期刊
JOURNAL OF NEUROCHEMISTRY
卷 140, 期 2, 页码 280-293出版社
WILEY-BLACKWELL
DOI: 10.1111/jnc.13886
关键词
cell free; dimerization; membrane protein; SV31; synaptic vesicles; Zn2+ binding and transport
资金
- Jurgen Manchot Stiftung
- Adolf Messer Stiftung
- Collaborative Research Center (SFB) 807 of the German Research Foundation (DFG)
- European Research Council under the European Union's Seventh Framework Programme (FP7)/ERC Grant [337567]
- Cluster of Excellence Frankfurt (Macromolecular Complexes)
- Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI)
- German Research Foundation (DFG)
The integral synaptic vesicle protein SV31 has been shown to bind divalent cations. Here, we demonstrate that SV31 protein synthesized within a cell-free system binds Zn2+ and to a lower extent Ni2+ and Cu2+ ions. Expression with Zn2+ stabilized the protein and increased solubility. SV31 was preferentially monomeric in detergent and revealed specific binding of Zn2+. When co-translationally inserted into defined nanodisc bilayers, SV31 assembled into dimeric complexes, resulting in increased binding of Zn2+. Putative Zn2+-binding motifs within SV31 comprise aspartic acid and histidine residues. Site-directed mutagenesis of two conserved aspartic acid residues leads to a potent decrease in Zn2+ binding but did not affect dimerization. Chemical modification of histidine residues abolished some of the Zn2+-binding capacity. We demonstrate proton-dependent transport of Zn2+ as by accumulation of fluorescent FluoZin-1 inside of SV31-containing proteoliposomes. Transport activity has a Km value of 44.3 mu M and required external Zn2+ and internal acidic pH. Our results demonstrate that the synaptic vesicle-integral protein SV31 functions as a proton-dependent Zn2+ transporter. SV31 may attribute specific and yet undiscovered functions to subsets of synapses.
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