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SPECIAL SECTION ON MITOCHONDRIAL MODELING AND FUNCTION
1Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland; 2Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, and 3Indiana Centers for Applied Protein Sciences, Indianapolis, Indiana
Submitted 11 March 2006 ; accepted in final form 15 August 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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structural proteins; oxidative phosphorylation; liquid chromatography; mass spectrometry; electrophoresis; histone; liver; heart; kidney; brain
It is appreciated that the functional emphasis of mitochondria differs between tissues, as well as potentially even within a given cell (29), depending on the nuclear proteins that are incorporated into the organelle. Before these experiments, a quantitative comparison of the mitochondrial proteins in heart, liver, kidney, and brain has not been performed. The data from these experiments may help elucidate the relative functional emphasis of mitochondria in different tissues and lend insight into the pathways and proteins that might be under similar expression control. These experiments sought to determine the protein expression distribution of mitochondria between brain, heart, liver, and kidney in rat. These experiments establish the quantitative differences in many mitochondrial proteins in different tissues. They provide information on the overall mitochondrial proteome, with 145 new proteins being identified as mitochondrial constituents of the rat proteome.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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180 g body wt) as previously described (4). All procedures were carried out at 4°C. All centrifugations were carried out with an SS-34 rotor spun in a refrigerated Sorvall RC5B centrifuge. Brain mitochondria were isolated as follows. Brain tissue from each rat was placed in 10 ml of cold isolation buffer (250 mM sucrose, 10 mM Tris·HCl, 0.5 mM EDTA, and 0.5 mM EGTA, pH 7.4) and washed three times before it was minced with scissors. The minced tissue was homogenized by 12 strokes of a loose-fitting Potter-Elvehjem homogenizer. The homogenate was diluted with 20 ml of isolation buffer and centrifuged at 4,000 rpm for 3 min. The supernatant was set aside. The pellet was suspended in 10 ml of isolation buffer and homogenized with four strokes of a tight-fitting pestle. After addition of 20 ml of isolation buffer, the homogenate was centrifuged at 4,000 rpm for 3 min. The supernatant was decanted and combined with the primary supernatant. The combined supernatant was centrifuged at 4,000 rpm for 3 min. After the pellet was discarded, the supernatant was spun at 15,000 rpm for 8 min. The supernatant was discarded. The fluffy light-colored portion of the pellet was separated from the darker-brown, bottom portion of the pellet. The brown portion of the pellet was suspended in 40 ml of isolation buffer, and the solution was centrifuged at 15,000 rpm. The supernatant was discarded, and the fluffy top portion of the pellet was again separated from the dark-brown pellet layer and discarded. This process was repeated twice to yield the final mitochondrial pellet. Mitochondria prepared from the six brains, livers, hearts, and kidneys were frozen at –80°C until proteomic analysis. All procedures were in accordance with the guidelines described in the Animal Care and Welfare Act (US Code 2142, Section 13) and approved by the University of Indiana IACUC. Sample preparation for mass spectrometry. Proteins were extracted with 8 M urea (incubated for 1 h with agitation at ambient temperature) and quantified by the Bradford assay according to the manufacturer's instructions (Bio-Rad). A solution (1 ml) containing 200 µg of protein in 8 M urea and 5 mM ammonium carbonate (pH 10.8) was prepared. Chicken lysozyme (1 µg) and 200 µl of a reduction/alkylation solution (97.5% acetonitrile, 2% iodoethanol, and 0.5% triethylphosphine) were added to each sample, and the solutions were incubated at 37°C for 1 h. Samples were placed in a Speedvac overnight to dry completely. Pellets were resuspended in 1 ml of 100 mM ammonium bicarbonate (pH 8) containing 4 µg of trypsin for 4 h at 37°C. After addition of 4 µg of trypsin, the samples were incubated overnight at 37°C. Samples were filtered through a 0.45-µm filter, and 100 µl (20 µg of protein) of sample were used for mass spectrometric (MS) analysis.
Protein identification and quantification by MS. Proteins were prepared and analyzed by liquid chromatography (LC) with tandem MS (LC-MS/MS), as previously described (18). There were four groups (4 different organs as described above), six samples per organ, and two HPLC injections per sample (i.e., 12 analyses per tissue type from 6 different animals). Samples were run on a Surveyor HPLC (Thermo Finnigan) with a C18 microbore column (Zorbax 300SB-C18, 1 mm x 5 cm). All tryptic peptides (20 µg in 100 µl) were injected in random order. Peptides were eluted with a linear gradient from 5% to 45% acetonitrile developed over 120 min at a flow rate of 50 µl/min. The effluent was introduced into a Thermo Finnigan LTQ linear ion-trap MS as it eluted off the column. The data were collected in the triple-play mode. The acquired data were filtered by proprietary software as previously described (18). Database searching against the International Protein Index rat database and nonredundant Rattus database was carried out using the SEQUEST algorithm. Protein quantification was carried out using the protein quantification software licensed from Eli Lilly (18). Briefly, after the raw files are acquired from the LTQ, all total ion chromatograms were aligned by retention time. Each aligned peak was matched by parent ion, charge state, daughter ions (MS/MS data), and retention time (within 1-min window). If any of these parameters were not matched, the peak was disqualified from the quantification. The area under the curve (31) from an individually aligned peak was measured, normalized, and compared for the abundance of that set of matched peptide peaks. A comparison of proteins across tissues is shown in Fig. 1.
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If multiple peptides had the same protein identification, their quantile normalized log2 intensities were averaged to obtain log2 protein intensities. The log2 protein intensity was the final quantity that was fit by a separate ANOVA statistical model for each protein: log2(intensity) = overall mean + group effect (fixed) + sample effect (random) + replicate effect (random), where group effect refers to the effect caused by the experimental conditions or treatments, sample effect represents the random effects from individual biological samples and also includes the random effects from sample preparations, and replicate effect refers to the random effects from replicate injections of the same sample. The workflow for these experiments is illustrated in Fig. 2.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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As a second validation, proteins known to exist in fixed ratios in the mitochondria were compared to determine how closely the measured ratios recapitulated those expected according to the literature if the proteins were identified in group 1* in these studies. The 
- and 
-subunits of the F1-ATPase have been shown to exist in mitochondria in a 1:1 ratio (33) (1, 30), and both proteins were identified in this screen with high confidence. The - and -subunits of the E1 component of the branched-chain -keto acid dehydrogenase complex have been shown to exist in a 1:1 ratio (an 22 complex) (14, 16, 27) and were identified in group 1*. Similarly, the - and -subunits of the E1 component of the pyruvate dehydrogenase complex also exist in a 1:1 ratio (an 22 complex) (20) and were identified in group 1*. The relative proportion of these subunits was determined between tissues. The amount of each was not expected to be the same, because the ionization efficiency of the subunits was not expected to be the same, but the ratio of one subunit to the other should be the same between tissues. In other words, the ratio of the liver signal for to the liver signal for should be similar to the ratio of the heart signal for to the heart signal for . The standard deviation of the ratio of subunits between tissues was 1.26 ± 0.08 for the E1 component of the pyruvate dehydrogenase complex, 1.09 ± 0.11 for the E1 component of the branched-chain -keto acid dehydrogenase complex, and 1.10 ± 0.15 for F1-ATPase. In this type of study, peptides can be compared with themselves, but cross-comparison is not possible, because no information about the relative ionization efficiency was collected. Relative amounts could be quantitated between tissues, but absolute quantitation and intraprotein relative quantitation were not possible.
Protein analysis. Although care was taken to isolate mitochondria from other cellular contaminates, any mitochondrial preparation with intact matrix space and outer membranes will be exposed to extramitochondrial contamination. Thus the isolation procedure used in this study was not adequate to confirm mitochondrial localization. Group 1* peptides were analyzed to determine which were associated with the mitochondria in the literature, the National Center for Biotechnology Information (NCBI) Entre Protein database, AmiGO, UniProt, and Swiss Prot databases. Our analysis yielded 236 proteins with documented association with the mitochondria (see black entries in supplemental table in online version of this article).
The NH2-terminal sequence analysis of Claros and Vincens (12) was used to provide a probability of translocase of the outer mitochondrial membrane (TOM) complex translocation (PTcT) with a threshold of 0.50 for the remaining group 1* proteins. This threshold was chosen as a conservative value compared with the PTcT of known mitochondrial matrix proteins (Table 2).
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0.05). The PTcT value can result in false negatives for matrix proteins as well as proteins that are in the outer membrane [voltage-dependent anion-selective channel type 1 (12)] or intermembrane space. In addition, internal mitochondrial localization sequences were also excluded from this analysis (10), inasmuch as these were very difficult to evaluate accurately. Among the group 1* proteins, 495 were not definitively localized in the literature and do not contain a high PTcT were identified without corroborating evidence (see the online version of this article for supplemental information). Many of these proteins may be localized to the mitochondria in future studies.
To visualize the overall heterogeneity of the mitochondrial proteins across tissues, pie charts were created at the 30% and 50% threshold levels of differences between tissues (Fig. 3).
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10% of the mitochondrial proteome that was enhanced by >50%. Novel mitochondrial protein analysis. The group of proteins that had not previously been localized to the mitochondria were subjected to an NCBI protein domain basic local alignment search tool (BLAST) search. Among these proteins, four histone family proteins and several structural elements were found in the group localized to the mitochondria. It would be unlikely that proteins that are contaminants in a mitochondrial preparation would be demonstrated consistently across samples and across tissues of individual animals. Histone quantitation values are shown in Fig. 4, which demonstrates the consistency of the measurement of the histones across samples and tissues. The structural elements and histone family proteins isolated in this screen, along with their likely PTcT and cleavage sequences, are listed in Table 3. [See supplemental information for complete peptide data (Appendix 1), complete protein data (Appendix 2), graphical data on intensity of individual samples, including replicates of proteins (Appendix 3), individual protein intratissue variation by intensity (Appendix 4), and proteins that are not definitively localized with respect to mitochondria (Appendix 5).]
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The relative mitochondrial protein contents of the tissues were distinctly different, consistent with the notion that the mitochondria were programmed by the nucleus to enhance different functions within a given tissue. Beyond the 5–10% statistical significance of the multiple measurements, what difference in protein content is physiologically significant? Without precise information about the protein content dependence on relative flux control or signaling pathway or structure, it is difficult to establish a hard "functional" threshold for a difference in protein content, especially in a screen of many proteins used in this study. Small protein changes can be significant clinically with compensatory changes in gene expression: <50% expression difference in multiple genes is responsible for conditions such as Down's syndrome and Turner's syndrome. In the case of Rubinstein-Taybi syndrome, loss of one allele of one gene is enough to cause serious disease (25). We assumed a threshold of 30% to include all proteins that may prove biologically significant. We also examined a 50% threshold to get a sense of the distribution of protein on the basis of the threshold selected. Stated another way, to view the overall heterogeneity of the mitochondrial proteome between tissues, we organized the proteins to show the percentage of the total proteins isolated in this screen that was expressed below or above the 30% threshold or above the 50% threshold. Figure 3 shows the overall protein expression difference between mitochondria: 49% of the proteins are homogenously distributed at the 50% difference threshold and only 19% at the 30% threshold. However, the mitochondrial proteins specifically enhanced in a given tissue remained essentially constant at 10% of the proteome at 30% and 50% thresholds, suggesting that this 10% imparted the unique functional enhancements associated with each tissue. Clearly, it is impossible to set a particular protein concentration to represent a physiologically significant threshold, since the actual protein dependence for each protein function is specific to each protein, and many of these remain unknown. However, we believe that the somewhat arbitrary 30% threshold is an excellent point at which to begin evaluation of these changes and did reveal consistent patterns of protein expression with known mitochondrial functional differences across tissues. In comparison across tissues, not surprisingly, the similar biochemical function of liver and kidney was revealed by a 14% and 9% overlap with 30% and 50% difference thresholds, respectively. All other specific overlaps between the different tissues were <5%, with the notable exception of the proteins common to all mitochondria. Thus a large group of mitochondrial proteins are similarly expressed across tissues, whereas specific functions are enhanced in each tissue by modification of only 5–10% of the mitochondrial proteome. In our companion article (18a), we describe the metabolic pathways and cross-correlations of the protein contents across this data set.
There were several limits of this approach of quantitative screening of the mitochondrial proteome. Identification of a protein is dependent on the several factors that limit coverage of detected proteins. A protein can be confidently identified only if at least two distinct peptides that are generated by tryptic digestion can be ionized at levels sufficient to trigger MS/MS analysis with an established pattern of fragmentation in MS/MS. Hydrophobic proteins present a particular problem, since membrane-spanning domains are often repetitive and are not unique between different proteins. These proteins are also more difficult to solubilize and ionize. Special methods are required to extract a quantity sufficient for generation of confident identifications. Inasmuch as a reverse-phase column is used for peptide separation before MS analysis, hydrophobic peptides are also more likely to be retained by the column, although a good coverage of membrane-associated proteins was detected in the present study. High-abundance peptides can mask very low-abundance proteins, and in a study that attempts to be quantitative, any attempt to perform subfractionation before analysis would make quantitation difficult to validate. Thus, beyond purification of the mitochondria, no further fractionation was attempted in this study.
Simple isolation of mitochondria via standard differential centrifugation techniques alone was not adequate to ensure the presence of only mitochondrial elements. Of special concern were peroxisomes, endoplasmic reticulum, and sarcoplasmic reticulum, which have a high affinity for the outer membrane of the mitochondrion and/or can be in particles of similar density. Thus other criteria were needed to ensure that the detected proteins were not just copurifying with mitochondria. In this study, we used the additional confirmations of previous literature citations, consistency in isolation between tissues and animals, and probability of the NH2-terminal sequence TOM complex translocation (PTcT) as additional criteria for mitochondrial localization. We set the threshold of PTcT at a very conservative 0.5 on the basis of comparison with known cytoplasmic and mitochondrial matrix proteins (see supplemental table). With regard to the confidence of protein identification with MS, we again used very conservative thresholds. Only proteins reaching our group 1* confidence level were analyzed in this study. In addition, only proteins that were detected in all samples were included, eliminating tissue-specific contaminants. Using these combined criteria, we obtained intratissue quantitative data of 228 known (i.e., literature cited) mitochondrial proteins, as well as 145 proteins that had a predicted NH2-terminal mitochondrial localization signal and were not previously localized to the mitochondria.
We found 145 proteins that had not been ascribed to the mitochondrial compartment in the rat. We searched several resources to determine localization, including Swiss Prot, Uniprot, Entre Protein, and AmiGO databases and the literature. Among these proteins, two classes of these proteins fell into groups that have not previously been localized to mitochondria: structural elements, such as actin-binding proteins and intermediate filaments, and histone-related proteins. We found four histone family proteins: proteins similar to H3 histone family 3B, CG31613-PA, histone H4, and histone H2A.l. These demonstrated very-high-value PTcT NH2-terminal sequences and also contained mitochondrial cleavage sequences, supporting the notion that these proteins localize to the mitochondrial matrix. The histones were isolated across 4 tissues in all 24 injections of purified mitochondria, and the identifications were remarkably consistent between animals, further reducing the likelihood that these represented contaminants. The ratio of the histones detected was not the same in all tissues but, as illustrated in Fig. 4, was very consistent within a given tissue. Further confidence that these histones were not from a nuclear source is that other nuclear proteins were not detected and are generally uncommon as contaminants in mitochondrial preparations. In the literature, there is some precedence for histone family proteins in mitochondria. Mitochondrial DNA (mtDNA) can form nucleoids with structure similar to chromatin in plants (13), and the yeast literature has demonstrated that histone family proteins are important in proper mitochondrial function (7, 9, 11). There are also evolutionary correlates of histone-like proteins that are important in DNA structure in the prokaryotic organisms that are thought to represent the predecessors of mitochondria (15, 19, 22). The electron micrographs of the mitochondrial nucleoid demonstrate a tightly wound ball of DNA (13) with protein associated, whereas proteins similar to the yeast histone HM protein have been isolated in bovine heart mitochondrial preparations (34). However, it is unclear whether these histones form some type of nucleosome within the matrix. The formation of the nuclear nucleosome requires five histones: histone H1, H2A, H2B, H3, and H4. Since only four histones were found in the matrix, with three "unique" histones containing a cleavage signal distinctly different from nuclear histones, it is unlikely that a standard repeating-structure nucleosome is formed. However, on the basis of the ability of these histone proteins to bind to DNA and the previous data suggesting a mitochondrial nucleoid formation, it is reasonable to speculate that the histones are coating the mtDNA in some fashion. Beyond a classic role of histones in regulating transcription, the unique environment of the mitochondrial matrix suggests another protective role for this coating and why it might be unique for mtDNA in the matrix. The mtDNA is within angstroms of the electron transport chain, which is the major producer of reactive oxygen species (12) in the cell. It is reasonable to suggest that a coating of histones would protect mtDNA from oxidative damage from this local source. The mtDNA is short (16,569 bp in human) and lacks introns or intragenic regions. The repair machinery is considerably less well developed in the mitochondria than the nuclear DNA repair machinery; thus this unique histone coating may be partially responsible for protecting this vulnerable molecule. Clearly, further work is needed on the interaction of these histones and mtDNA with regard to numerous structure-function relations as have been described in the nucleus.
While this manuscript was in review, Forner et al. (17), in a study of mitochondrial protein in rats, included 53 of the proteins listed here as newly localized to the mitochondria. They used criteria that were considerably more inclusive with regard to which proteins localize to the mitochondria. They were also considerably less stringent in assigning protein identification. On the basis of their criteria for a positive identification, we would have listed 2,226 as positively identified in the LC-MS. Also, several of the proteins that they localized to the mitochondria did not meet our criteria for localization following the criteria outlined by Carr et al. (8).
Multiple structural elements were identified in all the mitochondrial preparations with good PTcT scores and, in most cases, mitochondria-specific cleavage signals. These proteins included glial fibrillary acidic protein, glia maturation factor-, lamin A, protein similar to -tubulin, desmin, protein similar to trichohyalin, CORO1A protein, vimentin, cyclase-associated protein homolog, and keratin type II cytoskeletal 8. Many of the 145 proteins identified as mitochondria localized in this study, when subjected to an NCBI protein domain BLAST search, exhibited cytoskeletal binding domains by homology. The role of these structural proteins is unknown. The mitochondrial matrix space is highly ordered and not random in distribution, but structural elements have not been previously defined. It has been assumed that the high concentration of protein in the matrix space (40% of weight) creates a gel that is responsible for the structural organization of the mitochondria. Although this surely accounts for aspects of the matrix structure, it does not explain the mutability of the matrix space and the structural changes that accompany cell division or fission/fusion of the mitochondria or the multiple morphologies that mitochondria can assume even in the same cell type. This would also explain the ability of heart mitochondrial matrix preparations to contract in a hypotonic solution when substrate is added (4, 5). A set of structural proteins would help reconcile, as well as explain, why many proteins established to be in the matrix space have cytoskeletal binding domains. Structural elements have been demonstrated in the plant mitochondria literature. Dai and colleagues (13) showed that an actin homolog was present in all the mitochondrial nucleoids, and Lo et al. (35) showed an actin homolog in intact mitochondria. We speculate that the structural elements uncovered in this study represent those of a scaffolding network in the mitochondrion responsible for its morphology and, possibly, play a role in its division. Several of these proteins by homology contain DNA-binding domains, lending weight to the hypothesis that the regulation of the mitochondrial genome is considerably more complicated than is assumed in the literature. This work expands the proteome of the mitochondria and proposes functions not previously ascribed to the mammalian mitochondria.
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MS was performed at the Indiana Centers for Applied Protein Sciences, with support in part from the Indiana Genomics Initiative and the Indiana 21st Century Research and Technology Fund.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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