Functional Characterization of Two Novel Mammalian Electrogenic Proton-dependent Amino Acid Cotransporters

Abstract

We cloned two cDNAs encoding proton/amino acid cotransporters, designated as mPAT1 and mPAT2, from murine tissues. They were identified by sequence similarity to the amino acid/auxin permease family member of lower eukaryotes. We functionally characterized both transporters by flux studies and electrophysiology after expression in Xenopus laevis oocytes. Both mPAT1 and mPAT2 induced a pH-dependent electrogenic transport activity for small amino acids (glycine, alanine, and proline) that is altered by membrane potential. Direct evidence for amino acid/H+-symport was shown by intracellular acidification, and a flux coupling stoichiometry for proline/H+-symport of 1:1 was determined for both transporters. Besides small apolar l-amino acids, the transporters also recognize their d-enantiomers and selected amino acid derivatives such as γ-aminobutyric acid. The mPAT1 transporter, the murine orthologue of the recently cloned rat LYAAT-1 transporter, can be considered as a low affinity system when compared with mPAT2. The mRNA of mPAT1 is highly expressed in small intestine, colon, kidney, and brain; the mPAT2-mRNA is mainly found in heart and lung. Phenotypically, the PAT1 transporter possesses the same functional characteristics as the previously described proton-dependent amino acid transport process in apical membranes of intestinal and renal epithelial cells. We cloned two cDNAs encoding proton/amino acid cotransporters, designated as mPAT1 and mPAT2, from murine tissues. They were identified by sequence similarity to the amino acid/auxin permease family member of lower eukaryotes. We functionally characterized both transporters by flux studies and electrophysiology after expression in Xenopus laevis oocytes. Both mPAT1 and mPAT2 induced a pH-dependent electrogenic transport activity for small amino acids (glycine, alanine, and proline) that is altered by membrane potential. Direct evidence for amino acid/H+-symport was shown by intracellular acidification, and a flux coupling stoichiometry for proline/H+-symport of 1:1 was determined for both transporters. Besides small apolar l-amino acids, the transporters also recognize their d-enantiomers and selected amino acid derivatives such as γ-aminobutyric acid. The mPAT1 transporter, the murine orthologue of the recently cloned rat LYAAT-1 transporter, can be considered as a low affinity system when compared with mPAT2. The mRNA of mPAT1 is highly expressed in small intestine, colon, kidney, and brain; the mPAT2-mRNA is mainly found in heart and lung. Phenotypically, the PAT1 transporter possesses the same functional characteristics as the previously described proton-dependent amino acid transport process in apical membranes of intestinal and renal epithelial cells. amino acid/auxin permease proton/amino acid transporter γ-aminobutyric acid vesicular GABA transporter lysosomal amino acid transporter1 expressed sequence tag open reading frame 4-morpholineethanesulfonic acid group of overlapping clones enhanced green fluorescent protein One of the largest families of amino acid transporters identified so far is the amino acid/auxin permease (AAAP)1 family (1Saier M.H., Jr. Microbiology. 2000; 146: 1775-1795Crossref PubMed Scopus (170) Google Scholar, 2Young G.B. Jack D.L. Smith D.W. Saier M.H., Jr. Biochim. Biophys. Acta. 1999; 1415: 306-322Crossref PubMed Scopus (123) Google Scholar). This protein family is widespread in nature, with members found in yeast, plants, invertebrates, and mammals. Although their amino acid sequences appear very divergent, a common signature has been identified, and secondary structure predictions show conserved similarities (2Young G.B. Jack D.L. Smith D.W. Saier M.H., Jr. Biochim. Biophys. Acta. 1999; 1415: 306-322Crossref PubMed Scopus (123) Google Scholar). Accordingly, most members of the AAAP family possess 11 membrane-spanning domains with very similar hydropathy profiles. Functionally characterized members as AAP1 and AVT1 found inSaccharomyces cerevisiae and Arabidopsis thalianaoperate as proton symporters for amino acids and selected derivatives such as auxins (2Young G.B. Jack D.L. Smith D.W. Saier M.H., Jr. Biochim. Biophys. Acta. 1999; 1415: 306-322Crossref PubMed Scopus (123) Google Scholar, 3Boorer K.J. Frommer W.B. Bush D.R. Kreman M. Loo D.D. Wright E.M. J. Biol. Chem. 1996; 271: 2213-2220Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 4Russnak R. Konczal D. McIntire S.L. J. Biol. Chem. 2001; 276: 23849-23857Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Three subfamilies within the AAAP family that show low although significant similarities (20–30%) have been identified so far in mammalian organisms. They are represented by the VGAT transporter group, the system A/N transporter group, and the LYAAT branch (5Sagne C. Agulhon C. Ravassard P. Darmon M. Hamon M., El Mestikawy S. Gasnier B. Giros B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7206-7211Crossref PubMed Scopus (173) Google Scholar). The vesicular γ-aminobutyric acid (GABA) transporter VGAT was the first identified mammalian member of the AAAP family with an assigned function (6McIntire S.L. Reimer R.J. Schuske K. Edwards R.H. Jorgensen E.M. Nature. 1997; 389: 870-876Crossref PubMed Scopus (681) Google Scholar). It mediates the uptake of the inhibitory neurotransmitters GABA and glycine into synaptic vesicles (6McIntire S.L. Reimer R.J. Schuske K. Edwards R.H. Jorgensen E.M. Nature. 1997; 389: 870-876Crossref PubMed Scopus (681) Google Scholar, 7Sagne C., El Mestikawy S. Isambert M.F. Hamon M. Henry J.P. Giros B. Gasnier B. FEBS Lett. 1997; 417: 177-183Crossref PubMed Scopus (239) Google Scholar). The driving force of VGAT is the electrochemical proton gradient generated by the vesicular H+-ATPase (8Forgac M. J. Biol. Chem. 1999; 274: 12951-12954Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Cytoplasmic GABA and glycine can therefore be accumulated within the vesicles via a proton antiport mechanism (6McIntire S.L. Reimer R.J. Schuske K. Edwards R.H. Jorgensen E.M. Nature. 1997; 389: 870-876Crossref PubMed Scopus (681) Google Scholar). The system A/N transporter group also belong to the AAAP family. System N plays an important role especially in the homeostasis of glutamine in brain, where it serves as a delivery system for the precursor of the excitatory amino acid glutamate (9Chaudhry F.A. Reimer R.J. Krizaj D. Barber D. Storm-Mathisen J. Copenhagen D.R. Edwards R.H. Cell. 1999; 99: 769-780Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). The system N-mediated glutamine influx is coupled to the cotransport of one Na+ ion in antiport with one H+ ion (9Chaudhry F.A. Reimer R.J. Krizaj D. Barber D. Storm-Mathisen J. Copenhagen D.R. Edwards R.H. Cell. 1999; 99: 769-780Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Although system A transporters are highly homologous to system N, they show a very different substrate specificity as well as a different ion-substrate flux coupling ratio (10Reimer R.J. Chaudhry F.A. Gray A.T. Edwards R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7715-7720Crossref PubMed Scopus (163) Google Scholar). System A transporters prefer alanine and most of the other neutral amino acids and are considered to provide bulk quantities of these amino acids in most cell types. System A carriers act as Na+/amino acid symporters. Although transport by system A is sensitive to low pH, no proton exchanger activity has been shown in contrast to system N (11Yao D. Mackenzie B. Ming H. Varoqui H. Zhu H. Hediger M.A. Erickson J.D. J. Biol. Chem. 2000; 275: 22790-22797Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Recently, Sagne et al. (5Sagne C. Agulhon C. Ravassard P. Darmon M. Hamon M., El Mestikawy S. Gasnier B. Giros B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7206-7211Crossref PubMed Scopus (173) Google Scholar) report the cloning of the first mammalian proton/amino acid symporter from rat brain, which is also a member of the AAAP family (5Sagne C. Agulhon C. Ravassard P. Darmon M. Hamon M., El Mestikawy S. Gasnier B. Giros B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7206-7211Crossref PubMed Scopus (173) Google Scholar). This lysosomal amino acid transporter, designated as LYAAT-1, when transfected in CV-1 cells mediated the uptake of small amino acids such as alanine and proline and the neurotransmitter GABA. The observation of an enhanced transport activity by low extracellular pH suggested a proton-symport mechanism for amino acid influx. The LYAAT-1 transcripts were identified in glutamatergic and GABAergic neurons in rat brain. The LYAAT-1 protein was localized in the lysosomal membrane in these neurons, although when expressed heterologously, it mediated plasma membrane amino acid uptake (5Sagne C. Agulhon C. Ravassard P. Darmon M. Hamon M., El Mestikawy S. Gasnier B. Giros B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7206-7211Crossref PubMed Scopus (173) Google Scholar). The first members of the system A/N subfamily, the SN1 protein, and LYAAT-1 have been identified by data base search for mammalian homologues of the VGAT transporter (5Sagne C. Agulhon C. Ravassard P. Darmon M. Hamon M., El Mestikawy S. Gasnier B. Giros B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7206-7211Crossref PubMed Scopus (173) Google Scholar, 6McIntire S.L. Reimer R.J. Schuske K. Edwards R.H. Jorgensen E.M. Nature. 1997; 389: 870-876Crossref PubMed Scopus (681) Google Scholar, 9Chaudhry F.A. Reimer R.J. Krizaj D. Barber D. Storm-Mathisen J. Copenhagen D.R. Edwards R.H. Cell. 1999; 99: 769-780Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). We searched for mammalian AAAP family members based on the S. cerevisiae protein sequence YKL146wp, which had been identified by genome-scanning analysis as a putative amino acid transport protein. Recently it was shown that YKL146wp indeed codes for a protein that represents a vesicular amino acid transporter, designated as AVT3 (4Russnak R. Konczal D. McIntire S.L. J. Biol. Chem. 2001; 276: 23849-23857Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Here we report the identification and characterization of two new members, the murine orthologue of rLYAAT-1, designated as mPAT1, and an additional member of this subfamily, designated as mPAT2. Both transporters, when expressed in Xenopus laevis oocytes, show the functional characteristics of electrogenic H+/amino acid symporters. The mPAT2 transporter displays the characteristics of a high affinity system with a more restricted substrate specificity than mPAT1. The yeast protein sequence YKL146wp belonging to the AAAP family was blasted (tblastn method) against murine and human EST sequences. Two closely related cDNA sequences were identified that were so far not linked to any functionally known cDNA. The EST clone 1920302 (designated as mPAT2) from a murine 14 days-post-coital embryonic cDNA library was purchased from the Resource Center/Primary Data base of the German Human Genome Project (IMAGp998B154710Q2, RZPD, Berlin, Germany). The original cDNA in the pME18S-FL3 vector was cut with XhoI and EcoRI and ligated in pCRII to the The of the open reading frame of the was by the of EST sequences. The cDNA the was identified the of cDNA system The of mPAT1 was with a high the and with small intestinal The of the is to their in to the first base of the Two were cloned in the pCRII and both were the with an The were in the pCRII vector in which an of the the was This the for expression laevis oocytes. The expression of with the additional was more than than that of the the additional not on and protein the were of were with the and with a of amino sequence method) were with the were with A for in and to cells. of and were in and The were with of of and for functional functional characterization of mPAT1 of were into oocytes. The were in after uptake were for in uptake The was by the uptake with amino acid and the l-amino acid as a were for in amino acid uptake this not The were with of and to in was by for the of pH-dependent uptake were with were as described previously H. J. 2001; PubMed Scopus Google Scholar). the was in an open and with pH in the of amino were and were by in the to were and after substrate when The by mPAT1 mPAT2 a membrane was as the the in the and the of studies the of transport was by coupling stoichiometry was determined by of with in as described (11Yao D. Mackenzie B. Ming H. Varoqui H. Zhu H. Hediger M.A. Erickson J.D. J. Biol. Chem. 2000; 275: 22790-22797Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). were and with for with were and was by The was with to the and to a the were from different amino acid in pH with from two different for were to and after the substrate that transport activity were pH of with mPAT1 was with the proton The were with different pH and with a of and were of the for the the for H+ of was as the the membrane with a and the electrochemical of the from different murine were with the were with of and were by on a and to a membrane N, The was with by for in after high of the was on a and with a of the was with a cDNA The of mPAT2 was with the and the as the and both sequences was to the of the into the EcoRI of the vector which had the mPAT2 and the in the same of the mPAT2 in the was by The mPAT2 cDNA was on both cells were on to a of of the was transfected the the of the Two after the cells were with a for the of green of with was to the The of the protein was after of into laevis oocytes. the was cut with XhoI and into the pCRII vector the as also for enhanced expression of mPAT1. of alanine, and proline into was when compared with the uptake in oocytes. to mPAT2, proline uptake in the with an from in to influx was not alanine, and proline not also induced in not an in cloning we identified two closely related cDNA sequences from based on the yeast protein sequence YKL146wp, designated as and The is represented by an EST clone from a murine 14 embryonic cDNA library The of mPAT1 has been by the with from murine small The possess and The mPAT1 and mPAT2 show an of and a similarity of both to have 11 membrane-spanning domains the putative domains the amino acid sequences show the N most the is Three conserved putative were found the of the when that the N is to the The known mammalian protein that is closely related to the is the recently identified rat LYAAT-1 (5Sagne C. Agulhon C. Ravassard P. Darmon M. Hamon M., El Mestikawy S. Gasnier B. Giros B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7206-7211Crossref PubMed Scopus (173) Google Scholar). LYAAT-1 is the rat orthologue of the mPAT1 protein with a similarity of The and belong to the AAAP family (2Young G.B. Jack D.L. Smith D.W. Saier M.H., Jr. Biochim. Biophys. Acta. 1999; 1415: 306-322Crossref PubMed Scopus (123) Google Scholar). functionally identified AAAP family members are the vesicular GABA transporter VGAT and the system A/N transporters. The a new the VGAT and system A/N of the first of the human sequences and human EST sequences that the human orthologue are on within a than on the The human orthologue a similarity of and to the murine transporter the indeed function as amino acid transporters, we the and flux studies with amino acids in laevis mPAT1 mPAT2. A the uptake of selected amino acids in the in of The uptake of alanine and proline were highly in and oocytes, uptake was not enhanced when compared with uptake in oocytes. alanine, and proline induced The of the amino acids were in oocytes, mPAT2 the with glycine by alanine and proline and of glycine by the in glycine with of the that the membrane is a driving This of substrate was more in than in oocytes. generated by by the membrane from to in in the that the transport of mPAT1 and mPAT2, we first the of different and on the of Na+ by and by had no significant on mPAT1 and mPAT2 transport activity the other the extracellular pH to a in mPAT1 and mPAT2 mediated influx The mPAT1 transport activity was more by of the extracellular pH than that of mPAT2. The uptake of pH was than that pH in oocytes, in was by Direct evidence for a proton/amino acid mechanism was by intracellular pH by a with in membrane when in the of glycine shown in the of glycine and proline to the to an intracellular as well as to a of mPAT1. were by the amino The same was for glycine in oocytes, which is no substrate of the transporters, to an and membrane not to the of any amino acid not The stoichiometry was determined by with the in with the in a with a of and The were and and the were not different from the in a This that the transport via mPAT1 and mPAT2 with 1:1 Both transporters are very substrate We l-amino acids for their to in and of amino acids with such as alanine, and to a were to with the transporters substrate A and other amino acid was to the other the transporters are not very as shown by the induced by in and D. mPAT1 were as high as of in the of mPAT2, d-enantiomers induced than of the glycine and the neurotransmitter GABA induced more in than in and for in with were for selected amino acids and different were and after and the were the The amino acid with the affinity was with of and mPAT2 was found to have a more than affinity for acids when compared with the of mPAT1. The very low affinity of for both transporters the very as the of the group has the same affinity for both transporters of and for mPAT1 and mPAT2, that mPAT2 is more than mPAT1. GABA is a very substrate of mPAT1 a substrate of mPAT2 mPAT2 therefore represents a high transporter and is more in substrate when compared with the low affinity of selected amino acids and derivatives of proton/amino acid transport of mPAT1 and mPAT2 were determined by for the in uptake were to and were by Data the of oocytes. from the on transport as found in the apical membranes of cells and in renal membrane vesicles from are not in a new transport of mPAT1 and mPAT2 were determined by for the in uptake were to and were by Data the of oocytes. from the on transport as found in the apical membranes of cells and in renal membrane vesicles from are not that the are expressed in murine The mPAT1 be in small intestine, kidney, brain, and were also in other not The mPAT2 mRNA was most expressed in and also be in kidney, and Although functional expression in suggested the transporters to be plasma membrane we the to the of mPAT2. The mPAT2 protein, when with the protein was to the plasma membrane of cells was also found in the most represented by the and the we not have any evidence for this the rat orthologue of mPAT1, the protein, has been in rat to in neurons (5Sagne C. Agulhon C. Ravassard P. Darmon M. Hamon M., El Mestikawy S. Gasnier B. Giros B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7206-7211Crossref PubMed Scopus (173) Google we of the protein in transfected cells with the which in mainly of both was no significant was that mPAT2 is not an protein of lysosomal not when in cells We identified by in expressed sequence tag two closely related murine transporter designated as mPAT1 and mPAT2, which belong to the AAAP family (2Young G.B. Jack D.L. Smith D.W. Saier M.H., Jr. Biochim. Biophys. Acta. 1999; 1415: 306-322Crossref PubMed Scopus (123) Google Scholar). The two be functionally characterized as proton/amino acid symporters with a specificity for amino acids with small with the rat LYAAT-1 transporter orthologue of the a new to the VGAT and system A/N transporter subfamilies within the of the AAAP family. to (5Sagne C. Agulhon C. Ravassard P. Darmon M. Hamon M., El Mestikawy S. Gasnier B. Giros B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7206-7211Crossref PubMed Scopus (173) Google a human orthologue of mPAT2 can be identified in the first of the human the is indeed expressed in human in is by the of cDNA sequence in the EST data base and Both transporters similar functional when expressed in laevis oocytes. We show that the carriers the influx of small amino acids and for the first the electrogenic of the transport substrate amino acids are in the pH the plasma membrane is to H+ influx coupled to amino acid This was by a intracellular induced by transport evidence was found for of other that are the with amino acids by mPAT1 and mPAT2. activity of mPAT1 was highly on membrane and on extracellular pH in contrast to mPAT2, found to be by both This that activity of mPAT1, so mPAT2, be by in membrane and pH within the these the transport also substrate H+ has to be Both transporters a substrate amino acids with small apolar such as alanine, and is The mPAT2 protein has to the amino acids than mPAT1, with for and the same The of a group in the the substrate affinity for both transporters and in to for the of a group as in substrate The mPAT2 transporter a more substrate and a more with small neutral acids as high affinity GABA and are Besides these functional the mRNA transcripts of the transporters also show expression in murine tissues. The mPAT1 mRNA has been in most of the with expression in small intestine, colon, kidney, and brain, of the mPAT2 mRNA were in heart and lung. The of the of proton-dependent small amino acid transporters with different functional in the has to be The functionally known and characterized mammalian of the new murine transporters is the lysosomal amino acid transporter from It most represents the first lysosomal proton/amino acid symporter found in (5Sagne C. Agulhon C. Ravassard P. Darmon M. Hamon M., El Mestikawy S. Gasnier B. Giros B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7206-7211Crossref PubMed Scopus (173) Google Scholar). rLYAAT-1, which has been functionally characterized after in the cell displays a very similar substrate specificity and similar as mPAT1, which represents the murine two were functional not GABA uptake into CV-1 cells a of (5Sagne C. Agulhon C. Ravassard P. Darmon M. Hamon M., El Mestikawy S. Gasnier B. Giros B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7206-7211Crossref PubMed Scopus (173) Google in induced significant in oocytes. The most be the very low affinity of this amino acid to mPAT1, with an of which the GABA affinity for has been determined with a of (5Sagne C. Agulhon C. Ravassard P. Darmon M. Hamon M., El Mestikawy S. Gasnier B. Giros B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7206-7211Crossref PubMed Scopus (173) Google GABA a of in the of mPAT1. This be by the different such as pH and membrane the of the carriers in it be considered that the two described are as well lysosomal transporters for the of small amino acids from the lysosomal into the as by Sagne et al. (5Sagne C. Agulhon C. Ravassard P. Darmon M. Hamon M., El Mestikawy S. Gasnier B. Giros B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7206-7211Crossref PubMed Scopus (173) Google no functional evidence for such has been found in lysosomal the functionally characterized lysosomal small amino acid transport and have different substrate and and appear not to act as proton symporters J. Biol. Chem. Full Text PDF PubMed Google Scholar). the other in functional data with amino acid influx studies in cell and membrane we found a similarity mPAT1 and the proton-dependent amino acid transport previously identified in the apical membrane of the intestinal cells J. Biol. PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar, FEBS Lett. PubMed Scopus Google Scholar, J. 2000; PubMed Scopus Google Scholar) and renal membrane H. H. S. Jorgensen C. J. Google Scholar, J. PubMed Scopus Google Scholar, H. C. H. S. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, H. H. Jorgensen Biochim. Biophys. Acta. PubMed Scopus Google Scholar). transport in apical membranes of intestinal and renal epithelial cells have been well and it was shown that they are secondary for the same small amino acids and selected The substrate specificity and of mPAT1 is to the transport system described in which small apolar amino acids, acids the acid and the neurotransmitter GABA J. Biol. PubMed Scopus Google Scholar). the stoichiometry of the transport system was to be 1:1 J. Biol. PubMed Scopus Google as found for mPAT1. The substrate as from the are in the same as determined for mPAT1 for J. Biol. PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar, FEBS Lett. PubMed Scopus Google Scholar, J. 2000; PubMed Scopus Google Scholar, H. H. S. Jorgensen C. J. Google Scholar, J. PubMed Scopus Google Scholar, H. C. H. S. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, H. H. Jorgensen Biochim. Biophys. Acta. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). in be to different such as pH membrane potential. The mPAT1 is in expressed in the small and kidney, evidence for role as a transport system in the apical membrane of and cells. when with cell and human we a with the not of mPAT1 and mPAT2 in and of in CV-1 cells that the as plasma membrane this be an of with the protein not evidence for a lysosomal of this protein in cells. on this evidence we have the more for the two new proton amino acid to that they are not a although the protein was expressed in in of the rat (5Sagne C. Agulhon C. Ravassard P. Darmon M. Hamon M., El Mestikawy S. Gasnier B. Giros B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7206-7211Crossref PubMed Scopus (173) Google Scholar). studies in other than have so far not been is known the of the in such as small and it be that the protein is in a to the plasma membrane the cell has been shown for other membrane the epithelial transporter which is mainly found in the apical plasma membrane of intestinal and renal epithelial cells J. 1997; Google H. H. S. K. K. Biophys. 1996; PubMed Scopus Google it is also found in of cells H. Smith J. 1999; 276: Google Scholar). it be that the as both proton-dependent plasma membrane transporters and lysosomal for small amino we have identified, and functionally characterized two mammalian electrogenic H+/amino acid symporters with a high for amino acids with apolar and small as well as related Although their has not been they as plasma membrane and lysosomal The in of mPAT1, in of the of substrate substrate and the of apical membrane carriers identified in and for which the protein had not been identified