Rapid single-step affinity purification of HA-tagged plant mitochondria

Franziska Kuhnert, Anja Stefanski, Nina Overbeck, Leonie Drews, Andreas S. Reichert, Kai Stühler, Andreas P.M. Weber

Research output: Contribution to journalArticlepeer-review

23 Citations (Scopus)

Abstract

Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. However, isolating 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 (Arabidopsis thaliana) with a triple hemagglutinin 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 Glu transporter À BOUT DE SOUFFLE (BOU) and identified 27 differentially expressed mitochondrial proteins compared with tagged Col-0 controls. Our work sets the stage for the development of advanced mitochondria isolation protocols for distinct cell types.

Original languageEnglish
Pages (from-to)692-706
Number of pages15
JournalPlant Physiology
Volume182
Issue number2
DOIs
Publication statusPublished - Feb 2020
Externally publishedYes

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