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. 2009;4(4):e5315.
doi: 10.1371/journal.pone.0005315. Epub 2009 Apr 24.

SVOP Is a Nucleotide Binding Protein

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Free PMC article

SVOP Is a Nucleotide Binding Protein

Jia Yao et al. PLoS One. .
Free PMC article

Abstract

Background: Synaptic Vesicle Protein 2 (SV2) and SV2-related protein (SVOP) are transporter-like proteins that localize to neurotransmitter-containing vesicles. Both proteins share structural similarity with the major facilitator (MF) family of small molecule transporters. We recently reported that SV2 binds nucleotides, a feature that has also been reported for another MF family member, the human glucose transporter 1 (Glut1). In the case of Glut1, nucleotide binding affects transport activity. In this study, we determined if SVOP also binds nucleotides and assessed its nucleotide binding properties.

Methodology/principal findings: We performed in vitro photoaffinity labeling experiments with the photoreactive ATP analogue, 8-azido-ATP[gamma] biotin and purified recombinant SVOP-FLAG fusion protein. We found that SVOP is a nucleotide-binding protein, although both its substrate specificity and binding site differ from that of SV2. Within the nucleotides tested, ATP, GTP and NAD show same level of inhibition on SVOP-FLAG labeling. Dose dependent studies indicated that SVOP demonstrates the highest affinity for NAD, in contrast to SV2, which binds both NAD and ATP with equal affinity. Mapping of the binding site revealed a single region spanning transmembrane domains 9-12, which contrasts to the two binding sites in the large cytoplasmic domains in SV2A.

Conclusions/significance: SVOP is the third MF family member to be found to bind nucleotides. Given that the binding sites are unique in SVOP, SV2 and Glut1, this feature appears to have arisen separately.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Purified SVOP-FLAG fusion proteins are labeled with 8-azido-ATP-biotin.
Recombinant SVOP-FLAG fusion protein was purified from transfected HEK293 cells with Anti-FLAG M2 affinity gel. About 5 µg protein preparation was used in each photoaffinity labeling reaction with 100 µM 8-azido-ATP-biotin in the presence or absence of 1 mM non-photoreactive ATP. A control without UV photolysis was set up in parallel. The samples were resolved by SDS-PAGE and transferred to PVDF membrane for western blot analysis. The bound 8-azido-ATP was visualized by ExtrAvidin-HRP and anti-FLAG antibody was used to detect the proteins.
Figure 2
Figure 2. 8-azido-ATP binding to SVOP-FLAG is saturable and displays a binding affinity of 83 µM.
Purified SVOP-FLAG was labeled with the indicated concentrations of 8-azido-ATP-biotin and subjected to SDS-PAGE and western blot analysis. The net intensity of the regions of interest was quantified using a Kodak Image Station 440. A, Representative western blot result of SVOP-FLAG labeling as a function of 8-azido-ATP concentration. B, Quantification of the western data. The data were expressed as the intensity of 8-azido-ATP labeling normalized to SVOP protein signals. The error bars represent SEM (n = 4).
Figure 3
Figure 3. Nucleotide specificity of the 8-azido-ATP binding to SVOP.
SVOP-FLAG was labeled with 100 µM 8-azido-ATP in the absence or presence of 1 mM indicated competitive nucleotides. Samples were subjected to SDS-PAGE and western blot followed by quantitative analysis. Panel A shows a representative western blot result. Panel B shows the quantification of the western blot data. The error bars represent SEM, n = 5.
Figure 4
Figure 4. SVOP demonstrates highest affinity for NAD.
SVOP-FLAG was labeled with 100 µM 8-azido-ATP in the absence or presence of ATP (0.25–1.5 mM) or NAD (0.25–1.5 mM). Data were expressed as the percentage of 8-azido-ATP labeling according to control with no ATP or NAD in the reaction. A and B were representative western blot results of SVOP labeling in the presence of different concentration of ATP (A) or NAD (B). C shows the quantification of the western blot results. Error bars represent SEM, n = 3. The half maximum inhibition concentration of NAD and ATP on SVOP ATP binding is about 0.25 mM and 0.75 mM, respectively.
Figure 5
Figure 5. The nucleotide binding site in SVOP localizes to the C-terminal half of the protein.
A. 8-azido-ATP photoaffinity labeled SVOP-FLAG was cleaved by hydroxylamine and the samples were subjected to western blot analysis using anti-FLAG, anti-SVOP and ExtrAvidin-HRP. Arrowhead indicated an N-terminal fragment which is recognized by anti-SVOP antibody but not labeled by 8-azido-ATP. The Arrows indicate a C-terminal fragment which is labeled by 8-azido-ATP and anti-FLAG antibody. B. Only the C-terminal half of SVOP shows dominant nucleotide binding. N- and C-terminal halves of SVOP-FLAG were expressed and purified from HEK293 cells. Photoaffinity labeling was performed as described under Methods . C. Shown are the results of trypsin digestion of 8-azido-ATP labeled SVOP-FLAG. Labeled protein was digested at 37°C in the presence of trypsin. At the time periods indicated, an aliquot was withdrawn and subjected to analysis as described under Methods . The arrowheads indicate the smallest trypsinized 15 kDa fragment which is labeled by both 8-azido-ATP and anti-FLAG antibody. The proportion of total anti-FLAG and 8-azido-ATP labeling (undigested protein) was similar for the 15 KDa fragment (∼9% in both cases), consistent with it being labeled with the same efficiency as the full-length protein. Therefore the nucleotide-binding site is contained within this 15 KDa fragment. The image of 8-azido-ATP labeling blot was adjusted with contrast to show better results.
Figure 6
Figure 6. Mutants of SVOP show normal nucleotide binding.
Mutated SVOP constructs were generated by site-directed mutagenesis. Photoaffinity labeling with 8-azido-ATP was performed with the wildtype and the mutated proteins. All the mutants show similar binding capability as the wild type.
Figure 7
Figure 7. Comparison of the nucleotide binding sites in Glut1, SV2A and SVOP.
Comparison of the nucleotide binding sites in Glut1, SV2A and SVOP suggests convergent evolution of nucleotide binding. Predicted 12 transmembrane domains are depicted. Red lines represent the nucleotide binding domains. The amino and carboxy termini of the proteins are indicated with letter N and C.

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