Rapid single-step affinity purification of HA-tagged mitochondria from Arabidopsis thaliana

ABSTRACT Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. However, isolation of mitochondria from plant cells, for physiological and biochemical analyses, is a lengthy and tedious process. Established isolation protocols require multiple centrifugation steps and substantial amounts of starting material. To overcome these limitations, we tagged mitochondria in Arabidopsis thaliana with a triple haemagglutinin-tag for rapid purification via a single affinity purification step. This protocol yields a substantial quantity of highly pure mitochondria from 1 g of Arabidopsis seedlings. The purified mitochondria were suitable for enzyme activity analyses and yielded sufficient amounts of proteins for deep proteomic profiling. We applied this method for the proteomic analysis of the Arabidopsis bou-2 mutant deficient in the mitochondrial glutamate transporter À bout de souffle (BOU) and identified 27 differentially expressed mitochondrial proteins compared with transgenic Col-0 controls. Our work also sets the stage for the development of advanced mitochondria isolation protocols for distinct cell types. One-sentence summary Affinity-tagging of mitochondria in plant cells with a triple hemagglutinin-tag enables single-step affinity purification of mitochondria in less than 20 min.