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

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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.

Section snippets

Materials and methods

Materials. MitoTracker Red CMX-Ros and 2′,7′-bis(2-carboxyethyl)-5(6)carboxyfluorescein tetraacetoxy methylester (BCECF/AM) were purchased from Molecular Probes (Eugene, OR, USA); rotenone, oligomycin, monensin, and nigericin were from Sigma (St. Louis, MO, USA).

Construction of MIMS-EYFP. MIMS-EYFP has been constructed using the same cloning strategy employed for MIMS-aequorin [11]. Briefly, the ClaI/EcoRI fragment encoding HAI-tagged EYFP was generated by amplifying EYFP, using as template

Results and discussion

To obtain a direct measure of pH in IMS, we constructed a new EYFP chimera, denominated MIMS-EYFP. To this purpose, the cDNA encoding HAI-tagged EYFP was fused in-frame with encoding GPD [15], an integral protein of the inner mitochondrial membrane, with a large C-terminal tail protruding into the intermembrane space [16]. A schematic map of the final construct is shown in Fig. 1A. Based on the topology of GPD [16], the EYFP moiety is expected to be exposed in the mitochondrial intermembrane

Acknowledgments

This work was supported by grants from the University of Bologna “Progetto Dipartimentale: Meccanismi e segnali molecolari della sopravvivenza cellulare” to M.R., Telethon, Italy No. GGP02323 to A.G., No. 1285 and GTF02013 to R.R., the Italian Association for Cancer Research (AIRC), the Human Frontier Science Program, the Italian University Ministry (MIUR and FIRB), and the Italian Space Agency (ASI) to R.R. We are grateful to Dr. G. Venturoli, University of Bologna, for critical reading of the

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Abbreviations: BCECF, 2′,7′-bis(2-carboxyethyl)-5(6)carboxyfluorescein tetraacetoxy methyl ester; cyt-EYFP, cytosolic EYFP; EYFP, enhanced yellow fluorescent protein; GPD, glycerol-phosphate dehydrogenase; IMS, intermembrane space; MIMS-EYFP, EYFP targeted to mitochondrial intermembrane space; mt-EYFP, EYFP targeted to mitochondrial matrix; pHi, intracellular pH; ΔΨm, mitochondrial membrane potential; ΔpH, pH gradient.

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