@article{4237d512bebc476f8c9b2e52016b933c,
title = "Rapid single-step affinity purification of HA-tagged plant mitochondria",
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 {\`A} 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.",
author = "Franziska Kuhnert and Anja Stefanski and Nina Overbeck and Leonie Drews and Reichert, {Andreas S.} and Kai St{\"u}hler and Weber, {Andreas P.M.}",
note = "Funding Information: 1This work was supported by the Deutsche Forschungsgemein-schaft (CRC 1208; and funding under Germany{\textquoteright}s Excellence Strategy [EXC-2048/1 - project ID 390686111]). 2Author for contact:
[email protected]. 3Senior author. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Andreas P.M. Weber (
[email protected]). F.K. generated transgenic lines harboring affinity-tagged mitochondria, isolated mitochondria, analyzed the data, and drafted the manuscript; A.S., N.O., and K.S. performed the proteomics analysis and analyzed the data; L.D. and A.S.R. assisted with the Seahorse Analyzer measurements; A.P.M.W. conceived and supervised the experiments and contributed to the writing of the manuscript. [OPEN]Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.19.00732 Publisher Copyright: {\textcopyright} 2020 American Society of Plant Biologists. All Rights Reserved.",
year = "2020",
month = feb,
doi = "10.1104/pp.19.00732",
language = "English",
volume = "182",
pages = "692--706",
journal = "Plant Physiology",
issn = "0032-0889",
publisher = "American Society of Plant Biologists",
number = "2",
}