The synaptic vesicle protein <scp>SV</scp>31 assembles into a dimer and transports Zn²⁺

Abstract

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 K_m value of 44.3 μM 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.