Monitoring Mitochondrial Membrane Potential

Import of nuclear-encoded proteins into the mitochondrial matrix requires a normal mitochondrial membrane potential (ΔΨ) [69 (link)]. Thus, we used the chimeric fusion protein preCox4-mCherry that includes the inner mitochondrial membrane targeting pre-sequence of Cox4 fused to mCherry as a readout to monitor the mitochondrial membrane potential in vivo [43 (link)]. In our experiments, we designed the strain MCY2080 carrying OM45-GFP as a control mitochondrial marker and pre-COX4-mCherry which was integrated at an intergenic region near the centromere of chromosome IV. While pre-COX4-mCherry requires the ΔΨ for its import into mitochondria, OM45 is an outer membrane protein for which import is not affected by changes in the ΔΨ [70 (link)]. Prior to imaging, cells were grown to mid-log phase at 30 °C in SD medium and supplemented when required with 10 mM citrate (citric acid—Merck, C2404), 10 mM alpha-ketoglutarate (alpha-ketoglutaric acid—Merck,75890), or 10 mM glutamate (L-glutamic acid—Merck, G5889). The pH of the media was adjusted using few drops of KOH 10 M solution upon addition of citrate or alpha-ketoglutarate. Cells were subsequently fixed with 3.7% formaldehyde for at least 20 min at room temperature. Using conventional fluorescent microscopy, we counted cells which exhibited a diffuse cytoplasmic localization of preCox4-mCherry, reflecting defects in pre-COX4-mCherry import and in turn a drop in the ΔΨ, while the mitochondrial localization of OM45-GFP was unaffected. The localization of preCox4-mCherry was assessed in at least 100 cells. Data reported are the mean and SEM (error bars) from 3 independent experiments.