Identification of a Disulfide Bridge in Sodium-Coupled Neutral Amino Acid Transporter 2(SNAT2) by Chemical Modification

Chen Chen,Yanmeng Yuan,Jiahong Wang,Ruiping Cai,Zhou Zhang,Christof Grewer,Zhanyun Guo

doi:10.1371/journal.pone.0158319

Abstract

Sodium-coupled neutral amino acid transporter 2 (SNAT2) belongs to solute carrier 38 (SLC38) family of transporters, which is ubiquitously expressed in mammalian tissues and mediates transport of small, neutral amino acids, exemplified by alanine(Ala, A). Yet structural data on SNAT2, including the relevance of intrinsic cysteine residues on structure and function, is scarce, in spite of its essential roles in many tissues. To better define the potential of intrinsic cysteines to form disulfide bonds in SNAT2, mutagenesis experiments and thiol-specific chemical modifications by N-ethylmaleimide (NEM) and methoxy-polyethylene glycol maleimide (mPEG-Mal, MW 5000) were performed, with or without the reducing regent dithiothreitol (DTT) treatment. Seven single mutant transporters with various cysteine (Cys, C) to alanine (Ala, A) substitutions, and a C245,279A double mutant were introduced to SNAT2 with a hemagglutinin (HA) tag at the C-terminus. The results showed that the cells expressing C245A or C279A were labeled by one equivalent of mPEG-Mal in the presence of DTT, while wild-type or all the other single Cys to Ala mutants were modified by two equivalents of mPEG-Mal. Furthermore, the molecular weight of C245,279A was not changed in the presence or absence of DTT treatment. The results suggest a disulfide bond between Cys245 and Cys279 in SNAT2 which has no effect on cell surface trafficking, as well as transporter function. The proposed disulfide bond may be important to delineate proximity in the extracellular domain of SNAT2 and related proteins.

Highlights

The solute carrier 38 (SLC38) family of transporters represents a main branch of solute carrier families in mammals [1]
In order to identify the existence of potential disulfide bridges in Sodium-coupled neutral amino acid transporter 2 (SNAT2), a chemical modification approach was performed utilizing wild-type SNAT2-HA with or without the reducing reagent DTT
Followed by DTT treatment under denaturing conditions, a SNAT2-HA band with a larger molecular mass of about 110 kDa could be observed (Fig 1A, lane 3), suggesting that cysteine residues of SNAT2 may be engaged in disulfide bridges

Summary

Introduction

The SLC38 family of transporters represents a main branch of solute carrier families in mammals [1]. Most of the 11 transporters in this family are Na+-dependent and are able to carry out net transport of neutral amino acids except 5 orphan transporters [2]. The other six of these family members have been well-characterized to date and have been subdivided into System A (SNAT1, SNAT2 and SNAT4) [3,4,5,6].and System N (SNAT3, SNAT5 and SNAT7) [7,8,9,10,11] transporters, in terms of their functional properties and patterns of regulation. System A prefers small aliphatic amino acids, while System N has a much narrower substrate profiles of glutamine, asparagine and histidine [12, 13]. System A transports substrates coupled to the uptake of Na+ with a stoichiometry of 1:1 [15]

Methods

Results

Conclusion