Mitochondrial targeting of glycolysis in a major lineage of eukaryotes

Glycolysis is a major cytosolic catabolic pathway that provides ATP for many organisms1. Mitochondria play an even more important role in the provision of additional cellular ATP for eukaryotes2. Here, we show that in many stramenopiles, the C3 part of glycolysis is localised in mitochondria. We discovered genuine mitochondrial targeting signals on the six last enzymes of glycolysis. These targeting signals are recognised and sufficient to import GFP into mitochondria of a heterologous host. Analysis of eukaryotic genomes identified these targeting signals on many glycolytic C3 enzymes in a large group of eukaryotes found in the SAR supergroup3, in particular the stramenopiles. Stramenopiles, or heterokonts, are a large group of ecologically important eukaryotes that includes multi- and unicellular algae such as kelp and diatoms, but also economically important oomycete pathogens such as Phytophthora infestans. Confocal immunomicroscopy confirmed the mitochondrial location of glycolytic enzymes for the human parasite Blastocystis. Enzyme assays on cellular fractions confirmed the presence of the C3 part of glycolysis in Blastocystis mitochondria. These activities are sensitive to treatment with proteases and Triton X-100 but not proteases alone. Our work clearly shows that core cellular metabolism is more plastic than previously imagined and suggests new strategies to combat stramenopile pathogens such as the causative agent of late potato blight, P. infestans.