Membrane topological structure of neutral system N/A amino acid transporter 4 (SNAT4) protein

Qian Shi, Rugmani Padmanabhan, Carla J. Villegas, Sumin Gu, Jean X. Jiang

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8 Scopus citations

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 Cysnull 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.

Original languageEnglish (US)
Pages (from-to)38086-38094
Number of pages9
JournalJournal of Biological Chemistry
Volume286
Issue number44
DOIs
StatePublished - Nov 4 2011

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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