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J. Biol. Chem., Vol. 279, Issue 46, 48369-48375, November 12, 2004

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"Respirasome"-like Supercomplexes in Green Leaf Mitochondria of Spinach^*

Frank Krause, Nicole H. Reifschneider, Dirk Vocke, Holger Seelert, Sascha Rexroth, and Norbert A. Dencher

From the Physical Biochemistry, Department of Chemistry, Darmstadt University of Technology, Petersenstrasse 22, D-64287 Darmstadt, Germany

Higher plant mitochondria have many unique features compared with their animal and fungal counterparts. This is to a large extent related to the close functional interdependence of mitochondria and chloroplasts, in which the two ATP-generating processes of oxidative phosphorylation and photosynthesis, respectively, take place. We show that digitonin treatment of mitochondria contaminated with chloroplasts from spinach (Spinacia oleracea) green leaves at two different buffer conditions, performed to solubilize oxidative phosphorylation supercomplexes, selectively extracts the mitochondrial membrane protein complexes and only low amounts of stroma thylakoid membrane proteins. By analysis of digitonin extracts from partially purified mitochondria of green leaves from spinach using blue and colorless native electrophoresis, we demonstrate for the first time that in green plant tissue a substantial proportion of the respiratory complex IV is assembled with complexes I and III into "respirasome"-like supercomplexes, previously observed in mammalian, fungal, and non-green plant mitochondria only. Thus, fundamental features of the supramolecular organization of the standard respiratory complexes I, III, and IV as a respirasome are conserved in all higher eukaryotes. Because the plant respiratory chain is highly branched possessing additional alternative enzymes, the functional implications of the occurrence of respiratory supercomplexes in plant mitochondria are discussed.

Received for publication, June 1, 2004 , and in revised form, September 1, 2004.

^* This work was supported in part by Deutsche Forschungsgemeinschaft Grant SFB 472 (to N. A. D. and H. S.) as well as by Fonds der Chemischen Industrie and by Bundesministerium für Bildung und Forschung Grant 03-DE5DA1-8 (to N. A. D.). This article is part of the Ph.D. theses of F. Krause and N. H. Reifschneider, Technische Universität Darmstadt (D17). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-6151-165376; Fax: 49-6151-164171; E-mail: f_krause{at}pop.tu-darmstadt.de.

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