pH difference across the outer mitochondrial membrane measured with a green fluorescent protein mutant

Biochem Biophys Res Commun. 2005 Jan 28;326(4):799-804. doi: 10.1016/j.bbrc.2004.11.105.

Abstract

In this study we have generated a EYFP targeted to the mitochondrial intermembrane space (MIMS-EYFP) to determine for the first time the pH within this compartment. The fragment encoding HAI-tagged EYFP was fused with the C-terminus of glycerol-phosphate dehydrogenase, an integral protein of the inner mitochondrial membrane. Human ECV304 cells transiently transfected with MIMS-EYFP showed the typical mitochondrial network, co-localized with MitoTracker Red. Following the calibration procedure, an estimation of the pH value in the intermembrane space was obtained. This value (6.88+/-0.09) was significantly lower than that determined in the cytosol after transfection with a cytosolic EYFP (7.59+/-0.01). Further, the pH of the mitochondrial matrix, determined with a EYFP targeted to this subcompartment, was 0.9 pH units higher than that in the intermembrane space. In conclusion, MIMS-EYFP represents a novel powerful tool to monitor pH changes in the mitochondrial intermembrane space of live cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biosensing Techniques / methods*
  • Cell Line
  • Endothelial Cells / chemistry
  • Endothelial Cells / metabolism
  • Glycerolphosphate Dehydrogenase / analysis
  • Glycerolphosphate Dehydrogenase / chemistry
  • Glycerolphosphate Dehydrogenase / genetics
  • Glycerolphosphate Dehydrogenase / metabolism
  • Green Fluorescent Proteins / analysis
  • Green Fluorescent Proteins / chemistry*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • Humans
  • Hydrogen-Ion Concentration
  • Intracellular Membranes / chemistry*
  • Intracellular Membranes / ultrastructure
  • Microscopy, Fluorescence / methods*
  • Mitochondria / chemistry*
  • Mitochondria / genetics
  • Mitochondria / metabolism*
  • Mitochondria / ultrastructure
  • Molecular Probes / analysis
  • Molecular Probes / chemistry
  • Molecular Probes / genetics
  • Mutagenesis, Site-Directed
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics

Substances

  • Molecular Probes
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Glycerolphosphate Dehydrogenase