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Plant Physiol, December 2001, Vol. 127, pp. 1694-1710

Proteomic Approach to Identify Novel Mitochondrial Proteins in Arabidopsis1

Volker Kruft, Holger Eubel, Lothar Jänsch, Wolf Werhahn, and Hans-Peter Braun*

Institut für Angewandte Genetik, Universität Hannover, D-30419 Hannover, Germany (H.E., W.W., H.-P.B.); Applied Biosystems, D-63225 Langen, Germany (V.K.); and Gesellschaft für Biotechnologische Forschung, D-38124 Braunschweig, Germany (L.J.)

An Arabidopsis mitochondrial proteome project was started for a comprehensive investigation of mitochondrial functions in plants. Mitochondria were prepared from Arabidopsis stems and leaves or from Arabidopsis suspension cell cultures, and the purity of the generated fractions was tested by the resolution of organellar protein complexes applying two-dimensional blue-native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tricine) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Arabidopsis mitochondrial proteome was analyzed by two-dimensional isoelectric focusing/ Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 650 different proteins in a pI range of pH 3 to 10 were separated on single gels. Solubilization conditions, pH gradients for isoelectric focusing, and gel staining procedures were varied, and the number of separable proteins increased to about 800. Fifty-two protein spots were identified by immunoblotting, direct protein sequencing, and mass spectrometry. The characterized proteins cooperate in various processes, such as respiration, citric acid cycle, amino acid and nucleotide metabolism, protection against O2, mitochondrial assembly, molecular transport, and protein biosynthesis. More than 20% of the identified proteins were not described previously for plant mitochondria, indicating novel mitochondrial functions. The map of the Arabidopsis mitochondrial proteome should be useful for the analysis of knockout mutants concerning nuclear-encoded mitochondrial genes. Considerations of the total complexity of the Arabidopsis mitochondrial proteome are discussed. The data from this investigation will be made available at http://www.gartenbau.uni-hannover.de/genetik/AMPP.


1 This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.

* Corresponding author; e-mail braun{at}genetik.uni-hannover.de; fax 49-511-7623608.

© 2001 American Society of Plant Physiologists



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