VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications

Oncotarget. 2016 Jan 19;7(3):2249-68. doi: 10.18632/oncotarget.6850.


Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.

Keywords: VDACs; cysteine oxidation; disulfide bridge; mass spectrometry; mitochondrial intermembrane space.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cysteine / chemistry*
  • Electron Transport / physiology
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Humans
  • Liver / cytology
  • Liver / enzymology
  • Liver / metabolism*
  • Mass Spectrometry
  • Mitochondria / metabolism*
  • Mitochondrial Membrane Transport Proteins / genetics
  • Mitochondrial Membrane Transport Proteins / metabolism*
  • Molecular Dynamics Simulation
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Protein Isoforms / metabolism
  • Rats
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Voltage-Dependent Anion Channels / genetics
  • Voltage-Dependent Anion Channels / metabolism*


  • Mitochondrial Membrane Transport Proteins
  • Protein Isoforms
  • Vdac3 protein, rat
  • Voltage-Dependent Anion Channels
  • Cysteine