Reconstitution of the mia40-erv1 oxidative folding pathway for the small tim proteins

Mol Biol Cell. 2009 Aug;20(15):3481-90. doi: 10.1091/mbc.e08-10-1062. Epub 2009 May 28.


Mia40 and Erv1 execute a disulfide relay to import the small Tim proteins into the mitochondrial intermembrane space. Here, we have reconstituted the oxidative folding pathway in vitro with Tim13 as a substrate and determined the midpoint potentials of Mia40 and Tim13. Specifically, Mia40 served as a direct oxidant of Tim13, and Erv1 was required to reoxidize Mia40. During oxidation, four electrons were transferred from Tim13 with the insertion of two disulfide bonds in succession. The extent of Tim13 oxidation was directly dependent on Mia40 concentration and independent of Erv1 concentration. Characterization of the midpoint potentials showed that electrons flowed from Tim13 with a more negative midpoint potential of -310 mV via Mia40 with an intermediate midpoint potential of -290 mV to the C130-C133 pair of Erv1 with a positive midpoint potential of -150 mV. Intermediary complexes between Tim13-Mia40 and Mia40-Erv1 were trapped. Last, mutating C133 of the catalytic C130-C133 pair or C30 of the shuttle C30-C33 pair in Erv1 abolished oxidation of Tim13, whereas mutating the cysteines in the redox-active CPC motif, but not the structural disulfide linkages of the CX(9)C motif of Mia40, prevented Tim13 oxidation. Thus, we demonstrate that Mia40, Erv1, and oxygen are the minimal machinery for Tim13 oxidation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Circular Dichroism
  • Disulfides / chemistry
  • Disulfides / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Hydrogen Peroxide / chemistry
  • Hydrogen Peroxide / metabolism
  • Mitochondrial Membrane Transport Proteins / chemistry*
  • Mitochondrial Membrane Transport Proteins / genetics
  • Mitochondrial Membrane Transport Proteins / metabolism
  • Mitochondrial Precursor Protein Import Complex Proteins
  • Mitochondrial Proteins / chemistry*
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism
  • Oxidation-Reduction
  • Oxidoreductases Acting on Sulfur Group Donors / chemistry*
  • Oxidoreductases Acting on Sulfur Group Donors / genetics
  • Oxidoreductases Acting on Sulfur Group Donors / metabolism
  • Protein Folding
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Signal Transduction*
  • Toluene / analogs & derivatives
  • Toluene / chemistry
  • Toluene / metabolism


  • Disulfides
  • MIA40 protein, S cerevisiae
  • Mitochondrial Membrane Transport Proteins
  • Mitochondrial Precursor Protein Import Complex Proteins
  • Mitochondrial Proteins
  • Saccharomyces cerevisiae Proteins
  • TIM13 protein, S cerevisiae
  • Toluene
  • Hydrogen Peroxide
  • Oxidoreductases Acting on Sulfur Group Donors
  • ERV1 protein, S cerevisiae
  • dithiol