Porcelli Anna Maria, Ghelli Anna, Zanna Claudia, Pinton Paolo, Rizzuto Rosario, Rugolo Michela
Department of Biology, University of Bologna, Via Irnerio 42, 40126 Bologna, Italy.
Biochem Biophys Res Commun. 2005 Jan 28;326(4):799-804. doi: 10.1016/j.bbrc.2004.11.105.
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.
在本研究中，我们构建了一种靶向线粒体膜间隙的增强型黄色荧光蛋白（MIMS-EYFP），首次用于测定该区域的pH值。编码HAI标签的增强型黄色荧光蛋白的片段与线粒体内膜的整合蛋白甘油磷酸脱氢酶的C末端融合。瞬时转染了MIMS-EYFP的人ECV304细胞显示出典型的线粒体网络，与MitoTracker Red共定位。经过校准程序后，获得了膜间隙pH值的估计值。该值（6.88±0.09）显著低于用胞质增强型黄色荧光蛋白转染后在胞质溶胶中测定的值（7.59±0.01）。此外，用靶向该亚区域的增强型黄色荧光蛋白测定的线粒体基质pH值比膜间隙高0.9个pH单位。总之，MIMS-EYFP是监测活细胞线粒体膜间隙pH变化的一种新型有力工具。
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