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 alpha-ketoacid dehydrogenase and prohibitin 2. The association of several of these proteins with mtTFA-containing mtDNA nucleoids was confirmed by immunoprecipitation.
Highlights
Mitochondrial DNA is not packaged in nucleosomal particles, but has been reported to associate with the mitochondrial inner membrane
We have found that physical homogenization of mitochondria or lysis in the presence of Ͼ250 mM NaCl increased the fraction of free mitochondrial transcription factor A (mtTFA). mtTFA is completely removed from Mitochondrial DNA (mtDNA) by 0.5 M NaCl, a condition used in the experiments of VanTuyle and McPherson (23; data not shown)
We selected Xenopus oocytes as a starting material for this study because a single oocyte contains as many mitochondria as 105 somatic cells to provide a store of organelles for early embryonic development
Summary
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. Using electron microscopy, Albring et al [20] later identified an apparent sequence-specific attachment of mtDNA to the inner mitochondrial membrane but did not identify a protein anchor responsible for this association Such sequence-specific binding was not observed by Pinon et al [21] in their electron microscopic study of Xenopus mtDNA-protein complexes. Barat et al [24] suggested that the set of proteins associated with their preparations of Xenopus mtDNA nucleoids resembled proteins of the inner mitochondrial membrane. The novel association of these proteins with the mtDNA nucleoid was confirmed by their detection in mtDNA nucleoids immunoprecipitated with antibodies directed against mtTFA
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