Protein Components of Mitochondrial DNA Nucleoids in Higher Eukaryotes

doi:10.1074/mcp.M300035-MCP200

Protein Components of Mitochondrial DNA Nucleoids in Higher Eukaryotes*

From the ^‡Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794, and ^‖Cold Spring Harbor Laboratories, Cold Spring Harbor, NY 11724

§To whom correspondence should be addressed: Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651. Tel.: 631-444-3068; Fax: 631-444-3218; E-mail: dan{at}pharm.sunysb.edu.

Abstract

Mitochondrial DNA (mtDNA) is not packaged in nucleosomal particles, but has been reported to associate with the mitochondrial inner membrane. Gentle lysis of Xenopus oocyte mitochondria with nonionic detergent liberates a nucleoprotein complex containing mtDNA associated with a previously characterized DNA binding partner, mitochondrial transcription factor A (mtTFA), as well as a series of inner membrane proteins identified by sequencing. More extensive detergent treatment stripped most of these proteins from the DNA, leaving a limited number of proteins in a nucleoid core. Sequencing of the major proteins retained in association with mtDNA revealed the expected mtDNA binding proteins, mtTFA and mitochondrial single-stranded DNA binding protein (mtSSB), as well as four proteins not previously reported to associate with mtDNA. These include adenine nucleotide translocator 1, the lipoyl-containing E2 subunits of pyruvate dehydrogenase and branched chain α-ketoacid dehydrogenase and prohibitin 2. The association of several of these proteins with mtTFA-containing mtDNA nucleoids was confirmed by immunoprecipitation.

Footnotes

Published, MCP Papers in Press, September 26, 2003, DOI 10.1074/mcp.M300035-MCP200
↵1 The abbreviations used are: mtDNA, mitochondrial DNA; mtTFA, mitochondrial transcription factor A; mtSSB, mitochondrial single-stranded DNA binding protein; DTT, dithiothreitol; PVDF, polyvinyldifluoridine; ANT, adenine nucleotide translocator; PDC-E2, E2 subunit of pyruvate decarboxylase; BCKD-E2, E2 subunit of branched chain keto-acid dehydrogenase; PHB, prohibitin; VDAC, voltage-dependent anion channel or porin; COX1, subunit 1 of cytochrome oxidase; AD-PEO, autosomal dominant progressive external opthalmoplegia; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; LC-MS/MS, liquid chromatography-tandem mass spectroscopy.
↵* This work was supported by National Institutes of Health Research Grants ES04068, GM29681, and ES012039 (to D. F. B.). 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.
↵¶ Current address: University of California San Francisco, San Francisco, CA 94143.
↵** Current address: M.D. Anderson Cancer Center, Houston, TX 77030.
- Received April 18, 2003.
- Revision received September 17, 2003.