Identification of Two Proteins Associated with Mammalian ATP Synthase
Björn Meyer‡§, Ilka Wittig§, Elisabeth Trifilieff¶, Michael Karas‡ and Hermann Schägger§‖
Author Affiliations
From the ‡Institut für Pharmazeutische Chemie, Biozentrum, Centre of Excellence “Macromolecular Complexes,” Johann Wolfgang Goethe-Universität Frankfurt, Max-von-Laue-Str. 9, D-60439 Frankfurt am Main, Germany, §Molekulare Bioenergetik, Zentrum der Biologischen Chemie, Centre of Excellence “Macromolecular Complexes,” Fachbereich Medizin, Johann Wolfgang Goethe-Universität Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany, and ¶Chimie Organique des Substances Naturelles, CNRS, 5 Rue Blaise Pascal, 67084 Strasbourg Cedex, France
To whom correspondence should be addressed: Molekulare Bioenergetik, Zentrum der Biologischen Chemie, Fachbereich Medizin, Universität Frankfurt, Theodor-Stern-Kai 7, Haus 26, D-60590 Frankfurt am Main, Germany. Tel.: 49-69-6301-6927; Fax: 49-69-6301-6970; E-mail: schagger@zbc.kgu.de
Abstract
Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF1 inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.
Footnotes
Published, MCP Papers in Press, June 17, 2007, DOI 10.1074/mcp.M700097-MCP200
↵1 The abbreviations used are: complex I, NADH dehydrogenase complex; complex III, ubiquinol cytochrome c reductase; complex IV, cytochrome coxidase; complex V, ATP synthase; BN, blue native; CN, clear native; BNE, blue native electrophoresis; CNE, clear native electrophoresis; DDM, dodecylmaltoside; PMF, peptide mass fingerprint; DAPIT, diabetes-associated protein in insulin-sensitive tissue; 1-D, one-dimensional; 2-D, two-dimensional; dSDS, doubled SDS; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Su, subunit.
↵* This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 472 Project P11 (to H. S.) and Sonderforschungsbereich 628 Project P13 (to M. K. and H. S.). 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.
↵S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
Received March 6, 2007.
Revision received June 6, 2007.
© 2007 The American Society for Biochemistry and Molecular Biology
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