Document Type
Article
Publication Date
6-2016
Keywords
tissue expression pattern; system-a; glutamine transporter; functional-characteristics; cysteine residues; skeletal-muscle; families slc32; snat2; cell; subtype
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.
Publisher Attribution
Chen, C., Wang, J., Cai, R., Yuan, Y., Guo, Z., Grewer, C., & Zhang, Z. (2016). Identification of a Disulfide Bridge in Sodium-Coupled Neutral Amino Acid Transporter 2 (SNAT2) by Chemical Modification. PloS one, 11(6), e0158319.
Recommended Citation
Chen, Chen; Wang, Jiahong; Cai, Ruiping; Yuan, Yanmen M.; Guo, Zhanyun Y.; Grewer, Christof; and Zhang, Zhou, "Identification of a Disulfide Bridge in Sodium-Coupled Neutral Amino Acid Transporter 2(SNAT2) by Chemical Modification" (2016). Chemistry Faculty Scholarship. 1.
https://orb.binghamton.edu/chem_fac/1