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

J Biol Chem. 2011 Nov 4;286(44):38086-38094. doi: 10.1074/jbc.M111.220277. Epub 2011 Sep 14.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Transport System A / chemistry*
  • Amino Acid Transport System A / metabolism
  • Animals
  • Asparagine / chemistry
  • CHO Cells
  • Cell Line
  • Cricetinae
  • Cricetulus
  • Glycosylation
  • Humans
  • Membrane Proteins / chemistry
  • Membrane Transport Proteins / chemistry*
  • Mice
  • Mutagenesis, Site-Directed
  • Open Reading Frames
  • Protein Conformation
  • Protein Structure, Tertiary

Substances

  • Amino Acid Transport System A
  • Membrane Proteins
  • Membrane Transport Proteins
  • SLC38A4 protein, human
  • Slc38a4 protein, mouse
  • Asparagine