SVOP is a nucleotide binding protein.
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.


RESULTS
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
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.
Yao
Jia
J
Department of Pharmacology, University of Washington, Seattle, Washington, United States of America.
Bajjalieh
Sandra M
SM
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002478:Cells, Cultured; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D009243:NAD / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D033921:Vesicular Transport Proteins / Q000378:metabolism*
D001386:Azides; D009711:Nucleotides; C115845:Svop protein, rat; D033921:Vesicular Transport Proteins; D009243:NAD; C017040:8-azidoadenosine 5'-triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate