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Mitochondrial DNA (mtDNA) is packaged into bacterial nucleoid-like structures, each containing several mtDNA molecules. The distribution of nucleoids during mitochondrial fission and fusion events and during cytokinesis is important to the segregation of mitochondrial genomes in heteroplasmic cells bearing a mixture of wild-type and mutant mtDNA molecules. We report fractionation of HeLa cell mtDNA nucleoids into two subsets of complexes that differ in their sedimentation velocity and their association with cytoskeletal proteins. Pulse labeling studies indicated that newly replicated mtDNA molecules are evenly represented in the rapidly and slowly sedimenting fractions. Slowly sedimenting nucleoids were immunoaffinity purified using antibodies to either of two abundant mtDNA-binding proteins, TFAM or mtSSB. These two different immunoaffinity procedures yielded very similar sets of proteins, with 21 proteins in common, including most of the proteins previously shown to play roles in mtDNA replication and transcription. In addition to previously identified mitochondrial proteins, multiple peptides were observed for one novel DNA metabolic protein, the DEAH-box helicase DHX30. Antibodies raised against a recombinant fragment of this protein confirmed the mitochondrial localization of a specific isoform of DHX30. Mitochondrial DNA (mtDNA) is packaged into bacterial nucleoid-like structures, each containing several mtDNA molecules. The distribution of nucleoids during mitochondrial fission and fusion events and during cytokinesis is important to the segregation of mitochondrial genomes in heteroplasmic cells bearing a mixture of wild-type and mutant mtDNA molecules. We report fractionation of HeLa cell mtDNA nucleoids into two subsets of complexes that differ in their sedimentation velocity and their association with cytoskeletal proteins. Pulse labeling studies indicated that newly replicated mtDNA molecules are evenly represented in the rapidly and slowly sedimenting fractions. Slowly sedimenting nucleoids were immunoaffinity purified using antibodies to either of two abundant mtDNA-binding proteins, TFAM or mtSSB. These two different immunoaffinity procedures yielded very similar sets of proteins, with 21 proteins in common, including most of the proteins previously shown to play roles in mtDNA replication and transcription. In addition to previously identified mitochondrial proteins, multiple peptides were observed for one novel DNA metabolic protein, the DEAH-box helicase DHX30. Antibodies raised against a recombinant fragment of this protein confirmed the mitochondrial localization of a specific isoform of DHX30. Mammalian cells contain thousands of copies of mitochondrial DNA (mtDNA) 2The abbreviations used are: mtDNA, mitochondrial DNA; TFAM, transcription factor A mitochondrial; AAA, ATPases associated with various activities; ANT, adenine nucleotide transporter; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry; IMMT, mitofilin; mtSSB, mitochondrial single-stranded DNA-binding protein; E64, l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane; DTT, dithiothreitol; PBS, phosphate-buffered saline. organized in several hundred nucleoids (1Legros F. Malka F. Frachon P. Lombes A. Rojo M. J. Cell Sci. 2004; 117: 2653-2662Crossref PubMed Scopus (292) Google Scholar, 2Iborra F. Kimura H. Cook P. BMC Biol. 2004; 2: 9Crossref PubMed Scopus (246) Google Scholar). These nucleoid structures are dynamic and redistribute actively within mitochondria undergoing fission and fusion (3Garrido N. Griparic L. Jokitalo E. Wartiovaara J. van der Bliek A.M. Spelbrink J.N. Mol. Biol. Cell. 2003; 14: 1583-1596Crossref PubMed Scopus (274) Google Scholar). It is widely accepted that the segregation of nucleoids may control the inheritance of mutant mtDNAs, thus influencing the development of mitochondrial disorders. The packaging of multiple mtDNA molecules into a single nucleoid helps to explain the classical observation that the segregation of mtDNA mutants is faster than would be expected given the large number of mtDNA genomes in a cell. Although mtDNA-binding proteins are thought to play critical roles in mtDNA maintenance and segregation (4Battersby B. Loredo-Osti J. Shoubridge E. Nat. Genet. 2003; 33: 183-186Crossref PubMed Scopus (113) Google Scholar), the composition of mtDNA nucleoids is poorly understood, especially in higher organisms. The organization and inheritance of mtDNA nucleoids has very recently been reviewed (5Chen X.J. Butow R.A. Nat. Rev. Genet. 2005; 6: 815-825Crossref PubMed Scopus (360) Google Scholar, 6Malka F. Lombes A. Rojo M. Biochim. Biophys. Acta. 2006; 5-6: 463-472Crossref Scopus (45) Google Scholar). Studies of mtDNA nucleoids in the yeast, Saccharomyces cerevisiae, have progressed more rapidly than experiments in higher eukaryotes due to the simplicity of the yeast nuclear genome and the fact that bakers' yeast is a facultative anaerobe that can survive without mtDNA. Formaldehyde cross-linking studies have revealed several classes of proteins in association with S. cerevisiae mtDNA, including metabolic proteins such as aconitase and Ilv5 (7Chen X.J. Wang X. Kaufman B.A. Butow R.A. Science. 2005; 307: 714-717Crossref PubMed Scopus (9) Google Scholar). Other yeast proteins such as Mmm1p (8Hobbs A.E. Srinivasan M. McCaffery J.M. Jensen R.E. J. Cell Biol. 2001; 152: 401-410Crossref PubMed Scopus (169) Google Scholar), Mdm10p, and Mdm12p (9Boldogh I.R. Nowakowski D.W. Yang H.-C. Chung H. Karmon S. Royes P. Pon L.A. Mol. Biol. Cell. 2003; 14: 4618-4627Crossref PubMed Scopus (215) Google Scholar) may mediate contacts between nucleoids and cytoskeletal components important for mtDNA inheritance. Mmm1p is localized in close proximity to Mgm101p, which has also been characterized as a nucleoid protein (10Meeusen S. Tieu Q. Wong E. Weiss E. Schietz D. Yates J. Nunnari J. J. Cell Biol. 1999; 145: 291-304Crossref PubMed Scopus (90) Google Scholar, 11Meeusen S. Nunnari J. J. Cell Biol. 2003; 163: 503-510Crossref PubMed Scopus (146) Google Scholar). Other proteins such as Mmm2p (12Youngman M.J. Hobbs A.E. Burgess S.M. Srinivasan M. Jensen R.E. J. Cell Biol. 2004; 164: 677-688Crossref PubMed Scopus (119) Google Scholar), Mdm31, and Mdm32 (13Dimmer K.S. Jakobs S. Vogel F. Altmann K. Westermann B. J. Cell Biol. 2005; 168: 103-115Crossref PubMed Scopus (91) Google Scholar) genetically influence mtDNA nucleoid maintenance but are not as closely associated with mtDNA. There are two major reasons why yeast mtDNA nucleoids appear to differ from those in higher eukaryotes. First, individual yeast nucleoids contain only one to two mtDNA genomes, perhaps equivalent to one genome that may or may not be replicating, whereas vertebrate mtDNA nucleoids typically contain 5-7 entire genomes (1Legros F. Malka F. Frachon P. Lombes A. Rojo M. J. Cell Sci. 2004; 117: 2653-2662Crossref PubMed Scopus (292) Google Scholar, 2Iborra F. Kimura H. Cook P. BMC Biol. 2004; 2: 9Crossref PubMed Scopus (246) Google Scholar). Second, many of the yeast nucleoid proteins noted above lack homologs in higher eukaryotes. Biochemical studies of mtDNA nucleoids in higher eukaryotes have reported their association with the inner membrane (3Garrido N. Griparic L. Jokitalo E. Wartiovaara J. van der Bliek A.M. Spelbrink J.N. Mol. Biol. Cell. 2003; 14: 1583-1596Crossref PubMed Scopus (274) Google Scholar, 14Albring M. J. Sci. S. A. PubMed Scopus Google Scholar, M. D. Cell PubMed Scopus Google Scholar) and cytoskeletal structures D. J. Cook P. PubMed Scopus Google Scholar). nucleoid proteins identified to in are to a characterized DNA-binding proteins. The protein TFAM, to bacterial protein, is a major mtDNA packaging protein in yeast, and Mol. Cell. Biol. PubMed Scopus Google Scholar, 2001; PubMed Scopus Google Scholar, Science. PubMed Scopus Google Scholar, J. Biol. PubMed Google Scholar, K. H. K. N. D. 2003; PubMed Scopus Google Scholar). a and of bacterial is a single-stranded DNA-binding protein associated with mtDNA in eukaryotes J. Cell Biol. PubMed Scopus (45) Google Scholar, E. F. B. J. PubMed Scopus Google Scholar, B. J. S. M. PubMed Scopus Google Scholar). a mitochondrial DNA has been shown to with TFAM and (3Garrido N. Griparic L. Jokitalo E. Wartiovaara J. van der Bliek A.M. Spelbrink J.N. Mol. Biol. Cell. 2003; 14: 1583-1596Crossref PubMed Scopus (274) Google Scholar). molecules packaged in nucleoids are in a of dynamic including replication and transcription. the protein composition of nucleoids may be expected to as of mtDNA nucleoids Wang Mol. Cell 2003; 2: PubMed Scopus Google Scholar) revealed several novel mitochondrial nucleoid proteins, including adenine nucleotide and the of two large and The association of mtDNA nucleoids with proteins to in the mitochondrial inner such as and is a in the classical observation that mtDNA is In this report of mtDNA nucleoids from HeLa cells and of associated proteins. We that mitochondrial nucleoids are in two major associated with labeling that newly replicated DNA is in nucleoid The more rapidly sedimenting is closely associated with cytoskeletal proteins, of the yeast nucleoids The more slowly sedimenting with cytoskeletal We used this as the for immunoaffinity of nucleoids using antibodies against either of two abundant mtDNA-binding proteins, TFAM or mtSSB. A of proteins with of immunoaffinity including several proteins to be in mtDNA A novel of this is the which for the as a mitochondrial In addition to DNA metabolic proteins, which may be identified a of membrane proteins, and metabolic proteins similar in to the yeast nucleoid reported recently (7Chen X.J. Wang X. Kaufman B.A. Butow R.A. Science. 2005; 307: 714-717Crossref PubMed Scopus (9) Google Scholar). In to this protein or for the of aconitase in mtDNA Cell HeLa cells were in with with of and of Mitochondrial in of mitochondria and complexes were without the A a mixture containing E64, and were for and in were for and in and In experiments were with and in to of nucleoids with cytoskeletal proteins. were in and using a of for of the to the to were for each to the which of with containing different were of of and a of of were for in a The mitochondria were from the with and for of and of mitochondria from of were in of containing and were with mitochondria for of to to The mitochondria were and in a for to the nuclear DNA and to the Mitochondrial were in and the to purified The purified mitochondria were in with of of mitochondrial protein, and for with The were in for to the of Mitochondrial were protein in DTT, of for a of The for and for The of a of of above a of of and DTT, and were in for of to mtDNA cells in were with of for or from the cells were as were and with were a The in of for in for The in for and to that of of and of using antibodies against either TFAM or as K. H. K. N. D. 2003; PubMed Scopus Google Scholar, Wang Mol. Cell 2003; 2: PubMed Scopus Google Scholar), with control experiments using were and purified from the of with recombinant TFAM or purified in for the control were chromatography using a The antibodies were coupled to in the were with of a in mtDNA nucleoids in containing and a with the were a and the proteins were as a The of the in containing DTT, proteins were with were and with using Wang Mol. Cell 2003; 2: PubMed Scopus Google Scholar). were identified a mass with a The mass were with and with to from the protein of with were to in PubMed Scopus Google Scholar). were either with J. 117: PubMed Scopus Google Scholar), or to membrane to of proteins with specific the to and The antibodies used against TFAM, mtSSB, and ANT, and and Antibodies against components of and were from antibodies against aconitase were a from and antibodies against P. P. E. J. Cell Sci. PubMed Google were and Antibodies to were raised against a of in A containing isoform of number from used as for using and to a fragment with and These two were used to the fragment to to of a in in but in containing and purified chromatography chromatography in the of A of this used to a to a and a coupled to to of The purified antibodies were used for as above and for HeLa cells were in in with of and of in a of in were in in for and in for were in for and with either or in for The cells were with antibodies in for In experiments in the were in for and in in for with for the cells were with using a or a The antibodies used for were from the the from Mitochondrial into and Slowly mtDNA nucleoid reported to this In that mitochondria procedures were with of nuclear not observed in with mtDNA nucleoids cells contain more mtDNA than nuclear We that this be mitochondria purified with and using a from that of and S. J. Biol. PubMed Scopus Google Scholar). in a with metabolic to of the to a the of of mass observed of the mtDNA with DNA from nuclear The for mitochondrial nucleoid is shown in nucleoids were as with velocity sedimentation of mitochondrial in very in and can be the of a for DNA into rapidly and slowly sedimenting as shown in A of each for that confirmed that the DNA has the for mtDNA The of as that TFAM and mtSSB, characterized mtDNA-binding proteins, are associated with slowly and rapidly sedimenting nucleoid fractions. the the cytoskeletal proteins and are only in the rapidly sedimenting nucleoid In one to proteins in the rapidly sedimenting observed with cytoskeletal proteins including and proteins. We to the of cytoskeletal proteins in nucleoid including and in the to cell or in the cell but that not the reported used only the slowly sedimenting nucleoid the of mtDNA to for the of two classes of mtDNA nucleoids is that the association of nucleoids with a to the may be either or with mtDNA In yeast, and Nunnari S. Nunnari J. J. Cell Biol. 2003; 163: 503-510Crossref PubMed Scopus (146) Google Scholar) have shown that only a of nucleoids is in replication and that replication in association with a large In vertebrate cells in F. Kimura H. Cook P. BMC Biol. 2004; 2: 9Crossref PubMed Scopus (246) Google Scholar) that the entire mtDNA nucleoids not as a but that single mtDNA molecules within a nucleoid It is that mtDNA molecules may to be from nucleoid structures the DNA is to and newly replicated mtDNA molecules be in either the rapidly or slowly sedimenting We HeLa cell with for or to of is for replication of a single mtDNA J. Mol. Biol. PubMed Scopus Google Scholar), and several are for of in the labeling of into mtDNA replication and newly replicated molecules. Mitochondrial were sedimentation and mtDNA and with and and A of the distribution of mtDNA and mtDNA in labeling of either rapidly or slowly sedimenting of DNA and revealed in the specific of the two not We that nucleoid is in or newly replicated molecules. of or used in for of These experiments that the rapidly sedimenting a very of whereas the slowly sedimenting a of the rapidly sedimenting to the in of the proteins to a of the that the proteins were associated with mtDNA, as previously shown for mtDNA nucleoids Wang Mol. Cell 2003; 2: PubMed Scopus Google Scholar). the of nucleoids in with that of proteins, used purified antibodies against either TFAM or to nucleoids as that in complexes with and complexes containing and A control with to of proteins, that proteins were not to antibodies coupled to the We that with either or antibodies is a experiments this the of the sedimentation and reported with of and in the and In in the such as of with and of the in in the of nucleoid proteins, as shown in of used mass to the proteins in several of slowly sedimenting nucleoids sedimentation and immunoaffinity using either or from the with were The entire into to to major were in and peptides were were accepted only proteins were the in and peptides were identified with than We observed a large number of typically either or antibodies were used in proteins purified using antibodies and purified using the 21 proteins, were observed with We proteins to be associated with nucleoids were identified using and antibodies These proteins are in with a number of proteins to mtDNA that were observed with only one of the two of with protein are in in slowly sedimenting HeLa mtDNA and DNA and proteins with roles in mtDNA replication and Other protein this is a this is a Wang Mol. Cell 2003; 2: PubMed Scopus Google Scholar) and (5Chen X.J. Butow R.A. Nat. Rev. Genet. 2005; 6: 815-825Crossref PubMed Scopus (360) Google this is a in a proteins reported in are into several major First, as identified the of proteins to as in the replication and transcription of mtDNA. abundant DNA-binding proteins TFAM, mtSSB, as as proteins, including of DNA the DNA helicase J. F. K. Q. M. S. N. L. L. A. H. D. J. A. H. M. Nat. Genet. 2001; PubMed Scopus Google Scholar), and the transcription factor M. M. A. A. Nat. Genet. PubMed Scopus Google Scholar) were observed in but not These are in the studies have their roles in mtDNA We have confirmed the of of DNA in nucleoid using proteins with helicase were observed and are in a large number of proteins in is We also a of proteins in membrane and the as in proteins have been in mtDNA nucleoid from and yeast as It is that not aconitase in the mtDNA the (7Chen X.J. Wang X. Kaufman B.A. Butow R.A. 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We antibodies against proteins identified mass as as several proteins that to such as The experiments shown in several First, as a not of proteins to the in used a large of to the of many proteins in the are also in the Second, TFAM, mtSSB, and are in fractions. proteins, and were in whereas and were in the the aconitase in the of the not the the of in the purified with the of two proteins mass whereas from complexes were not to with the of a of of as a of the DNA metabolic proteins observed in nucleoids in this the DEAH-box which has not previously been as a mitochondrial The this has been to protein in the of of and of The two of are to a protein of the identified but proteins not have with a of mitochondrial In the is to have a mitochondrial of with a of mitochondrial of the for the and protein and using the revealed that the to lack several in the as in the of also as helicase has a very similar in this the that the containing the mitochondrial localization to the of the We that the be in a similar to a protein with the shown in protein would the mitochondrial and peptides identified of the the of this protein in a fragment of in and raised against this of that this nuclear and mitochondrial of DHX30. the mitochondrial isoform of is associated with nucleoids in HeLa cells were and with a against DNA with purified The two were with a a similar with and The in that mtDNA nucleoids are with the as as reported previously (1Legros F. Malka F. Frachon P. Lombes A. Rojo M. J. Cell Sci. 2004; 117: 2653-2662Crossref PubMed Scopus (292) Google Scholar). and antibodies a similar TFAM a of with the mtDNA is that as a DEAH-box may as that with the nucleoid may be than that of the DNA-binding protein It that has a with mtDNA We that the is of a with in mitochondria and studies be to the protein isoform shown in this mitochondrial localization or with mitochondrial localization the mitochondrial of to is a recently mitochondrial protein that is important for of L. L. M.J. J. Mol. Biol. Cell. 2005; PubMed Scopus Google Scholar). a as as a of proteins. thought to be for protein have within that is not to be is to the inner mitochondrial membrane with the of to the in within mitochondria with P. P. E. J. Cell Sci. PubMed Google Scholar). in nucleoid used to is with TFAM as a nucleoid of within mitochondria with to but not with is of the previously for Mmm2p and yeast mtDNA nucleoids (12Youngman M.J. Hobbs A.E. Burgess S.M. 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Other proteins, including DNA and the transcription factor were observed in but not the of (3Garrido N. Griparic L. Jokitalo E. Wartiovaara J. van der Bliek A.M. Spelbrink J.N. Mol. Biol. Cell. 2003; 14: 1583-1596Crossref PubMed Scopus (274) Google Scholar) has helicase as a of identified two in the nucleoid the helicase and helicase DHX30. The helicase has recently been as a mitochondrial protein with DNA helicase M. J. K. S. A. A. E. K. P. PubMed Scopus Google Scholar), in mtDNA replication has not been In observation of multiple peptides from in nucleoids the that this protein can be into of the in and to a for a for a mitochondrial isoform of in the of in raised a to the protein and that isoform of the protein with mitochondria and is in the of mtDNA is to the of in mtDNA replication transcription. proteins previously shown to have were observed in of has been shown to van der L. H. J. Biol. 2003; PubMed Scopus Google Scholar). 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Wang et al. (Fri,) studied this question.