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
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Research Support, Non-U.S. Gov't
MeSH terms
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Biosensing Techniques / methods*
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Cell Line
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Endothelial Cells / chemistry
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Endothelial Cells / metabolism
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Glycerolphosphate Dehydrogenase / analysis
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Glycerolphosphate Dehydrogenase / chemistry
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Glycerolphosphate Dehydrogenase / genetics
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Glycerolphosphate Dehydrogenase / metabolism
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Green Fluorescent Proteins / analysis
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Green Fluorescent Proteins / chemistry*
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism*
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Humans
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Hydrogen-Ion Concentration
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Intracellular Membranes / chemistry*
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Intracellular Membranes / ultrastructure
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Microscopy, Fluorescence / methods*
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Mitochondria / chemistry*
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Mitochondria / genetics
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Mitochondria / metabolism*
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Mitochondria / ultrastructure
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Molecular Probes / analysis
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Molecular Probes / chemistry
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Molecular Probes / genetics
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Mutagenesis, Site-Directed
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Recombinant Fusion Proteins / analysis
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
Substances
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Molecular Probes
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Recombinant Fusion Proteins
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Green Fluorescent Proteins
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Glycerolphosphate Dehydrogenase