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

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

Qian Shi  1 , Rugmani Padmanabhan  1 , Carla J Villegas  1 , Sumin Gu  1 , Jean X Jiang  2
Affiliations

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

Qian Shi et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Predicted membrane topological models of SNAT4. A, two putative topological models of SNAT4 were constructed using TMpred and TopPred and confirmed by other methods listed in Table 1. The major difference between these two models is the orientation of the largest loop domain, which spans from amino acid residue ∼242 to ∼335. This results in the different membrane orientations of the last four extra- or intracellular loops. The mutations as indicated were generated based on these two models. B, three-dimensional structure models of SNAT4 were generated by HHpred or SWISS-MODEL, using Arg-bound AdiC protein (Protein Data Bank code 3L1L) as a template. NT, N terminus; CT, C terminus. C, SNAT4-c-myc construct was generated by fusing c-myc tag with SNAT4 in pcDNA 3.1 vector. The Cys-null mutant was generated by replacing all endogenous cysteines with alanines. Expression of wild-type and the Cys-null mutant of SNAT4 in CHO cells was examined by immunolabeling with anti-c-myc antibody. Scale bar, 20 μm.
FIGURE 2.
FIGURE 2.
N terminus of SNAT4 is oriented at the extracellular side of the cell determined by immunofluorescence and MTSEA-biotin chemical labeling. A, SNAT4 protein was immunolabeled by an antibody against the N terminus of SNAT4 expressed in HepIR cells in the absence of Triton X-100. The SNAT4 signals were detected by FITC-conjugated goat anti-rabbit IgG secondary antibody. Nucleus was counterstained with DAPI. Scale bar, 20 μm. B, using the Cys-null mutant as a template, cysteine was individually introduced back to generate 18C and T54C mutants. MTSEA-biotin labeling was performed in CHO cells expressing exogenous WT, Cys-null, 18C, and T54C mutants of SNAT4. The lysates of the cell labeled with MTSEA-biotin were loaded on NeutrAvidin beads. Biotinylated proteins (upper) and the preloaded cell lysates (lower) were immunoblotted with affinity-purified anti-SNAT4 antibody or anti-GAPDH antibody.
FIGURE 3.
FIGURE 3.
C terminus of SNAT4 faces the extracellular side of the cell. A, CHO cells expressing exogenous c-myc-tagged SNAT4 were immunolabeled with anti-c-myc antibody in the absence of Triton X-100. Nuclei were counterstained with DAPI. Scale bar, 20 μm. B, Cys-null mutant was generated by replacing all endogenous cysteines with alanines. The N543C mutant was generated using the Cys-null mutant as a template. Cells expressing WT, N543C, and vector control were labeled with MTSEA-biotin. The isolated biotinylated proteins (upper) and the preloaded cell lysates (lower) were immunoblotted with anti-SNAT4 antibody or anti-GAPDH antibody.
FIGURE 4.
FIGURE 4.
Identification of extracellular loop domains of SNAT4 using MTSEA-biotin chemical labeling. DNA constructs containing single cysteine (T129C, A183C, L219C, 232C, 249C, 321C, 345C, T366C, A401C, T440C, I474C, and L507C) on the Cys-null template of SNAT4 were generated and transfected into CHO cells. The cells were labeled with MTSEA-biotin, and the isolated biotinylated proteins (upper) and the preloaded cell lysates (lower) were immunoblotted with anti-SNAT4 antibody or anti-GAPDH antibody. A, amino acid residues at positions 183, 249, and 321 are located at the extracellular loop domains. B, amino acid residue at 401 is located at an extracellular loop domain.
FIGURE 5.
FIGURE 5.
MTSEA-biotin reacts with all single cysteine mutants of SNAT4 in the presence of detergent. A, CHO cells expressing exogenous WT or Cys-null mutant were pretreated with or without 0.25% Triton X-100 prior to MTSEA-biotin labeling. The isolated biotinylated proteins or the preloaded cell lysates were immunoblotted with anti-SNAT4 antibody (upper) or anti-β-actin/anti-GAPDH antibody (lower). B–D, CHO cells expressing exogenous WT, single cysteine mutants generated based on the Cys-null template, the Cys-null mutant or SNAT1, another member of SNAT family, were pretreated with or without 0.25% Triton X-100 prior to MTSEA-biotin labeling. The isolated biotinylated proteins were immunoblotted with anti-SNAT4 antibody (upper) or anti-β-actin antibody as control (lower).
FIGURE 6.
FIGURE 6.
Asn-260 and Asn-264 located in the largest loop domain are modified by N-linked glycosylation. A, the N-linked glycosylated sites of SNAT4 protein were predicted by NetNGlyc 1.0 server. B, two predicted N-glycosylated sites, Asn-260 and Asn-264, were mutated to Gln. WT and single and double mutants of SNAT4 were expressed in CHO cells. Isolated membrane samples were immunoblotted with anti-SNAT4 antibody. Membrane protein samples isolated from the cell expressing WT SNAT4 were treated with PNGase-F and immunoblotted with anti-SNAT4 antibody.
FIGURE 7.
FIGURE 7.
Schematic illustration of SNAT4 topological structure and the location of mutation sites used. Based on the data we obtained, the diagram of topological structure of SNAT4 was generated using the TOPO2 server and modified by Photoshop software (Adobe Systems, San Jose, CA). Five endogenous cysteines are labeled in black up arrows; other single cysteine mutation sites are represented as down arrows; two glycosylation sites, Asn-260 and Asn-264, are indicated in hexagons.
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