenopus oocytes (25), the PAT4 degradation that results from overexpression of Dennd3 is likely to contribute to the reduced mTORC1 activity. Moreover, it should be noted that overexpression of Dennd3 had no effect on Akt activity (Fig.…”

Recent Advances in Understanding Amino Acid Sensing Mechanisms that Regulate mTORC1

“…The pH dependence of PAT4 is distinct from other members of PATs: it has the maximal transport rate at pH 7.4 and a lower transport rate at pH 5.5 and pH 8.4 [46]. …”

“…More recently, in HCT116 colorectal cancer cells, PAT4 was found to respond to two rapidly metabolized AAs (glutamine and serine) to drive mTORC1 activation by interacting with Rab1A (a small GTPase) and mTORC1 on the Golgi [50]. Since glutamine and serine bind to PAT4 with a lower affinity, and can compete with high-affinity PAT4 substrates [46], PAT4-mediated activation of mTORC1 may not depend on the transport function of PAT4.…”