Rapid Affinity Purification of Tagged Plant Mitochondria (Mito-AP) for Metabolome and Proteome Analyses

authored by: Markus Niehaus, Henryk Straube, Patrick Künzler, Nils Rugen, Jan Hegermann, Patrick Giavalisco, Holger Eubel, Claus Peter Witte, Marco Herde
Abstract: The isolation of organelles facilitates the focused analysis of subcellular protein and metabolite pools. Here we present a technique for the affinity purification of plant mitochondria (Mito-AP). The stable ectopic expression of a mitochondrial outer membrane protein fused to a GFP:Strep tag in Arabidopsis (Arabidopsis thaliana) exclusively decorates mitochondria, enabling their selective affinity purification using magnetic beads coated with Strep-Tactin. With Mito-AP, intact mitochondria from 0.5 g plant material were highly enriched in 30-60 min, considerably faster than with conventional gradient centrifugation. Combining gradient centrifugation and Mito-AP techniques resulted in high purity of .90% mitochondrial proteins in the lysate. Mito-AP supports mitochondrial proteome analysis by shotgun proteomics. The relative abundances of proteins from distinct mitochondrial isolation methods were correlated. A cluster of 619 proteins was consistently enriched by all methods. Among these were several proteins that lack subcellular localization data or that are currently assigned to other compartments. Mito-AP is also compatible with mitochondrial metabolome analysis by triple-quadrupole and orbitrap mass spectrometry. Mito-AP preparations showed a strong enrichment with typical mitochondrial lipids like cardiolipins and demonstrated the presence of several ubiquinones in Arabidopsis mitochondria. Affinity purification of organelles is a powerful tool for reaching higher spatial and temporal resolution for the analysis of metabolomic and proteomic dynamics within subcellular compartments. Mito-AP is small scale, rapid, economic, and potentially applicable to any organelle or to organelle subpopulations.
Organisation(s): Institute of Plant Nutrition
Section Plant Molecular Biology and Plant Proteomics
External Organisation(s): Hannover Medical School (MHH)
Max Planck Institute for Biology of Ageing
Type: Article
Journal: Plant physiology
Volume: 182
Pages: 1194-1210
No. of pages: 17
ISSN: 0032-0889
Publication date: 03.2020
Publication status: Published
Peer reviewed: Yes
ASJC Scopus subject areas: Physiology, Genetics, Plant Science
Electronic version(s): https://doi.org/10.15488/17903 (Access: Open)
https://doi.org/10.1104/PP.19.00736 (Access: Closed)