Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter.
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 tra...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3579933?pdf=render |
| _version_ | 1828261734322798592 |
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| author | Rugmani Padmanabhan Iyer Sumin Gu Bruce J Nicholson Jean X Jiang |
| author_facet | Rugmani Padmanabhan Iyer Sumin Gu Bruce J Nicholson Jean X Jiang |
| author_sort | Rugmani Padmanabhan Iyer |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T03:44:16Z |
| format | Article |
| id | doaj.art-0849e18172ce40ec91c19b557dd8139e |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-13T03:44:16Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-0849e18172ce40ec91c19b557dd8139e2022-12-22T03:04:03ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0182e5679210.1371/journal.pone.0056792Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter.Rugmani Padmanabhan IyerSumin GuBruce J NicholsonJean X JiangSNAT4 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.http://europepmc.org/articles/PMC3579933?pdf=render |
| spellingShingle | Rugmani Padmanabhan Iyer Sumin Gu Bruce J Nicholson Jean X Jiang Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter. PLoS ONE |
| title | Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter. |
| title_full | Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter. |
| title_fullStr | Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter. |
| title_full_unstemmed | Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter. |
| title_short | Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter. |
| title_sort | identification of a disulfide bridge important for transport function of snat4 neutral amino acid transporter |
| url | http://europepmc.org/articles/PMC3579933?pdf=render |
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