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/10.1104/PP.19.00736 (Access: Closed)