Padmanabhan Iyer R et al. (2013), Identification of a disulfide bridge important ...

XB-ART-48187

PLoS One 2013 Jan 01;82:e56792. doi: 10.1371/journal.pone.0056792.

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Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter.

Padmanabhan Iyer R , Gu S , Nicholson BJ , Jiang JX .

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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.

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Species referenced: Xenopus laevis
Genes referenced: aanat actl6a nhs slc38a4

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	Figure 2. Cys-null mutant of SNAT4 completely loses transport function, but is capable of expressing on the cell surface.(A) The locations of the 5 cysteine residues are indicated (arrows) in the determined topological structure of SNAT4 [12]. (B) cRNA of the wild type and Cys-null mutant was injected into the oocytes and subjected to [3H] L-alanine uptake assays. Water injected oocytes were used as a negative control. The transporter activity obtained was normalized with the protein expression data. Data is presented as mean ± SEM, n = 3 (∼10 oocytes/sample). Cys-null versus WT, *, P<0.001. (C) Xenopus oocytes injected with wild type and Cys-null mutant cRNA were surface biotinylated with NHS-SS-Biotin. Biotinylated proteins and the pre-loaded cell lysates (pre-loading) were also immunoblotted with anti-SNAT4 antibody or anti-pan-actin antibody. The ratio of biotinylated versus total SNAT4 was quantified and the data is presented as mean ± SEM, n = 20. The levels of SNAT4 expressed on the cell surface and corresponding pre-loaded SNAT4 were quantified by Scion Image software and the percentage of biotinylated versus total SNAT4 was calculated. Data is presented as mean ± SEM, n = 3 (∼ 20 oocytes/sample). All mutants versus WT, *, P<0.001.
	Figure 3. Replacing any four cysteines fails to recover transporter activity.DNA constructs containing 4 cysteine to alanine mutations with a single cysteine remaining, Cys-18 (18C), Cys-232(232C), Cys-249 (249C), Cys-321 (321C) or Cys-345 (345C) were generated by PCR using Cys-null SNAT4 as a DNA template. The cRNAs were injected in Xenopus oocytes. The transport activity was determined and the data was normalized with the SNAT4 protein level. Data is presented as mean ± SEM, n = 3 (∼10 oocytes/sample). All mutants versus WT, ***, P<0.001.
	Figure 4. Residues Cys-249 and Cys-321 are functionally involved in transport function of SNAT4.(A) DNA constructs containing single cysteine site mutants, C18A, C232A, C249A, C321A and C345A were generated by PCR using WT SNAT4 as a template. cRNAs were injected in Xenopus oocytes. The transport activity was determined and the data was normalized with the protein expression. The transport activity of both C249A and C321A mutants was completely abolished. Data is presented as mean ± SEM, n = 3 (∼ 10 oocytes/sample). All mutants versus WT, *, P<0.001. (B) DNA construct containing 3 cysteine to alanine mutations with retained Cys-249 and Cys-321 residues (C18A, C232A, C345A) was generated by site directed mutagenesis and the transporter activity was determined by uptake assay. After normalization with total protein expression of SNAT4 variants in oocyte (left panel), the mutant showed partial recovery in L-alanine transport as compared to the water injected control. Data is presented as mean ± SEM, n = 3 (∼10 oocytes/sample). The mutant versus WT, *, P<0.001.
	Figure 5. Residues Cys-249 and Cys-321 are linked by disulfide bridge.Xenopus oocytes injected with cRNAs of WT (lane 1), Cys-null mutant (lane 2), or mutants with retained 2 cysteine residues, Cys-249 and Cys-321 (C18A, C232A, C345A) (lane 3), 1 cysteine residue Cys-249 (249C) (lane 4) or 1 cysteine residue, Cys-321 (321C) (lane 5) were surface biotinylated by cysteine labeling with MTSEA-Biotin. Preloaded cell lysates (Pre-loading) and biotinylated samples were immunoblotted with anti-SNAT4 antibody or anti-pan-actin antibody (∼ 80 oocytes/sample).
	Figure 6. Transporter activity of cysteine mutant with only intact disulfide bridge (Cys-249 and Cys-321 residues) decreases in the presence of DTT, TCEP and GSH.Wild type and mutant SNAT4 expressing oocytes were incubated with DTT (0–10 mM) (A), TCEP (0–10 mM) (B) and 10 mM GSH (D). [3H]-alanine uptake assay was performed. Under reducing conditions, L-alanine transport in mutant SNAT4 significantly decreased. (C) Mutant SNAT4 expressing oocytes were incubated with 1 mM TCEP in the presence and absence of 0.02% H2O2. Under oxidative conditions, L-alanine transport in mutant SNAT4 was significantly recovered. Data is presented as mean ± SEM, n = 3 (∼ 10 oocytes/sample). Treated versus untreated control of the mutant, ***, P<0.001.
	Figure 7. Disruption of the disulfide bridge partially loses L-arginine transport function of SNAT4.cRNA of the wild type, C18A, C232A, C345A or C321A mutant was injected into Xenopus oocytes and subjected to [3H] L-arginine uptake assays. Water injected oocytes were used as a negative control. Data is presented as mean ± SEM, n = 3 (∼10 oocytes/sample). Mutants versus WT, *, P<0.05.
	Figure 1. Dose-dependent Inhibition of SNAT4 Transport Activity by DTT and TCEP.Xenopus oocytes expressing wild type SNAT4 were preincubated with DTT (0–10 mM) (A) or TCEP (0–10 mM) (B) for 30 min. [3H] L-alanine uptake assay was then performed in the presence of DTT or TCEP. Water injected oocytes were used as a negative control. L-alanine uptake was significantly decreased in a dose-dependent manner compared to untreated control. Data is presented as mean ± SEM, n = 3 (∼ 10 oocytes/sample). DTT or TCEP at 1 and 10 mM versus untreated control of SNAT4, ***, P<0.001.

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