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Originally published In Press as doi:10.1074/mcp.M300035-MCP200 on September 26, 2003.
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Molecular & Cellular Proteomics 2:1205-1216, 2003.
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Protein Components of Mitochondrial DNA Nucleoids in Higher Eukaryotes*

Daniel F. Bogenhagen{ddagger},§, Yousong Wang{ddagger}, Ellen L. Shen{ddagger}, and Ryuji Kobayashi||,**

From the {ddagger} 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


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

The hypothesis that a primitive eukaryote first arose through fusion of two preexisting life forms is widely accepted. This model holds that mitochondria evolved from an {alpha}-proteobacterial partner in this fusion (1). Presumably, a process of directional transfer of genes from mitochondria to the nucleus led to the progressive loss of genes from the mitochondrial genome to the nucleus, leaving only a small set of genes in contemporary mitochondrial DNA (mtDNA).1 Relatively little is known of the packaging of animal mtDNA, in contrast to the wealth of information on the protein packaging of nuclear DNA in chromatin. The endosymbiotic hypothesis suggests that the DNA-protein complexes in mitochondria may resemble those in their bacterial ancestors. While eubacteria do not package their DNA in nucleosomes, they do contain abundant basic proteins like HU and INT that compact the DNA and exert a significant influence on gene expression (2).

mtDNA is associated with at least two basic proteins with clear bacterial homologs. The HMG-box protein, mitochondrial transcription factor A (mtTFA), which has been conserved from yeast to humans, is structurally related the bacterial HU protein. Indeed, the yeast mtTFA and Escherichia coli HU proteins can genetically substitute for one another (3). mtTFA plays a role as a transcription factor in vertebrates (4–6), but apparently not in yeast (7). Thus, it appears that the essential role that mtTFA plays in yeast mtDNA maintenance is as an architectural DNA binding protein, which accounts for its relative abundance (8). mtTFA is remarkably abundant in mitochondria of Xenopus oocytes and human HeLa cells, with hundreds of copies of the protein per mtDNA (5, 9–11). A second mtDNA binding protein that is relatively well-characterized is mitochondrial single-stranded DNA binding protein (mtSSB) (12–14). mtSSB is a tetramer of ~16-kDa subunits with DNA binding characteristics and structure closely related to those of E. coli SSB (15–17). The helicase, twinkle, has also recently been shown to reside in the mtDNA nucleoid (18).

While mtTFA and mtSSB appear to be universal mtDNA binding proteins, the structure of mtDNA-protein complexes, or nucleoids, remains poorly understood. Early observations of mtDNA extruded from osmotically ruptured mitochondria revealed circular DNA fibers apparently attached to membrane fragments (19). 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.

Studies of mtDNA nucleoprotein structure have been hampered by the fact that mtDNA constitutes less than 1% of the total DNA in most somatic cells. Various investigators using different tissue sources and different methods have failed to define a consistent set of protein components of mtDNA nucleoids. The notion that mtDNA was attached to the inner mitochondrial membrane was supported by the similar membrane association of the bacterial chromosome and by biochemical studies of complexes isolated from sarkosyl lysates of rat liver mitochondria (22). However, in a later publication, Van Tuyle and McPherson (23), using a very different high salt extraction procedure to isolate rat liver mtDNA-protein complexes, did not find a great similarity between nucleoid proteins and inner membrane proteins. 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. To date, no inner membrane proteins have been positively identified in mtDNA nucleoids.

The composition of mtDNA nucleoids has received more attention in yeast (25). Newman et al. (26) reported purification of a complex containing yeast mtTFA (Abf2p) along with a limited set of polypeptides. Continued experimentation in the Butow laboratory involving sequencing of polypeptides cross-linked to mtDNA identified some of these novel associated proteins as mitochondrial hsp60 and the E2 subunit of yeast {alpha}-ketoglutarate dehydrogenase, KGD2 (27). Interestingly, Sato et al. (28) recently identified KGD2 in their yeast nucleoid preparations as well. Kaufman et al. (27) confirmed that deletion of yeast KGD2 in an abf2-deficient strain increases the instability of the mtDNA genome.

Recent advances in the sensitivity and efficiency of protein sequencing technology have made it possible to reinvestigate the set of proteins associated with mtDNA in higher organisms. We employed mtTFA as a marker to follow nucleoids during purification from Xenopus oocyte mitochondria by adapting methods developed by Barat et al. (24). This approach employs both a glycerol gradient sizing step and a buoyant separation in nonionic metrizamide gradients. We found that the set of proteins associated with mtDNA and the apparent sedimentation properties and buoyant density of nucleoids depends critically on the extent of treatment with nonionic detergents during lysis of mitochondria. The proteins in the crude nucleoid fraction appear to reside in a relatively detergent-resistant complex, because prolonged detergent treatment is required to release less-tightly bound proteins. We have observed a set of proteins that remains persistently associated with mtDNA following two rounds of treatment with Triton X-100. The persistent components of the mtDNA nucleoid include adenine nucleotide translocator, prohibitin2, and the E2 subunits of two large dehydrogenase complexes, pyruvate dehydrogenase and branched chain keto acid dehydrogenase. The novel association of these proteins with the mtDNA nucleoid was confirmed by their detection in mtDNA nucleoids immunoprecipitated with antibodies directed against mtTFA.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Adult female Xenopus laevis were obtained from Xenopus I (Ann Arbor, MI). Metrizamide was obtained from Crescent Biochemicals (Islandia, NY). Other reagent grade chemicals were obtained from Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Sequencing grade trypsin was obtained from Roche (Basel, Switzerland). Antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), Molecular Probes (Eugene, OR) or Calbiochem (La Jolla, CA).

Purification of Xenopus Oocyte Mitochondria
All steps in the purification of mitochondria and DNA-protein complexes were conducted at 4 °C without freezing of the sample at intermediate steps. Ovaries (25–30 g) were excised from two mature Xenopus laevis females, washed extensively in ice-cold 0.5x SSC, and homogenized in 50 ml of MSH buffer (0.21 M mannitol, 0.07 M sucrose, 20 mM HEPES, pH 8, 2 mM EDTA, 2 mM dithiothreitol (DTT)). This and all buffers contained a protease inhibitor mix including 2 mM benzamidine HCl, 0.2 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 5 µg/ml leupeptin, and 0.2 µg/ml L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane (E-64)). Yolk and pigment granules were pelleted by two rounds of centrifugation at 1000 x g in a Sorvall SS34 rotor. Mitochondria were pelleted by centrifugation at 20,000 x g in the SS34 rotor and resuspended in 40 ml of MSH buffer from the light-colored layer of the pellet, leaving the black pigment pellet as undisturbed as possible. Mitochondria were repelleted under the same conditions and resuspended in 30 ml of KMHED buffer (125 mM KCl, 7 mM MgCl2, 30 mM HEPES, pH 8, 1 mM EDTA, 2 mM DTT). This salt wash was intended to reduce contamination by material adherent to the mitochondrial outer membranes. The resuspended mitochondria were layered over four preformed sucrose step gradients containing 15 ml of 1 M sucrose over 12 ml of 1.5 M sucrose in SW28 Ultraclear (Beckman, Fullerton, CA) centrifuge tubes. All sucrose gradient solutions were prepared with a general buffer containing 20 mM HEPES, pH 8, 2 mM DTT, 2 mM EDTA, and protease inhibitors as described above. Gradients were spun at 92,600 x g in the SW28 rotor for 30 min to sediment mitochondria to the 1 M/1.5 M sucrose interface. The solution above this layer was removed by suction, and the mitochondrial layer was gently removed with a Pasteur pipette, leaving the 1.5 M sucrose layer behind. Mitochondria were diluted with three volumes of 0.5x MSH buffer and repelleted as described above. Mitochondria were resuspended in 6 ml of 1.25x lysis buffer (1x lysis buffer is 20 mM HEPES, pH 8, 2 mM EDTA, 2 mM DTT). A sample was removed for determination of the protein concentration, which generally varied from 6 to 7 mg/ml in the Bradford Assay (BioRad, Hercules, CA) or Advanced Protein Assay (Cytoskeleton, Denver, CO) using BSA as a standard. One-fifth volume of 6% Triton X-100 (Surfact-Amps; Pierce, Rockford, IL) was added to adjust the solution to 1.2% Triton X-100. The suspension was inverted gently several times and was observed to clarify and darken upon addition of detergent. The lysate was clarified by centrifugation at 2000 x g in a Sorvall RT6000 bench top centrifuge.

Purification of mtDNA Nucleoids
A total of 700 µl of the 1.2% Triton X-100 lysate was layered onto each of several 15–40% glycerol gradients poured with a gradient maker over a 500-µl pad of 30% glycerol/30% metrizamide. All glycerol gradient solutions contained 30 mM HEPES, pH 8, 2 mM EDTA, 2 mM DTT, 20 mM NaCl, and protease inhibitors as above. Following centrifugation at 186,000 x g in an SW41 rotor for 2 h, 700-µl fractions were collected. In the initial experiments, Triton X-100 was not included in the glycerol gradients, resulting in association of additional inner membrane proteins with nucleoids as discussed in the text. In later experiments, preformed glycerol gradients contained 0.2% Triton X-100. mtDNA was detected in gradient fractions by mixing 50 µl of gradient fractions with 150 µl of a 1:300 dilution of Picogreen dye (Molecular Probes) in the wells of a 96-well microtiter dish. Fluorescence was detected using a Fluorimager 595 (Amersham Pharmacia Biotech, Piscataway, NJ) with excitation at 488 nm and a 530-nm emission filter. As noted in "Results," mtDNA was found in a broad zone in the center of the gradient and in a tight layer at the interface between the gradient and the 30% metrizamide/30% glycerol pad. To confirm that the peak of Picogreen fluorescence in gradient fractions represented mtDNA, 100 µl samples of gradient fractions were mixed with 300 µl of 0.5 M NaAc in TE (10 mM Tris, pH 8, 1 mM EDTA) containing 10 µg of glycogen carrier, extracted with phenol-CHCl3, ethanol precipitated and subjected to electrophoresis on 1% agarose gels. Gels were stained either with ethidium bromide (Calbiochem) or Vistra green (Amersham Pharmacia Biotech) and imaged with a Fluorimager 595 (Amersham Pharmacia Biotech). In some cases, the identity of the mtDNA species was confirmed by transfer of DNA to a Nytran+ filter and hybridization with a probe derived from pXlm32 (29).

Fractions containing mtDNA at the 30% metrizamide/30% glycerol pad or in the center of the glycerol gradient were layered over preformed 25–50% metrizamide (Serva, Heidelberg, Germany) gradients containing 20 mM HEPES, pH 8, 2 mM EDTA, 2 mM DTT, and 10 mM NaCl. Unless indicated otherwise, the glycerol gradient fractions were retreated with 0.5% Triton X-100 before loading on the metrizamide gradient. Gradients were spun for 16 h at 186,000 x g in an SW41 rotor and collected into 600-µl fractions. DNA was detected by Picogreen fluorescence or by agarose gel electrophoresis as described above. In later experiments, the metrizamide gradients contained 0.2% Triton X-100. In the experiment shown in Fig. 6, the metrizamide gradient fractions containing mtDNA nucleoids were pooled, diluted 3-fold with 20 mM HEPES, pH 8, 2 mM DTT, 2 mM EDTA, and layered over a 0.5-ml pad of 50% metrizamide in an SW60Ti centrifuge tube. Samples were centrifuged at 257,000 x g for 3 h and the concentrated mtDNA nucleoids were collected from the pad.



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  		FIG. 6. Identification of proteins in the metrizamide gradient peak fraction. mtDNA nucleoids were prepared as in Fig. 5 except that the glycerol gradient and metrizamide gradient contained 0.5% Triton X-100. The metrizamide fractions containing the peak of DNA fluorescence were concentrated as described under "Experimental Procedures" and proteins were analyzed by SDS-PAGE. Duplicate samples were stained either with Sypro Ruby (lane SR) or with silver (lane Ag). The positions of marker proteins are shown on the left. Major bands were excised, and proteins were digested with trypsin. The peptides were analyzed by LC-MS/MS resulting in the identification of species labeled on the right.

 
Protein Analysis
Ten- or 15-µl samples of gradient fractions were mixed with an equal volume of sample loading solution and subjected to electrophoresis on 10% acrylamide gels in Tris-glycine buffer (30). Gels were either stained with silver (31), or electrophoretically transferred to polyvinyldifuoridene membrane (Immobilon PVDF; Millipore, Billerica, MA) to permit detection of proteins by immunoblotting with specific primary antiserum followed by the appropriate secondary antibody conjugated to alkaline phosphatase. Polyclonal serum directed against Xenopus mtTFA prepared in our laboratory was used at a dilution of 1:5000. Monoclonal antibodies 31HL directed against human porin, and 1D6-E1-A8 against human cytochrome oxidase subunit 1 were obtained from Calbiochem and Molecular Probes, respectively. Polyclonal adenine nucleotide translocator (ANT) (Q-18) antibodies directed against human adenine nucleotide transporter 1 were obtained from Santa Cruz Biotechnology. Antibodies directed against human the E2 subunit of pyruvate decarboxylase (PDC-E2) and prohibitin were obtained from Neomarkers (Fremont, CA) and Molecular Probes, respectively. Commercial antisera were used at the dilutions suggested by the manufacturer. Most secondary antibodies and colorimetric nitroblue-tetrazolium reagents were obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Alkaline phosphatase-labeled bovine anti-goat antibody was obtained from Santa Cruz Biotechnology.

Silver stained proteins selected for sequencing were excised from the gel and destained (32). Proteins were digested with sequencing grade trypsin in situ (33). Peptides recovered from the gel pieces were analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) using a Voyager DE-Str or by liquid chromatography (LC) tandem MS (MS/MS) using a ThermoFinnigan LCQ ion trap mass spectrometer (ThermoFinnigan, San Jose, CA). Proteins were identified by automated matching of ion patterns to a library of protein sequences using SEQUEST software (34) or SONAR (35). A number of proteins were identified with both MS methods, but the data reported here refer to hits observed with LC-MS/MS.

Immunopurification of Nucleoids
A magnetic bead-based affinity column with covalently bound antibodies directed against mtTFA was used to immunopurify nucleoids essentially as described by Alam et al. (11). Immunoglobulins were prepared from the serum of a rabbit immunized with Xenopus mtTFA by protein A Sepharose chromatography. Following desalting, the antibodies were coupled to magnetic tosylactivated M-280 Dynabeads (Dynal, Oslo, Norway) in 0.1 M sodium phosphate, pH 7.5, under conditions recommended by the manufacturer. Approximately 5 x 108 anti-mtTFA-coated beads were incubated with 0.6 ml of a glycerol gradient fraction enriched in mtDNA nucleoids in glycerol gradient buffer containing 70 mM NaCl and 0.5% Triton X-100. Following a 90-min binding, the beads were collected on a magnet, and the unbound proteins were removed as a supernatant fraction. The column was washed three times by resuspension of the beads in buffer containing 20 mM HEPES, pH 7.5, 70 mM NaCl, 2 mM EDTA, 2 mM DTT, 0.5% Triton X-100. Bound proteins were eluted with 10 M urea, 50 mM glycine, pH 2.4, containing 0.05% sarkosyl.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Glycerol Gradient Sedimentation of a Crude Mitochondrial Lysate Reveals Heterogeneous Rapidly Sedimenting mtDNA-Protein Complexes
We adopted the same strategy employed in previous studies of mitochondrial nucleoids to use glycerol gradient sedimentation to take advantage of the large size of these nucleoprotein complexes. However, in previous studies by Barat et al. (24) and by VanTuyle and McPherson (23), mtDNA was radioactively labeled either in vivo or in vitro in isolated mitochondria to permit monitoring of mtDNA. To avoid this radioactive labeling, we detected mtDNA by fluorescent staining with Picogreen dye. This permitted rapid identification of fractions containing mtDNA. To confirm that DNA fluorescence represented mtDNA, samples from gradient fractions were deproteinized and analyzed by gel electrophoresis, with mtDNA detected by staining with EtBr or Vistra green dye, or by hybridization with probes derived from cloned mtDNA. Fifteen to 40% glycerol gradient sedimentation of Triton X-100 lysates of oocyte mitochondria revealed the same sort of heterogeneous, rapidly sedimenting behavior noted in previous studies (Fig. 1). The presence of mitochondrial rRNA near the top of the gradient shows that the nucleoids sedimented much more rapidly than mitochondrial ribosomes. Because we were interested in determining the protein composition of nucleoids, we used mtTFA as a positive control during purification. Fig. 1 shows that the large majority of mtTFA is retained in these complexes. This indicates that most, if not all, of the abundant mtTFA in Xenopus oocyte mitochondria is associated with mtDNA. However, there is no assurance that some proteins may not be lost during sedimentation of nucleoids. Binding of proteins to DNA is generally a reversible reaction, and dissociation of individual protein molecules from the complex would result in trailing of protein behind the major nucleoid species in glycerol gradients. Thus, some mtTFA is observed at the top of the gradient in Fig. 1. We have found that physical homogenization of mitochondria or lysis in the presence of >250 mM NaCl increased the fraction of free mtTFA. mtTFA is completely removed from mtDNA by 0.5 M NaCl, a condition used in the experiments of VanTuyle and McPherson (23; data not shown).



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  		FIG. 1. Glycerol gradient sedimentation of mtDNA nucleoids. A sample of a 2% Triton X-100 lysate of mitochondria was layered on a 15–40% glycerol gradient and centrifuged as indicated under "Experimental Procedures." Eighteen fractions were collected from the tube bottom (Fraction 1). A, 10 µl of the indicated fractions were mixed with sample loading buffer, and mitochondrial proteins were resolved by SDS-PAGE. Proteins were blotted onto PVDF membrane and probed with a polyclonal serum specific for Xenopus mtTFA (9) followed by incubation with phosphatase-labeled secondary antibodies and colorimetric detection. B, 100 µl of indicated fractions were extracted with phenol-CHCl3. Nucleic acids were collected by ethanol precipitation and resolved by agarose gel electrophoresis. A photograph of the EtBr-stained gel is shown.

 
In the experiment shown in Fig. 1, we noted a pellet of material at the bottom of the gradient with an estimated volume less than 1% of the initial mitochondrial pellet. Subsequent analysis showed that this contained approximately one-third of the mtDNA. Therefore, the protocol was modified to include a pad of 30% metrizamide, 30% glycerol under the 15–40% glycerol gradient. Metrizamide is a nonionic density gradient medium that does not promote dissociation of complexes by disruption of ionic interactions (36). Because DNA has a relatively low buoyant density in metrizamide, nucleoids do not sediment through this pad. The sedimentation behavior of mitochondrial nucleoids was observed to depend critically on the detergent concentration used for mitochondrial lysis (data not shown). Triton X-100 at 1.2% was found to provide a larger fraction of mtDNA in the center of the glycerol gradients than either 0.5% or 2% Triton X-100. Nucleoids prepared by lysis with 25 mM octyl glucoside behaved similarly to those prepared with 1.2% Triton X-100. Because this concentration of octyl glucoside is below the critical micelle concentration, we conclude that aggregation within micelles is not a factor in the rapid sedimentation of nucleoids. We also found that the sedimentation depended critically on ionic strength. As shown in Fig. 2, a larger fraction of nucleoids sedimented to the pad below the glycerol gradient at low salt, and the nucleoid fraction containing mtDNA and mtTFA sedimented at a progressively slower rate at 70 mM and 120 mM NaCl. In this study, we report only results obtained with the nucleoid fraction retained in the gradient, not with the more rapidly sedimenting complex.



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  		FIG. 2. Sedimentation properties of mtDNA nucleoids depend on ionic strength. Mitochondria were lysed with 1.2% Triton X-100 in the presence of 20 mM NaCl (A), 70 mM NaCl (B), or 120 mM NaCl (C). Lysates were layered on 15–40% glycerol gradients containing the same NaCl concentrations, centrifuged, and fractionated as described in Fig. 1. DNA content was determined by Picogreen fluorescence, and mtTFA was identified by Western blotting as in Fig. 1.

 
Isopycnic Centrifugation of Glycerol Gradient-purified Nucleoids in Nonionic Metrizamide Gradients Shows a Distinct Protein:DNA Peak
mtDNA nucleoids are not highly purified after only a single purification step using glycerol gradient centrifugation. Therefore, we followed the procedure of Barat et al. (24) to employ isopycnic centrifugation of nucleoids in metrizamide gradients to provide additional purification. Fig. 3 shows that the combination of glycerol gradient sedimentation and banding in metrizamide gradients provided a nucleoid fraction with a protein composition very similar to that reported by Barat et al. (24). When the glycerol gradient-purified nucleoids were treated with DNase I before centrifugation in the metrizamide gradient, these proteins shifted to a higher position in the gradient. This nuclease sensitivity confirms that the proteins are associated with mtDNA.



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  		FIG. 3. The buoyant densities of nucleoid proteins in metrizamide gradient fractions are dependent on mtDNA. Glycerol gradient fractions containing nucleoids were identified by Picogreen fluorescence and pooled. Next, 700-µl samples were incubated with 30 µg/ml DNase I at 20 °C for 5 min or mock-incubated under the same conditions without nuclease. These samples were layered on metrizamide gradients and centrifuged as described under "Experimental Procedures." A and B show silver-stained SDS-PAGE analyses of proteins in the metrizamide gradients of the mock-treated sample (A) or the DNase I-treated sample (B). The arrow on the right of B indicates DNase I. C shows the Picogreen fluorescence in the metrizamide gradients of DNase I- and mock-treated samples.

 
To identify proteins in the nucleoid fraction, individual silver-stained protein bands were excised, digested in situ with trypsin, and analyzed by LC-MS/MS. The set of proteins we identified by sequencing included cytochrome oxidase subunits I and II, ATPase A and B, VDAC (voltage-dependent anion channel, or porin), and the adenine nucleotide translocator 1 (ANT 1) in addition to the nucleoid marker, mtTFA (Table I). These results provided the first identification of individual inner membrane proteins associated with vertebrate mtDNA nucleoids. No attempt was made to sequence all of the protein species in this fraction, because we assumed many inner membrane proteins would be represented. Finding VDAC (porin) in the mtDNA nucleoid fraction was not expected, because this is actually an outer membrane protein. However, VDAC is known to associate tightly with ANT to form the mitochondrial permeability transition pore that spans both membranes at contact sites and may be retained in nucleoids on this basis. The presence of VDAC in this nucleoid preparation suggested that all of the proteins in the preparation were not in direct contact with mtDNA.


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  		TABLE I Proteins detected by LC-MS/MS in mitochondrial nucleoids

 
In order to determine whether proteins detected in the nucleoid preparation truly copurified with mtDNA, proteins in the metrizamide gradient fractions were blotted and probed with antibodies directed either against Xenopus mtTFA or against several human mitochondrial membrane proteins. The immunoblots shown in Fig. 4 revealed that the mtTFA cobanded with mtDNA as expected and confirmed the presence of mitochondrial membrane proteins in this fraction. Remarkably, essentially all of the ANT protein detected in the metrizamide gradient was found in fractions containing mtDNA. Two other membrane proteins identified by sequencing, COX1 and porin (VDAC), showed a bimodal distribution with some protein associated in gradient fractions containing nucleoids, but with a substantial amount found in the upper regions of the gradient as well. This suggested that some proteins might be more loosely associated with mtDNA or might dissociate from complexes during the 16-h metrizamide centrifugation step.



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  		FIG. 4. Metrizamide gradient analysis of mtDNA nucleoids. A 700-µl sample of the glycerol gradient region containing mtDNA was layered on a 25–50% metrizamide gradient as described under "Experimental Procedures." Following centrifugation, 600-µl fractions were collected from the tube bottom. A, EtBr-stained gel of nucleic acids in the metrizamide gradient fractions. B, silver-stained protein gel of gradient fractions. C–F, immunoblots of proteins in gradient fractions probed with antisera directed against mtTFA (C), ANT1 (D), VDAC (or porin, E), and COX1 (F) antiserum followed by phosphatase-labeled secondary antibodies.

 
We suspected that the retention of some of the proteins in mtDNA nucleoid preparations in Figs. 3 and 4 might vary with the extent of detergent treatment. Therefore, we performed another purification in which additional Triton X-100 was included in the glycerol and metrizamide gradients. As shown in Fig. 5, this modified procedure produced a dramatic change in the protein composition of the nucleoids. Most silver-stained proteins, including VDAC and COX1, were stripped from mtDNA by more aggressive treatment with nonionic detergent. In contrast, ANT1 remained tightly associated in the nucleoid. The metrizamide-gradient purified nucleoids were concentrated and proteins were resolved by SDS-PAGE (Fig. 6). This fraction included five major silver-stained bands along with additional minor polypeptides. Protein sequencing confirmed the presence of mtTFA and ANT1 in the 30-kDa region and mtSSB at 16.8 kDa. Three higher molecular mass proteins were identified as pyruvate dehydrogenase subunit E2 (PDC-E2), branched chain ketoacid dehydrogenase subunit E2 (BCKD-E2), and the large subunit of prohibitin (PHB2). For the case of BCKD-E2 and PHB-2, the initial search of the GenBank nonredundant database by SONAR software identified the mouse homologs of the Xenopus proteins, because the authentic Xenopus proteins were not contained in the database. Following these identifications, BLAST searches using the mouse sequences as queries were performed to identify expressed sequence tags in the database. The sequences were used to construct contigs to predict the sequences of the Xenopus proteins. These complete coding sequences were analyzed to confirm that all of the peptides identified by the SONAR algorithm were represented in the Xenopus proteins.



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  		FIG. 5. Metrizamide gradient analysis of mtDNA nucleoids retreated with Triton X-100. A 700-µl aliquot from the same glycerol gradient preparation as in Fig. 3 was mixed with one-fourth volume of 10% Triton X-100 (final 2% Triton X-100) and layered on a metrizamide gradient. From this point, all steps in centrifugation and sample handling were performed in parallel with the control shown Fig. 4 without detergent retreatment. As in Fig. 4, A shows an analysis of the DNA content of gradient fractions, stained with Vistra green dye in this case. B shows the silver-stained proteins in gradient fractions, and C–F show immunoblots of protein fractions probed with the same antibodies used in Fig. 4, mtTFA, ANT1, VDAC, and Cox1, respectively.

 
The observation that ANT, PHB, PDC-E2, and BCKD-E2 are associated with mtDNA was unexpected. We took advantage of the fact that antibodies were available to the first three of these proteins to confirm their association with mtDNA nucleoids. First, we showed that each of these proteins cobanded in metrizamide or nycodenz gradients with mtTFA and mtDNA as shown in Fig. 5 for ANT and Fig. 7 for PHB and PDC-E2. Second, to confirm these results using an independent method, we immunopurified mtDNA nucleoids from the glycerol gradient fraction using a magnetic-bead column coated with antibodies to mtTFA. This method to purify mtDNA nucleoids has been used previously by Alam et al. (11). Under the conditions we employed, we found that ~60% of input mtDNA bound the column. The column was washed three times with buffer containing 70 mM NaCl and 0.5% Triton X-100, and bound proteins were eluted with a buffer containing 10 M urea as described under "Experimental Procedures." Equal fractions of the unbound supernatant, the wash fraction, and the final eluate were subjected to electrophoresis, blotted, and probed with antibodies directed against proteins of interest. As can be seen in Fig. 8, mtTFA, PDC-E2, PHB, and ANT were detected in the urea eluate, while porin was principally detected in the unbound and wash fractions, providing a negative control. We conclude that ANT, PDC-E2, PHB, and ANT are specifically associated with the mtTFA-mtDNA nucleoid.



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  		FIG. 7. Novel nucleoid proteins coband in metrizamide gradients with mtDNA. A, the relative DNA content of selected fractions of a metrizamide gradient analysis of mtDNA nucleoids is shown to illustrate the distribution of DNA surrounding the peak in fraction 6. B, 20-µl samples of fractions 4–8 surrounding the DNA peak were subjected to SDS-PAGE. Proteins were transferred to PVDF membrane, and mtTFA, PDC-E2, and PHB were identified by immunoblot analysis.

 


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  		FIG. 8. Novel nucleoid proteins immunoprecipitated with mtDNA nucleoids. A 600-µl sample of a glycerol gradient fraction containing mtDNA nucleoids was incubated with an affinity column containing tosylactivated M280 Dynabeads coupled to polyclonal antibodies directed against mtTFA. The unbound supernatant, detergent wash fraction, and the urea eluate were obtained by magnetic separation of the beads from solution as described under "Experimental Procedures." Samples of each fraction were subjected to SDS-PAGE, and proteins were detected by immunoblotting with antibodies directed against mtTFA, PDC-E2, ANT, PHB, and VDAC, as shown.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Operational Definition of mtDNA Nucleoids
Our study is the first to systematically analyze the protein composition of mtDNA nucleoids in a higher eukaryote during various stages of purification. 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. Thus, the concern that nuclear chromatin will contaminate a preparation of mtDNA nucleoids is reduced by the use of a cell type in which most of the DNA is mitochondrial in origin. We employed the two-step isolation procedure of Barat et al. (24) as a starting point. This method permits nucleoids to be purified on the basis of their rapid sedimentation in glycerol gradients and unusual density in metrizamide or nycodenz gradients. We used the well-characterized mtDNA binding protein mtTFA as a marker to monitor the isolation procedures. The polypeptide composition we obtained for the relatively crude nucleoids prepared by detergent lysis, sedimentation, and isopycnic centrifugation in metrizamide gradients showed excellent agreement with the results of Barat et al. (24). Sequencing of a number of major proteins revealed the presence of ATPase A and B, cytochrome oxidase subunits I and II, as well as porin and ANT1 in addition to mtTFA in this initial preparation. This provides the most complete evidence to date that mtDNA nucleoids isolated from vertebrate mitochondria are in fact membrane-associated. Indeed, it was quite surprising to see the wide range of membrane proteins retained in nucleoids liberated by a single treatment with nonionic detergent. The behavior of this fraction is reminiscent of detergent-resistant lipid rafts (37). However, because the raft structure is thought to depend on high cholesterol content, it seems more likely that the detergent-resistant behavior of the nucleoid may reflect the unusual structure of the mitochondrial inner membrane, which is thought to contain 75% protein and only 25% lipid. Many proteins may copurify with a core mtDNA nucleoid based on protein-protein interactions or due to the difficulty of removing lipid quantitatively from the hydrophobic inner membrane protein domains. We found that more aggressive detergent treatment, achieved by including 0.2–0.5% Triton X-100 in the glycerol and metrizamide gradients, removed the majority of loosely associated membrane proteins. In this study, we sought to identify proteins that remain associated with mtDNA following extensive treatment with nonionic detergents under low ionic strength conditions. However, we caution that the mtDNA nucleoid must be defined in operational terms that depend on the salt and detergent conditions used to prepare these complexes. Further experiments will be required with different handling conditions and in entirely different cell types to determine the generality of the nucleoid composition reported here and the roles of individual proteins.

Identification of Novel Proteins in mtDNA Nucleoids
We began these experiments with the goal of identifying novel mtDNA binding proteins that could anchor the nucleoid to the mitochondrial inner membrane. Our protein sequencing identified several proteins that could conceivably serve in this role. These will be discussed individually below. However, it is important to point out some common attributes shared by the novel proteins we observed in nucleoid preparations. First, they are all remarkably abundant and only a minor fraction of each protein is retained in mtDNA nucleoids. Second, each of the four novel mtDNA nucleoid proteins reported here have other well-characterized roles in mitochondria. Unfortunately, this implies that it will be difficult to use genetic methods to study these associations because all of the proteins we have identified are likely to be required for their critical roles in mitochondrial function. Third, in each case, the nature of the interaction of these proteins with mtDNA is uncertain. We have not determined whether any of these proteins has direct DNA binding activity or may instead associate with the mtDNA through interactions with other authentic mtDNA binding proteins such as mtTFA. These proteins are found in large complexes that could help account for the proteinaceous "knob" identified in association with mtDNA by Albring et al. (20). We have no data addressing the issue of whether the proteins we have observed bind mtDNA in a sequence-specific manner as suggested by Albring et al. (20). Finally, each of the novel mtDNA nucleoid proteins identified here present tantalizing connections to the general issue of the inheritance and/or stability of mtDNA, as summarized below.

The fact that the novel mtDNA nucleoid proteins we have identified are abundant may suggest that their association with mtDNA is artifactual. It is indeed difficult to rule out this possibility because the mitochondrial matrix compartment has an extraordinarily high protein concentration, estimated at ~500 mg/ml, and no point in the matrix is generally more than 200 Å away from inner membrane (38, 39). Given this potential complexity, two considerations suggest that our observation of novel proteins in mtDNA nucleoids is significant. First, the retention of known abundant mtDNA binding proteins, mtTFA and mtSSB, in the nucleoids provides a valuable positive control. Second, the proteins retained in these complexes are relatively tightly associated because other abundant proteins in the crude nucleoid preparation, such as ATPase subunits and porin, are effectively removed by more extensive washing of the complexes with nonionic detergents. Finally, we have used two very different procedures, buoyant density gradient centrifugation and immunoprecipitation, to show that ANT, PDC-E2, and PHB are retained in association with mtDNA nucleoids under conditions in which more indirectly associated membrane proteins such as porin are removed.

Adenine Nucleotide Translocator 1—
We have consistently observed that ANT1 is physically associated with mtDNA, having sequenced this protein in four independent nucleoid preparations. We typically obtain better sequence coverage for this protein than for the mtTFA, which comigrates with it on our SDS-polyacrylamide gels. ANT1 plays a major role in exchanging ADP for ATP at the mitochondrial inner membrane. Recently, Kaukonen et al. (40) have shown that the ANT1 gene is mutated in some patients with a genetic disorder leading to multiple mtDNA deletions, autosomal dominant progressive external opthalmoplegia (AD-PEO). Because ANT1 is important for the exchange of adenine nucleotides, it has been suggested that this mutation affects mtDNA replication through an indirect effect on nucleotide pools. Mice lacking the heart/muscle-specific Ant1 also accumulate tissue-specific deletions in mtDNA, which has been thought to be secondary to increased oxidative stress (41). The observation that ANT1 is in physical contact with mtDNA nucleoids raises the novel possibility that mutations may interfere with mtDNA replication in a manner that induces the multiple deletions observed in AD-PEO patients. ANT is a small protein with six membrane-spanning regions, yet has a very basic pI of 9.78, with clusters of positively charged residues exposed to the mitochondrial matrix (42). Interestingly, a primary sequence similarity between ANT and the DNA binding domain of estrogen receptor has been reported (43).

ANT is one of a large family of metabolite transporters in the mitochondrial membrane. Genetic studies in yeast have suggested that other members of this family, namely Yhm2p and Rim2p, may contribute to mtDNA maintenance. Cho et al. (44) showed that the YHM2 gene is a multicopy suppressor of a null mutant of ABF2, the yeast mtTFA. Interestingly, another suppressor of ABF2 deficiency is the ILV5 gene, which plays a role in branched chain amino acid metabolism, providing a potential link to a second of our novel proteins, BCKD-E2. Rim2p is a second member of the family of mitochondrial transporters that has a role in mtDNA maintenance in yeast. VanDyck et al. (45) showed that overexpression of the RIM2/MRS12 gene product can suppress the mtDNA loss phenotype observed in cells deficient in the Pif1 DNA helicase.

Dihydrolipoyl Acetyl- and Acyl-Transferases—
Our observation that both the E2 subunits of pyruvate dehydrogenase and branched chain keto acid dehydrogenase are associated in mtDNA nucleoids provides an interesting parallel to the work summarized in the introduction showing that the E2 subunit of {alpha}-keto glutarate dehydrogenase is associated with the yeast mtDNA nucleoid (27, 28). These three massive enzyme complexes each contain either 24 or 60 E2 subunits in a core surrounded by E1 subunits that provide the substrate specificity for their respective 2-oxo-acid dehydrogenase activities and by a common E3 subunit that regenerates the lipoic acid carrier. In each case, the E2 subunit employs a mobile lipoyl-conjugated domain to transfer the product of decarboxylation from the E1 subunit to acetyl CoA. All three complexes utilize a common E3 subunit to regenerate the E2 carrier (46). It is surely not coincidental that these E2 subunits are found in mtDNA nucleoids from highly divergent organisms and that disruption of the yeast KGD2 gene influences mtDNA stability. The nature of the association of these proteins with mtDNA nucleoids is presently obscure. It is noteworthy that binding of PDC-E2 proteins to DNA has been reported in two bacterial systems as well (47, 48).

It is striking that we have not seen other subunits of pyruvate dehydrogenase or branched chain keto acid dehydrogenase in nucleoid preparations. This implies that the E2 subunits of these large complexes are selectively isolated with mtDNA nucleoids without apparent association with the E1, E3, or other minor components of the large dehydrogenase complexes. The E2 subunits associate tightly in trimers stabilized by an extensive buried surface (49). These trimers are, in turn, assembled less tightly at the vertices of either a cube or a dodecahedron to form the 24- or 60-subunit core of a dehydrogenase. If mtDNA nucleoids associated with the exterior surface of intact pyruvate dehydrogenase, for example, one would expect that the principle contacts would be with the E1{alpha}2ß2 tetramers surrounding the E2 core, and that these subunits would tend to block access to the E2 subunits. Following importation into mitochondria, the E2 subunits are modified by addition of the lipoic acid cofactor to an exposed lysine residue in a flexible N-terminal domain. It is tempting to suggest that mtDNA nucleoids interact with E2 subunits selectively, not necessarily with the fully assembled complex. The lipoic acid cofactor covalently linked to the E2 subunits could provide an association with the mitochondrial inner membrane in much the same way that farnesylation helps anchor proteins to membranes. Stanley et al. (50) have suggested that the 2-oxo acid dehydrogenases may be associated with the inner membrane.

The E2 subunit of pyruvate dehydrogenase has additional clinical importance as an antigen in the autoimmune disease biliary cirrhosis (51). The E2 subunits of alpha-ketoglutarate dehydrogenase and branched chain amino acid dehydrogenase can also elicit this autoimmune disorder (52). In other autoimmune disorders, such as systemic lupus erythematosis, nucleic acid binding proteins are targeted by the immune system. It is tempting to speculate that the association of these E2 subunits with mtDNA may factor in their inappropriate presentation to the immune system as auto-antigens.

Prohibitin 2—
Several highly significant peptide hits were observed for the larger of two prohibitin proteins, establishing PHB2 as a component of the mtDNA nucleoid. PHB1 and PHB2 are associated in a large chaperone-like complex bound to the inner membrane though intrinsic transmembrane domains in PHB2. Co-immunoprecipitation experiments have shown that neither PHB1 not PHB2 is found without its binding partner (53). After we observed PHB2 sequences in our nucleoid preparation, we used immunoblotting to detect PHB1 among nucleoid proteins at a position migrating slightly faster than mtTFA (Fig. 7).

Several possible functions have been ascribed to the prohibitin complex in the scientific literature. A recent review clearly establishes its major role as a chaperone-like regulator of the AAA protease in the mitochondrial matrix that assists in the assembly of inner membrane complexes (54). The prohibitin complex is thought to help fold nascent membrane proteins synthesized within mitochondria. Because transcription and translation occur in the same intra-mitochondrial compartment, the association of PHB with mtDNA nucleoids may result from coupled transcription, translation, and folding of nascent membrane proteins. Alternatively, prohibitin may bind to dihydrolipoyl acetyl- and acyltransferase (E2) subunits of dehydrogenases that are in the process of being assembled into higher molecular mass complexes. This would help to account for the presence of both the E2 proteins and prohibitin in the mtDNA nucleoids. While these large dehydrogenase complexes are capable of self-assembly in vitro, prohibitin may participate in the assembly process in vivo. Prohibitin has been shown to influence cellular senescence (53, 55) and mitochondrial inheritance in Saccharomyces cerevisiae (56). The involvement of mtDNA mutations in aging is a subject of active research, and it is a high priority to understand the mechanisms whereby segregation and amplification of mutant mtDNAs permits the accumulation of high levels of mutant mtDNA genomes in tissues affected by mitochondrial disorders. Further experiments will be required to determine whether the presence of prohibitin in association with the mtDNA nucleoid is important to these processes.

Implications for mtDNA-Protein Dynamics
Our results show that mtDNA binds tightly to a limited number of proteins that help anchor it to the mitochondrial inner membrane. The identity of one of these as ANT1 establishes that mtDNA is attached to the membrane near the major pore involved in adenine nucleotide exchange and in the loss of membrane potential during apoptosis. Whether this association has implications with regard to mtDNA dynamics during apoptosis will be an interesting subject for future research. The three classes of novel proteins we have identified, ANT1, 2-oxo-acid dehydrogenase E2 subunits, and prohibitin, all have potentially interesting clinical implications in mitochondrial diseases and aging. Because mitochondria undergo continuous fusion and fission events, we suggest that the association of mtDNA with key metabolic proteins and with the ANT pore complex may prevent the mtDNA from becoming segregated in nonfunctional submitochondrial complexes.

A final interesting aspect of our results is that, with the exception of abundant proteins mtTFA and mtSSB, we did not detect proteins known to be involved in mtDNA replication and transcription. These regulatory proteins may be lost from the nucleoids during purification or may be present at levels below our current detection limits. Our use of Xenopus oocytes as the source for nucleoid purification may also be an important variable because many of the mtDNA genomes in our preparation may be derived from mature oocytes and may be relatively inactive in replication and transcription. Efforts are underway to extend the methods employed in this study to other cell systems.


   ACKNOWLEDGMENTS
 
We thank Stacey Lopez and Robert Rieger for assistance with protein sequencing and Thomas Roche for helpful discussions.


   FOOTNOTES
 
Received, April 18, 2003, and in revised form, September 17, 2003.

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. Back

* 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. Back

Current address: University of California San Francisco, San Francisco, CA 94143. Back

** Current address: M.D. Anderson Cancer Center, Houston, TX 77030. Back

§ 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.


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