SV31 is a Zn2+-binding synaptic vesicle protein

J Neurochem. 2011 Aug;118(4):558-70. doi: 10.1111/j.1471-4159.2011.07344.x. Epub 2011 Jul 1.

Abstract

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 Zn(2+) and Ni(2+) and to a minor extent Cu(2+), but not Fe(2+), Co(2+), Mn(2+), or Ca(2+). Zn(2+)-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-transfected 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 Zn(2+) -binding protein that in PC12 cells is targeted to endosomes and subpopulations of synaptic-like microvesicles.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Carrier Proteins / metabolism
  • Carrier Proteins / physiology*
  • Cations, Divalent / metabolism
  • Centrifugation, Density Gradient
  • Chromatography, Affinity
  • Cloning, Molecular
  • Computer Simulation
  • Cricetinae
  • Cricetulus
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism
  • Membrane Proteins / physiology*
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / metabolism
  • Nerve Tissue Proteins / physiology*
  • PC12 Cells
  • Polycyclic Compounds
  • Protein Binding
  • Protein Conformation
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Subcellular Fractions / metabolism
  • Synaptic Vesicles / metabolism
  • Synaptic Vesicles / physiology*
  • Transfection
  • Zinc / metabolism*

Substances

  • Carrier Proteins
  • Cations, Divalent
  • FluoZin-3
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Polycyclic Compounds
  • Recombinant Proteins
  • Tmem163 protein, rat
  • zinc-binding protein
  • Zinc