pH difference across the outer mitochondrial membrane measured with a green fluorescent protein mutant.
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.
Porcelli
Anna Maria
AM
Department of Biology, University of Bologna, Via Irnerio 42, 40126 Bologna, Italy. porcelli@alma.unibo.it
Ghelli
Anna
A
Zanna
Claudia
C
Pinton
Paolo
P
Rizzuto
Rosario
R
Rugolo
Michela
M
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
D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005993:Glycerolphosphate Dehydrogenase