title: Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter.
abstract: SNAT4 is a member of system N/A amino acid transport family that primarily expresses in liver and muscles and mediates the transport of L-alanine. However, little is known about the structure and function of the SNAT family of transporters. In this study, we showed a dose-dependent inhibition in transporter activity of SNAT4 with the treatment of reducing agents, dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine (TCEP), indicating the possible involvement of disulfide bridge(s). Mutation of residue Cys-232, and the two highly conserved residues Cys-249 and Cys-321, compromised the transport function of SNAT4. However, this reduction was not caused by the decrease of SNAT4 on the cell surface since the cysteine-null mutant generated by replacing all five cysteines with alanine was equally capable of being expressed on the cell surface as wild-type SNAT4. Interestingly, by retaining two cysteine residues, 249 and 321, a significant level of L-alanine uptake was restored, indicating the possible formation of disulfide bond between these two conserved residues. Biotinylation crosslinking of free thiol groups with MTSEA-biotin provided direct evidence for the existence of a disulfide bridge between Cys-249 and Cys-321. Moreover, in the presence of DTT or TCEP, transport activity of the mutant retaining Cys-249 and Cys-321 was reduced in a dose-dependent manner and this reduction is gradually recovered with increased concentration of H2O2. Disruption of the disulfide bridge also decreased the transport of L-arginine, but to a lesser degree than that of L-alanine. Together, these results suggest that cysteine residues 249 and 321 form a disulfide bridge, which plays an important role in substrate transport but has no effect on trafficking of SNAT4 to the cell surface.
authors: Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas, United States of America.
authors: Rugmani
authors: R
authors: Padmanabhan Iyer
authors: Sumin
authors: S
authors: Gu
authors: Bruce J
authors: BJ
authors: Nicholson
authors: Jean X
authors: JX
authors: Jiang
mesh_terms: D026924:Amino Acid Transport System A / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D019921:Biotinylation; D004220:Disulfides / Q000737:chemistry*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D026924:Amino Acid Transport System A; D004220:Disulfides