SV31 is a Zn2+-binding synaptic vesicle protein

We recently identified in a proteomic screen a novel synaptic vesicle membrane protein of 31 kDa (SV31) of unknown function. According to its membrane topology and its phylogenetic relation SV31 may function as a vesicular transporter. Based on its amino acid sequence similarity to a prokaryotic heavy metal ion transporter we analyzed its metal ion-binding properties and show that recombinant SV31 binds the divalent cations Zn2+ and Ni2+ and to a minor extent Cu2+, but not Fe2+, Co2+, Mn2+, or Ca2+. Zn2+-binding of SV31 in viable cells was verified following heterologous transfection of pheochromocytoma cells 12 (PC12) with recombinant red fluorescent SV31 (SV31-RFP) and the fluorescent zinc indicator FluoZin-3. Sucrose density gradient fractionation of SV31-RFP-trans-fected PC12 cells revealed a partial overlap of SV31-RFP with synaptic-like vesicle markers and the early endosome marker rab5. Immunocytochemical analysis demonstrated a punctuate distribution in the cell soma and in neuritic processes and in addition in a compartment in vicinity to the plasma membrane that was immunopositive also for synaptosomal-associated protein 25 (SNAP-25) and syntaxin1A. Our data suggest that SV31 represents a novel Zn2+-binding protein that in PC12 cells is targeted to endosomes and subpopulations of synaptic-like microvesicles.