Rapid Single-Step Affinity Purification of HA-Tagged Plant Mitochondria

Plant Physiol. 2020 Feb;182(2):692-706. doi: 10.1104/pp.19.00732. Epub 2019 Dec 9.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics
  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism
  • Chromatography, Affinity / methods*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Glutamine / metabolism
  • Hemagglutinin Glycoproteins, Influenza Virus / genetics
  • Membrane Transport Proteins / genetics
  • Microscopy, Confocal
  • Mitochondria* / enzymology
  • Mitochondria* / metabolism
  • Mitochondrial Membrane Transport Proteins / genetics
  • Mitochondrial Membrane Transport Proteins / metabolism
  • Peptide Fragments / genetics
  • Plants, Genetically Modified
  • Proteome / genetics
  • Proteome / metabolism
  • Proteomics
  • Seedlings / metabolism*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism


  • Arabidopsis Proteins
  • CONSTANS protein, Arabidopsis
  • DNA-Binding Proteins
  • Hemagglutinin Glycoproteins, Influenza Virus
  • Membrane Transport Proteins
  • Mitochondrial Membrane Transport Proteins
  • Peptide Fragments
  • Proteome
  • Transcription Factors
  • a bout de souffle protein, Arabidopsis
  • tyrosyl-prolyl-tyrosyl-aspartyl-valyl-prolyl-aspartyl-tyrosyl-alanine
  • Glutamine