title: Membrane topological structure of neutral system N/A amino acid transporter 4 (SNAT4) protein.
abstract: Members of system N/A amino acid transporter (SNAT) family mediate transport of neutral amino acids, including l-alanine, l-glutamine, and l-histidine, across the plasma membrane and are involved in a variety of cellular functions. By using chemical labeling, glycosylation, immunofluorescence combined with molecular modeling approaches, we resolved the membrane topological structure of SNAT4, a transporter expressed predominantly in liver. To analyze the orientation using the chemical labeling and biotinylation approach, the "Cys-null" mutant of SNAT4 was first generated by mutating all five endogenous cysteine residues. Based on predicted topological structures, a single cysteine residue was introduced individually into all possible nontransmembrane domains of the Cys-null mutant. The cells expressing these mutants were labeled with N-biotinylaminoethyl methanethiosulfonate, a membrane-impermeable cysteine-directed reagent. We mapped the orientations of N- and C-terminal domains. There are three extracellular loop domains, and among them, the second loop domain is the largest that spans from amino acid residue ∼242 to ∼335. The orientation of this domain was further confirmed by the identification of two N-glycosylated residues, Asn-260 and Asn-264. Together, we showed that SNAT4 contains 10 transmembrane domains with extracellular N and C termini and a large N-glycosylated, extracellular loop domain. This is the first report concerning membrane topological structure of mammalian SNAT transporters, which will provide important implications for our understanding of structure-function of the members in this amino acid transporter family.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900.
authors.forename: Qian
authors.initials: Q
authors.lastname: Shi
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900.
authors.forename: Rugmani
authors.initials: R
authors.lastname: Padmanabhan
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900.
authors.forename: Carla J
authors.initials: CJ
authors.lastname: Villegas
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900.
authors.forename: Sumin
authors.initials: S
authors.lastname: Gu
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900. Electronic address: jiangj@uthscsa.edu.
authors.forename: Jean X
authors.initials: JX
authors.lastname: Jiang
mesh_terms: D026924:Amino Acid Transport System A / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D026924:Amino Acid Transport System A; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C507352:SLC38A4 protein, human; C420522:Slc38a4 protein, mouse; D001216:Asparagine