title: pH difference across the outer mitochondrial membrane measured with a green fluorescent protein mutant.
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.
authors.affiliation: Department of Biology, University of Bologna, Via Irnerio 42, 40126 Bologna, Italy. porcelli@alma.unibo.it
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Porcelli
authors.forename: Anna
authors.initials: A
authors.lastname: Ghelli
authors.forename: Claudia
authors.initials: C
authors.lastname: Zanna
authors.forename: Paolo
authors.initials: P
authors.lastname: Pinton
authors.forename: Rosario
authors.initials: R
authors.lastname: Rizzuto
authors.forename: Michela
authors.initials: M
authors.lastname: Rugolo
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D042783:Endothelial Cells / Q000737:chemistry / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000737:chemistry* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence / Q000379:methods*; D008928:Mitochondria / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D015335:Molecular Probes / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics
chemical_list: D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005993:Glycerolphosphate Dehydrogenase