Topology of NAT2, a Prototypical Example of a New Family of Amino Acid Transporters

Amino acids are the predominant form of nitrogen available to the heterotrophic tissues of plants. These essential organic nutrients are transported across the plasma membrane of plant cells by proton-amino acid symporters. Our lab has cloned an amino acid transporter from Arabidopsis, NAT2/AAP1, that represents the first example of a new class of membrane transporters. We are investigating the structure and function of this porter because it is a member of a large gene family in plants and because its wide expression pattern suggests it plays a central role in resource allocation. In the results reported here, we investigated the topology of NAT2 by engineering a c-myc epitope on either the N or C terminus of the protein. We then used in vitro translation, partial digestion with proteinase K, and immunoprecipitation to identify a group of oriented peptide fragments. We modeled the topology of NAT2 based on the lengths of the peptide fragments that allowed us to estimate the location of protease accessible cleavage sites. We independently identified the location of the N and C termini using immunofluorescence microscopy of NAT2 expressed in COS-1 cells. We also investigated the glycosylation status of several sites of potentialN-linked glycosylation. Based on the combined data, we propose a novel 11 transmembrane domain model with the N terminus in the cytoplasm and C terminus facing outside the cell. This model of protein topology anchors our complementary investigations of porter structure and function using site-directed and random mutagenesis. Amino acids are the predominant form of nitrogen available to the heterotrophic tissues of plants. These essential organic nutrients are transported across the plasma membrane of plant cells by proton-amino acid symporters. Our lab has cloned an amino acid transporter from Arabidopsis, NAT2/AAP1, that represents the first example of a new class of membrane transporters. We are investigating the structure and function of this porter because it is a member of a large gene family in plants and because its wide expression pattern suggests it plays a central role in resource allocation. In the results reported here, we investigated the topology of NAT2 by engineering a c-myc epitope on either the N or C terminus of the protein. We then used in vitro translation, partial digestion with proteinase K, and immunoprecipitation to identify a group of oriented peptide fragments. We modeled the topology of NAT2 based on the lengths of the peptide fragments that allowed us to estimate the location of protease accessible cleavage sites. We independently identified the location of the N and C termini using immunofluorescence microscopy of NAT2 expressed in COS-1 cells. We also investigated the glycosylation status of several sites of potentialN-linked glycosylation. Based on the combined data, we propose a novel 11 transmembrane domain model with the N terminus in the cytoplasm and C terminus facing outside the cell. This model of protein topology anchors our complementary investigations of porter structure and function using site-directed and random mutagenesis. Amino acids are actively transported into plant cells by proton-coupled symporters (1Bush D.R. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1993; 44: 513-542Crossref Scopus (312) Google Scholar). These proteins link translocation across the plasma membrane to the proton-motive force generated by a P-type, H+-ATPase (2Briskin D.P. Biochem. Biophys. Acta. 1990; 1019: 95-109Crossref Scopus (109) Google Scholar, 3Serrano R. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1989; 40: 61-94Crossref Google Scholar). In plants, there are many heterotrophic tissue systems that are dependent upon carbon and nitrogen import for growth and development. Since amino acids are the primary form of nitrogen available to the heterotrophic plant tissues, the amino acid symporters are responsible for the systemic distribution of organic nitrogen, and therefore, they are essential contributors to plant growth (4Pate J.S. Stewart F.C. Bidwell R.G.S. Plant Physiology: A Treatise, Vol. VIII: Nitrogen Metabolism. Academic Press, New York1983: 335-401Google Scholar, 5Bush D.R. Singh B. Plant Amino Acids: Biochemistry and Biotechnology. Marcel Dekker, NY1998Google Scholar). Detailed investigations of the transport properties and bioenergetics of these symporters using isolated plasma membrane vesicles and imposed proton electrochemical potential differences have shown that they are electrogenic transporters that are driven by either transmembrane proton or electrical potential differences (6Li Z.-C. Bush D.R. Plant Physiol. 1990; 94: 268-277Crossref PubMed Scopus (66) Google Scholar). These transporters are inhibited by chemical modification of histidine residues by diethyl pyrocarbonate (6Li Z.-C. Bush D.R. Plant Physiol. 1990; 94: 268-277Crossref PubMed Scopus (66) Google Scholar), and substrate protection experiments suggest the sensitive residue is at or near the substrate binding site (5Bush D.R. Singh B. Plant Amino Acids: Biochemistry and Biotechnology. Marcel Dekker, NY1998Google Scholar, 7Bush D.R. Li Z.-C. Bonnemain J.L. Delrot S. Lucas W.J. Dainty J. Recent Advances in Phloem Transport and Assimilate Compartmentation. Presses Academiques, France1991: 148-153Google Scholar). Several classes of symporters were initially resolved based on expression patterns and substrate specificity (6Li Z.-C. Bush D.R. Plant Physiol. 1990; 94: 268-277Crossref PubMed Scopus (66) Google Scholar,8Li Z.-C. Bush D.R. Plant Physiol. 1991; 96: 1338-1344Crossref PubMed Scopus (44) Google Scholar, 9Li Z.-C. Bush D.R. Arch. Biochem. Biophys. 1992; 294: 519-526Crossref PubMed Scopus (32) Google Scholar, 10Williams L.E. Nelson S.J. Hall J.L. Planta. 1990; 182: 540-545Crossref PubMed Scopus (58) Google Scholar, 11Williams L.E. Nelson S.J. Hall J.L. Planta. 1992; 186: 541-550Crossref PubMed Scopus (43) Google Scholar). Transport competition experiments showed that binding sites are stereo-specific and identified the carboxylic acid, the alpha amino group, and substitutions at the β-carbon as important determinants in governing substrate binding (8Li Z.-C. Bush D.R. Plant Physiol. 1991; 96: 1338-1344Crossref PubMed Scopus (44) Google Scholar, 9Li Z.-C. Bush D.R. Arch. Biochem. Biophys. 1992; 294: 519-526Crossref PubMed Scopus (32) Google Scholar). Recently, one of these symporters was expressed in Xenopus oocytes, and electrophysiological methods allowed for a high resolution investigation of transport kinetics that suggests these transporters operate by a simultaneous binding mechanism (12Boorer K.J. Frommer W.B. Bush D.R. Kreman M. Loo D.D.F. Wright E.M. J. Biol. Chem. 1996; 271: 2213-2220Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar).The first plant amino acid symporter cloned (NAT2/AAP1) was identified by two groups using functional complementation of yeast amino acid transport mutants with different Arabidopsis cDNA expression libraries (13Hsu L.-C. Chiou T.-J. Chen L. Bush D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7441-7445Crossref PubMed Scopus (110) Google Scholar, 14Frommer W.B. Hummel S. Riesmeier J.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5944-5948Crossref PubMed Scopus (159) Google Scholar). The deduced amino acid sequence of the encoded protein contains 485 amino acid residues with a calculated molecular mass of 52.9 kDa and three sites of potentialN-linked glycosylation. Hydropathy analysis suggested this is an integral membrane protein with 10–12 membrane-spanning regions. A search of the non-redundant protein data bases did not identify any strong homologies, suggesting NAT2/AAP1 represented a new class of transport protein (13Hsu L.-C. Chiou T.-J. Chen L. Bush D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7441-7445Crossref PubMed Scopus (110) Google Scholar).Several amino acid transporter genes have now been isolated fromArabidopsis using functional complementation of yeast transport mutants (15Tanner W. Caspari T. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 595-626Crossref PubMed Scopus (78) Google Scholar). These include five clones that are closely related to NAT2/AAP1 (AAP2–6) (16Kwart M. Hirner B. Hummel S. Frommer W.B. Plant J. 1993; 4: 993-1002Crossref PubMed Scopus (96) Google Scholar, 17Fischer W.-N. Kwart M. Hummel S. Frommer W.B. J. Biol. Chem. 1995; 270: 16315-16320Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, Hirner B. Frommer W.B. Plant 1996; PubMed Scopus Google Scholar), a amino acid transporter W.B. Hummel S. M. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), two transporters and Hirner B. Frommer W.B. Plant 1996; PubMed Scopus Google Scholar), and a and histidine transporter L. Bush D.R. Plant PubMed Scopus Google Scholar). of the plant amino acid transporters have substrate specificity they or for related groups of amino The of genes for amino acid suggests there is in the function of these transporters in nitrogen in plants D.R. Chiou T.-J. Chen L. J. 1996; 47: PubMed Google the of amino acid transporters in plant is these transport proteins at the molecular This is a of the of with membrane proteins and the of these membrane proteins expressed in Chiou and R. Chiou and R. the molecular structure and function of plant amino acid our has NAT2/AAP1 as a example for We this symporter because it is a member of a large family of because it amino acids that are in the translocation and also because it is expressed in plant tissues, suggesting it plays an important role in nitrogen In the results reported here, we have investigated the topology of NAT2 in the plasma membrane as an important first in plant amino acid symporters at the molecular We its membrane topology by engineering a c-myc epitope the N or C and then we expressed the proteins in a in and in COS-1 the results here, we showed that the N terminus of NAT2/AAP1 is on the of the plasma and the C terminus outside the cell. This was by the to by the and NAT2 proteins and by of NAT2 in COS-1 cells. In partial of the in vitro protein peptide suggesting NAT2 has protein that are accessible to proteinase model of NAT2 is based on the and of the from the of protein and of the N and C termini of NAT2 on of the plasma we these data that NAT2 contains 11 novel 11 transmembrane domain model we propose is different from the model of the that contains a of transmembrane with N and C termini Biol. 1992; 4: PubMed Scopus Google Scholar, Biol. 1993; PubMed Scopus Google Scholar, Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, Sci. 1995; 4: PubMed Scopus Google Scholar). This contains many plasma membrane transport systems identified in plants, and function as and for a of organic and Biol. 1992; 4: PubMed Scopus Google Scholar, Biol. 1993; PubMed Scopus Google Scholar, Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, Sci. 1995; 4: PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, B. Plant J. PubMed Scopus Google Scholar). NAT2 is also with the family of and amino acid sequence across the Rev. 1995; PubMed Google Scholar, B. 1995; PubMed Scopus Google Scholar). This family contains PubMed Scopus Google Scholar), W. J. Biol. Chem. Full Text PDF PubMed Google Scholar), M. M. 1989; PubMed Scopus (79) Google Scholar), M. J. Biochem. 1990; PubMed Scopus Google Scholar), 1993; PubMed Scopus Google Scholar), and and A. Hall A. Mol. Biol. PubMed Scopus Google in S. and J. J. J. 1992; PubMed Google Scholar), 1990; PubMed Scopus Google Scholar), and J. J. 1991; PubMed Google in The amino acid in this family that are to have transmembrane Rev. 1995; PubMed Google Scholar, B. 1995; PubMed Scopus Google Scholar). the topology of was shown by analysis to have transmembrane J. A. J. J. 1995; PubMed Scopus Google Scholar). The related amino acid transporters of as the family of amino acid 1993; PubMed Scopus Google and the amino acid transporters W. J. Biol. Chem. Full Text PDF PubMed Google are also to have membrane-spanning the and topology is the of the N and C termini on either of the plasma The C terminus of several transport proteins has been in transport R. S. A. T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, M. R. J. Biol. Chem. 1991; Full Text PDF PubMed Google and in protein L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). a of NAT2/AAP1 transport of this Chen and R. the for this is this suggests that the C terminus an important role in transport Since the C terminus of NAT2/AAP1 is on the outside of the plasma this also the that this by NAT2 is also with amino acid transporters as Hirner B. Frommer W.B. Plant 1996; PubMed Scopus Google Scholar), and amino acid W.B. Hummel S. M. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). the topology is to the Arabidopsis of the of the family that they are J. J. Mol. Biol. PubMed Scopus Google Scholar). This our on NAT2 as a example of this there is a and of the to the topology this is oriented to the outside of the cell. This the that this in substrate we the amino acid in this of amino acid and have three amino acid and they transport the group of amino and in and substrate patterns W.-N. Kwart M. Hummel S. Frommer W.B. J. Biol. Chem. 1995; 270: 16315-16320Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, Hirner B. Frommer W.B. Plant 1996; PubMed Scopus Google Scholar). The of these a of amino acid transporters are identified in plants, it important to the molecular of these amino acid transporters in the of nitrogen across the plant as a The topology of NAT2/AAP1 is the of investigations of the structure and function of Amino acids are actively transported into plant cells by proton-coupled symporters (1Bush D.R. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1993; 44: 513-542Crossref Scopus (312) Google Scholar). These proteins link translocation across the plasma membrane to the proton-motive force generated by a P-type, H+-ATPase (2Briskin D.P. Biochem. Biophys. Acta. 1990; 1019: 95-109Crossref Scopus (109) Google Scholar, 3Serrano R. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1989; 40: 61-94Crossref Google Scholar). In plants, there are many heterotrophic tissue systems that are dependent upon carbon and nitrogen import for growth and development. Since amino acids are the primary form of nitrogen available to the heterotrophic plant tissues, the amino acid symporters are responsible for the systemic distribution of organic nitrogen, and therefore, they are essential contributors to plant growth (4Pate J.S. Stewart F.C. Bidwell R.G.S. Plant Physiology: A Treatise, Vol. VIII: Nitrogen Metabolism. Academic Press, New York1983: 335-401Google Scholar, 5Bush D.R. Singh B. Plant Amino Acids: Biochemistry and Biotechnology. Marcel Dekker, NY1998Google Scholar). Detailed investigations of the transport properties and bioenergetics of these symporters using isolated plasma membrane vesicles and imposed proton electrochemical potential differences have shown that they are electrogenic transporters that are driven by either transmembrane proton or electrical potential differences (6Li Z.-C. Bush D.R. Plant Physiol. 1990; 94: 268-277Crossref PubMed Scopus (66) Google Scholar). These transporters are inhibited by chemical modification of histidine residues by diethyl pyrocarbonate (6Li Z.-C. Bush D.R. Plant Physiol. 1990; 94: 268-277Crossref PubMed Scopus (66) Google Scholar), and substrate protection experiments suggest the sensitive residue is at or near the substrate binding site (5Bush D.R. Singh B. Plant Amino Acids: Biochemistry and Biotechnology. Marcel Dekker, NY1998Google Scholar, 7Bush D.R. Li Z.-C. Bonnemain J.L. Delrot S. Lucas W.J. Dainty J. Recent Advances in Phloem Transport and Assimilate Compartmentation. Presses Academiques, France1991: 148-153Google Scholar). Several classes of symporters were initially resolved based on expression patterns and substrate specificity (6Li Z.-C. Bush D.R. Plant Physiol. 1990; 94: 268-277Crossref PubMed Scopus (66) Google Scholar,8Li Z.-C. Bush D.R. Plant Physiol. 1991; 96: 1338-1344Crossref PubMed Scopus (44) Google Scholar, 9Li Z.-C. Bush D.R. Arch. Biochem. Biophys. 1992; 294: 519-526Crossref PubMed Scopus (32) Google Scholar, 10Williams L.E. Nelson S.J. Hall J.L. Planta. 1990; 182: 540-545Crossref PubMed Scopus (58) Google Scholar, 11Williams L.E. Nelson S.J. Hall J.L. Planta. 1992; 186: 541-550Crossref PubMed Scopus (43) Google Scholar). Transport competition experiments showed that binding sites are stereo-specific and identified the carboxylic acid, the alpha amino group, and substitutions at the β-carbon as important determinants in governing substrate binding (8Li Z.-C. Bush D.R. Plant Physiol. 1991; 96: 1338-1344Crossref PubMed Scopus (44) Google Scholar, 9Li Z.-C. Bush D.R. Arch. Biochem. Biophys. 1992; 294: 519-526Crossref PubMed Scopus (32) Google Scholar). Recently, one of these symporters was expressed in Xenopus oocytes, and electrophysiological methods allowed for a high resolution investigation of transport kinetics that suggests these transporters operate by a simultaneous binding mechanism (12Boorer K.J. Frommer W.B. Bush D.R. Kreman M. Loo D.D.F. Wright E.M. J. Biol. Chem. 1996; 271: 2213-2220Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The first plant amino acid symporter cloned (NAT2/AAP1) was identified by two groups using functional complementation of yeast amino acid transport mutants with different Arabidopsis cDNA expression libraries (13Hsu L.-C. Chiou T.-J. Chen L. Bush D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7441-7445Crossref PubMed Scopus (110) Google Scholar, 14Frommer W.B. Hummel S. Riesmeier J.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5944-5948Crossref PubMed Scopus (159) Google Scholar). The deduced amino acid sequence of the encoded protein contains 485 amino acid residues with a calculated molecular mass of 52.9 kDa and three sites of potentialN-linked glycosylation. Hydropathy analysis suggested this is an integral membrane protein with 10–12 membrane-spanning regions. A search of the non-redundant protein data bases did not identify any strong homologies, suggesting NAT2/AAP1 represented a new class of transport protein (13Hsu L.-C. Chiou T.-J. Chen L. Bush D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7441-7445Crossref PubMed Scopus (110) Google Scholar). Several amino acid transporter genes have now been isolated fromArabidopsis using functional complementation of yeast transport mutants (15Tanner W. Caspari T. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 595-626Crossref PubMed Scopus (78) Google Scholar). These include five clones that are closely related to NAT2/AAP1 (AAP2–6) (16Kwart M. Hirner B. Hummel S. Frommer W.B. Plant J. 1993; 4: 993-1002Crossref PubMed Scopus (96) Google Scholar, 17Fischer W.-N. Kwart M. Hummel S. Frommer W.B. J. Biol. Chem. 1995; 270: 16315-16320Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, Hirner B. Frommer W.B. Plant 1996; PubMed Scopus Google Scholar), a amino acid transporter W.B. Hummel S. M. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), two transporters and Hirner B. Frommer W.B. Plant 1996; PubMed Scopus Google Scholar), and a and histidine transporter L. Bush D.R. Plant PubMed Scopus Google Scholar). of the plant amino acid transporters have substrate specificity they or for related groups of amino The of genes for amino acid suggests there is in the function of these transporters in nitrogen in plants D.R. Chiou T.-J. Chen L. J. 1996; 47: PubMed Google Scholar). the of amino acid transporters in plant is these transport proteins at the molecular This is a of the of with membrane proteins and the of these membrane proteins expressed in Chiou and R. Chiou and R. the molecular structure and function of plant amino acid our has NAT2/AAP1 as a example for We this symporter because it is a member of a large family of because it amino acids that are in the translocation and also because it is expressed in plant tissues, suggesting it plays an important role in nitrogen In the results reported here, we have investigated the topology of NAT2 in the plasma membrane as an important first in plant amino acid symporters at the molecular We its membrane topology by engineering a c-myc epitope the N or C and then we expressed the proteins in a in and in COS-1 cells. the results here, we showed that the N terminus of NAT2/AAP1 is on the of the plasma and the C terminus outside the cell. This was by the to by the and NAT2 proteins and by of NAT2 in COS-1 cells. In partial of the in vitro protein peptide suggesting NAT2 has protein that are accessible to proteinase model of NAT2 is based on the and of the from the of protein and of the N and C termini of NAT2 on of the plasma we these data that NAT2 contains 11 novel 11 transmembrane domain model we propose is different from the model of the that contains a of transmembrane with N and C termini Biol. 1992; 4: PubMed Scopus Google Scholar, Biol. 1993; PubMed Scopus Google Scholar, Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, Sci. 1995; 4: PubMed Scopus Google Scholar). This contains many plasma membrane transport systems identified in plants, and function as and for a of organic and Biol. 1992; 4: PubMed Scopus Google Scholar, Biol. 1993; PubMed Scopus Google Scholar, Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, Sci. 1995; 4: PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, B. Plant J. PubMed Scopus Google Scholar). NAT2 is also with the family of and amino acid sequence across the Rev. 1995; PubMed Google Scholar, B. 1995; PubMed Scopus Google Scholar). This family contains PubMed Scopus Google Scholar), W. J. Biol. Chem. Full Text PDF PubMed Google Scholar), M. M. 1989; PubMed Scopus (79) Google Scholar), M. J. Biochem. 1990; PubMed Scopus Google Scholar), 1993; PubMed Scopus Google Scholar), and and A. Hall A. Mol. Biol. PubMed Scopus Google in S. and J. J. J. 1992; PubMed Google Scholar), 1990; PubMed Scopus Google Scholar), and J. J. 1991; PubMed Google in The amino acid in this family that are to have transmembrane Rev. 1995; PubMed Google Scholar, B. 1995; PubMed Scopus Google Scholar). the topology of was shown by analysis to have transmembrane J. A. J. J. 1995; PubMed Scopus Google Scholar). The related amino acid transporters of as the family of amino acid 1993; PubMed Scopus Google and the amino acid transporters W. J. Biol. Chem. Full Text PDF PubMed Google are also to have membrane-spanning the and topology is the of the N and C termini on either of the plasma The C terminus of several transport proteins has been in transport R. S. A. T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, M. R. J. Biol. Chem. 1991; Full Text PDF PubMed Google and in protein L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). a of NAT2/AAP1 transport of this Chen and R. the for this is this suggests that the C terminus an important role in transport Since the C terminus of NAT2/AAP1 is on the outside of the plasma this also the that this by NAT2 is also with amino acid transporters as Hirner B. Frommer W.B. Plant 1996; PubMed Scopus Google Scholar), and amino acid W.B. Hummel S. M. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). the topology is to the Arabidopsis of the of the family that they are J. J. Mol. Biol. PubMed Scopus Google Scholar). This our on NAT2 as a example of this there is a and of the to the topology this is oriented to the outside of the cell. This the that this in substrate we the amino acid in this of amino acid and have three amino acid and they transport the group of amino and in and substrate patterns W.-N. Kwart M. Hummel S. Frommer W.B. J. Biol. Chem. 1995; 270: 16315-16320Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, Hirner B. Frommer W.B. Plant 1996; PubMed Scopus Google Scholar). The of these a of amino acid transporters are identified in plants, it important to the molecular of these amino acid transporters in the of nitrogen across the plant as a The topology of NAT2/AAP1 is the of investigations of the structure and function of In the results here, we showed that the N terminus of NAT2/AAP1 is on the of the plasma and the C terminus outside the cell. This was by the to by the and NAT2 proteins and by of NAT2 in COS-1 cells. In partial of the in vitro protein peptide suggesting NAT2 has protein that are accessible to proteinase model of NAT2 is based on the and of the from the of protein and of the N and C termini of NAT2 on of the plasma we these data that NAT2 contains 11 The novel 11 transmembrane domain model we propose is different from the model of the that contains a of transmembrane with N and C termini Biol. 1992; 4: PubMed Scopus Google Scholar, Biol. 1993; PubMed Scopus Google Scholar, Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, Sci. 1995; 4: PubMed Scopus Google Scholar). This contains many plasma membrane transport systems identified in plants, and function as and for a of organic and Biol. 1992; 4: PubMed Scopus Google Scholar, Biol. 1993; PubMed Scopus Google Scholar, Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, Sci. 1995; 4: PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, B. Plant J. PubMed Scopus Google Scholar). NAT2 is also with the family of and amino acid sequence across the Rev. 1995; PubMed Google Scholar, B. 1995; PubMed Scopus Google Scholar). This family contains PubMed Scopus Google Scholar), W. J. Biol. Chem. Full Text PDF PubMed Google Scholar), M. M. 1989; PubMed Scopus (79) Google Scholar), M. J. Biochem. 1990; PubMed Scopus Google Scholar), 1993; PubMed Scopus Google Scholar), and and A. Hall A. Mol. Biol. PubMed Scopus Google in S. and J. J. J. 1992; PubMed Google Scholar), 1990; PubMed Scopus Google Scholar), and J. J. 1991; PubMed Google in The amino acid in this family that are to have transmembrane Rev. 1995; PubMed Google Scholar, B. 1995; PubMed Scopus Google Scholar). the topology of was shown by analysis to have transmembrane J. A. J. J. 1995; PubMed Scopus Google Scholar). The related amino acid transporters of as the family of amino acid 1993; PubMed Scopus Google and the amino acid transporters W. J. Biol. Chem. Full Text PDF PubMed Google are also to have membrane-spanning regions. The the and topology is the of the N and C termini on either of the plasma The C terminus of several transport proteins has been in transport R. S. A. T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, M. R. J. Biol. Chem. 1991; Full Text PDF PubMed Google and in protein L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). a of NAT2/AAP1 transport of this Chen and R. the for this is this suggests that the C terminus an important role in transport Since the C terminus of NAT2/AAP1 is on the outside of the plasma this also the that this by NAT2 is also with amino acid transporters as Hirner B. Frommer W.B. Plant 1996; PubMed Scopus Google Scholar), and amino acid W.B. Hummel S. M. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). the topology is to the Arabidopsis of the of the family that they are J. J. Mol. Biol. PubMed Scopus Google Scholar). This our on NAT2 as a example of this there is a and of the to the topology this is oriented to the outside of the cell. This the that this in substrate we the amino acid in this of amino acid and have three amino acid and they transport the group of amino and in and substrate patterns W.-N. Kwart M. Hummel S. Frommer W.B. J. Biol. Chem. 1995; 270: 16315-16320Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, Hirner B. Frommer W.B. Plant 1996; PubMed Scopus Google Scholar). The of these a of amino acid transporters are identified in plants, it important to the molecular of these amino acid transporters in the of nitrogen across the plant as a The topology of NAT2/AAP1 is the of investigations of the structure and function of We for the and Chen of and of for the in experiments and with COS-1 and