Universität zu Köln

Kuhnert, Franziska, Stefanski, Anja, Overbeck, Nina, Drews, Leonie, Reichert, Andreas S., Stuehler, Kai and Weber, Andreas P. M. (2020). Rapid Single-Step Affinity Purification of HA-Tagged Plant Mitochondria. Plant Physiol., 182 (2). S. 692 - 707. ROCKVILLE: AMER SOC PLANT BIOLOGISTS. ISSN 1532-2548

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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 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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Kuhnert, Franziska UNSPECIFIED UNSPECIFIED UNSPECIFIED Stefanski, Anja UNSPECIFIED UNSPECIFIED UNSPECIFIED Overbeck, Nina UNSPECIFIED UNSPECIFIED UNSPECIFIED Drews, Leonie UNSPECIFIED UNSPECIFIED UNSPECIFIED Reichert, Andreas S. UNSPECIFIED UNSPECIFIED UNSPECIFIED Stuehler, Kai UNSPECIFIED UNSPECIFIED UNSPECIFIED Weber, Andreas P. M. UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-347208
DOI:	10.1104/pp.19.00732
Journal or Publication Title:	Plant Physiol.
Volume:	182
Number:	2
Page Range:	S. 692 - 707
Date:	2020
Publisher:	AMER SOC PLANT BIOLOGISTS
Place of Publication:	ROCKVILLE
ISSN:	1532-2548
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language GLYCINE DECARBOXYLASE; ARABIDOPSIS-THALIANA; PROTEIN IMPORT; TOM COMPLEX; DEHYDROGENASE; MEMBRANE; IDENTIFICATION; EXPRESSION; GLUTAMATE; MUTANT Multiple languages Plant Sciences Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/34720