HomeLarge Type Edition
HOME ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62:223-234 (2007)
© 2007 The Gerontological Society of America

Effect of Aging and Caloric Restriction on the Mitochondrial Proteome

Jinsook Chang, John E. Cornell, Holly Van Remmen, Kevin Hakala, Walter F. Ward and Arlan Richardson

Departments of 1 Physiology, 2 Cellular and Structural Biology, and3 Medicine and 4 Biochemistry and the 5 Barshop Institute for Longevity and Aging Studies at the University of Texas Health Science Center at San Antonio.
6 Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System, San Antonio.

Address correspondence to Walter F. Ward, PhD, Department of Physiology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive-MSC 7756, San Antonio, TX 78229-3900. E-mail: wardw{at}uthscsa.edu


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The rat mitochondrial proteome was analyzed using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), and proteins altered by age or caloric restriction (CR) were identified using mass spectrometry. Of 2061 mitochondrial proteins analyzed in the three tissues, a significant change with age occurred in 25 liver proteins (19 increased, 6 decreased), 3 heart proteins (1 increased, 2 decreased), and 5 skeletal muscle proteins (all increased). CR prevented the age-related change in the level of one liver mitochondrial protein, altered the levels of four proteins (one increased, three decreased) from heart, and one protein (decreased) from skeletal muscle. Identification of the proteins that changed with age or CR revealed that they were varied among the three tissues, that is, not one mitochondrial protein was changed, in common, by age or CR in any tissue studied. Thus, the effect of age on the mitochondrial proteome appears to be tissue-specific, and CR has a minor effect on age-related protein changes.

MITOCHONDRIA play many important roles in the function of cells. For example, through oxidative phosphorylation, mitochondria are responsible for the production of ~90% of the adenosine triphosphate (ATP) used for cellular functions (1); mitochondria are involved in the initiation of the intrinsic pathway of apoptosis (2); and mitochondria modulate calcium homeostasis involved in cell signaling (3). During the production of ATP, which is generated from the reduction of molecular oxygen, mitochondria are a major source of reactive oxygen species (ROS) in cells, and ROS have been implicated in a variety of chronic diseases such as atherosclerosis, emphysema, heart disease, neurodegenerative disease, and cancer (4,5).

Over the past three decades, a large amount of evidence has accumulated indicating that mitochondria play a role in aging (6–9). For example, the activities of the mitochondrial electron transport chain complexes isolated from a variety of tissues in several species are reported to decrease with age (10–21). Relevant to this alteration in electron transport chain activity, tissue-specific ATP content and production also are reported to decrease with age in mammals (22). In addition, both the rate and the sensitivity of induction of the permeability transition in response to calcium are shown to increase with age in mitochondria isolated from several tissues (23–25). Furthermore, mitochondria are observed to change with age by a decline in the level of mitochondrial transcription (26–29), a decline in metabolite transport (30,31), a decline in proteolytic activity (32,33), and an increase in mitochondrial DNA mutations (34,35). In addition to alterations in function, the generation of ROS by mitochondria appears to increase with age. For example, an age-related increase in ROS production by mitochondrial and submitochondrial particles has been observed in insects and mammals (36–38).

Caloric restriction (CR), where rodents are fed 30%–40% fewer calories than their ad libitum-fed littermates, is shown to extend the maximum life span 30%–50% and to retard both the rate of biological aging and the development of age-associated diseases (39–42). Although the molecular mechanism underlying effects of CR on aging remains to be determined, it has been argued that the alteration of mitochondrial structure and function by CR could play an important role in the anti-aging mechanism of CR (43). For example, CR retards the age-related decrease in mitochondrial electron transport chain activities (14), and several investigators report that CR reduces superoxide and/or hydrogen peroxide generation by mitochondrial and submitochondrial particles (37,44–46).

Although there is a great deal of evidence for changes in mitochondrial function with age and for ability of CR to retard some of these changes, there is very little information on the mechanism responsible for the age-related changes in mitochondrial function. Because proteins play a critical role in the structure and function of cells and organelles, age-related alterations in the mitochondrial proteome could be important to mitochondrial function. The mitochondrial proteome is thought generally to consist of 1000–3000 proteins (47). Using high-resolution 2-D PAGE, we have undertaken a comprehensive analysis of the mitochondrial proteome to study the effects of aging and CR on mitochondrial proteins obtained from rat liver, heart, and skeletal muscle. It should be noted that we have focused on the soluble mitochondrial proteins, and have successfully identified ~25%–50% of the mitochondrial proteome, based on the predicted range of the total proteins in the mitochondrial proteome. It is well known that current 2-D PAGE technology is unable to resolve large hydrophobic proteins, such as membrane proteins, and is also limited in the detection of very low abundance proteins. Resolution and identification of these proteins will require further technological advances in proteomics research.


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Materials
Immobiline DryStrip Gels (IPG; pH 3-10, 13 cm), IPG buffer, urea, DryStrip cover fluid, bromophenol blue, PlusOne 2-D Quant Kit, and DTT (dithiothreitol) were purchased from GE Healthcare (Piscataway, NJ). SDS (sodium dodecyl sulfate), Tris-base, glycine, 30% acrylamide/bis solution (37.5:1), TEMED (N, N, N', N'-tetramethylethylenediamine), and ammonium persulfate were purchased from Bio-Rad (Hercules, CA). CHAPS (3-[(3-cholamidopropyl)-dimethyammonio]-1-propane sulfate)) was obtained from Anatrace (Maumee, OH). Iodoacetamide, mannitol, sucrose, HEPES (N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid), trifluoroacetic acid, and glycerol were obtained from Sigma (St. Louis, MO). Sypro Ruby protein gel stain was obtained from Molecular Probes (Eugene, OR), and trypsin was purchased from Promega (modified; Madison, WI).

Animals and Diet
Female Fischer 344 rats at 6–7 months (young) and 24–25 months (old) of age, fed ad libitum or a CR diet, were obtained from the National Institute on Aging colony maintained by Harlan Sprague Dawley (Indianapolis, IN). CR was initiated at 14 weeks of age at 10% restriction, changed to 25% restriction at 15 weeks, and to 40% restriction at 16 weeks, at which time it was maintained throughout the life of the animal. The rats were housed one per cage and were maintained under barrier conditions on a 12-hour dark/light cycle. Rats were decapitated in the morning of each collecting date to minimize variations, and then the hearts, livers, and skeletal muscles (gastrocnemius muscle) were excised immediately. All procedures for handling the animals were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and the Subcommittee for Animal Studies at the Audie L. Murphy Memorial Veterans Hospital.

Isolation of Liver Mitochondria
Mitochondria were isolated according to the method of Johnson and Lardy (48). The liver was homogenized in ice-cold homogenization buffer consisting of 250 mM mannitol, 75 mM sucrose, 0.5 mM EGTA, 0.1 mM EDTA, and 10 mM HEPES by using a Potter-Elvehjem glass homogenizer with a teflon pestle. Homogenates were centrifuged at 1000 x g for 12 minutes to remove cell debris, and the supernatants were centrifuged at 10,000 x g for 15 minutes to obtain mitochondrial pellets. The resulting mitochondrial pellets were resuspended in homogenization buffer with 0.02% bovine serum albumin and centrifuged at 10,000 x g for 15 minutes. The pellets were then resuspended in homogenization buffer without EGTA and centrifuged at 10,000 x g for 15 minutes. This step was repeated once more. All isolation procedures were performed at 4°C. The resulting mitochondrial pellets were solubilized in lysis buffer consisting of 8 M urea, 4% CHAPS, and 40 mM Tris base, and then were centrifuged at 70,000 x g for 10 minutes. The supernatant, which contained the liver mitochondrial proteins, was stored at –80°C in various aliquots. Aliquots of supernatant were taken for 2-D PAGE, and the protein concentration of the mitochondrial aliquots was determined using a PlusOne 2-D Quant kit.

Isolation of Heart Mitochondria
Mitochondria were isolated as previously described (49). Heart tissue was homogenized in the same homogenization buffer used for isolation of liver mitochondria, with the addition of 0.02% nagarase, using a Potter-Elvehjem glass homogenizer. Bovine serum albumin (0.5%) was added to the homogenate, and the mixture was centrifuged at 1000 x g for 5 minutes. The resulting supernatant was centrifuged at 10,000 x g for 5 minutes to obtain mitochondrial pellets, which were then resuspended in homogenization buffer without EGTA. This step was repeated again, and the pellets were solubilized in lysis buffer as previously described for isolation of liver mitochondria.

Isolation of Skeletal Muscle Mitochondria
Mitochondria from skeletal muscle of rats were isolated as described by Chang and colleagues (50). Briefly, the gastrocnemius muscles from rats were excised and incubated with 3 mg of nagarase/g tissue for 5 minutes in Chappell–Perry buffer containing 100 mM KCl, 50 mM Tris-HCl, 5 mM MgCl2, 1 mM EDTA, and 1 mM ATP. The muscles were homogenized in prechilled Chappell–Perry buffer using a Potter-Elvehjem glass homogenizer with a teflon pestle. The homogenate was diluted with Chappell–Perry buffer and centrifuged at 600 x g for 10 minutes. The resulting supernatant was centrifuged at 14,000 x g for 10 minutes. The mitochondrial pellets were suspended in modified Chappell–Perry buffer containing 100 mM KCl, 50 mM Tris (pH 7.5), 1 mM MgCl2, 0.2 mM EDTA, and 0.2 mM ATP, and were centrifuged at 7000 x g for 10 minutes. Mitochondrial pellets were then resuspended in a half volume of modified Chappell–Perry buffer and centrifuged at 3500 x g for 10 minutes. The resulting mitochondrial pellets were dissolved in lysis buffer and handled as described above for liver.

2-D PAGE
The procedure used for 2-D PAGE was previously described by Chang and colleagues (50). Mitochondria extracts (100 µg of protein) were rehydrated in rehydration buffer containing 0.5% IPG buffer, 8 M urea, 0.5% CHAPS, a trace of bromophenol blue, and 20 mM DTT. Then, isoelectric focusing was performed using the IPGphor IEF System (GE Healthcare). The gel strips were then equilibrated twice for 15 minutes with gentle shaking in 7 mL of equilibration solution containing 50 mM Tris–HCl buffer, 6 M urea, 30% glycerol, 2% SDS, and a trace of bromophenol blue. DTT (1% wt/vol) was added to the first step, and iodoacetamide (2.5% wt/vol) was added to the second step. Electrophoresis in the second dimension was carried out using 12.5% polyacrylamide gels and the Ettan Dalt II large vertical system (GE Healthcare).

Visualization of Proteins and Analysis of 2-D Gels
The gels were stained using SYPRO Ruby gel staining solution, and the intensity of the protein spots on the gels was determined by scanning the gels with excitation at 532 nm and emission filter of 610BP30 using a Typhoon 9400 variable mode imager (GE Healthcare). The detection of spots on gel images, background subtraction, and matching of spots over gels were performed using the ImageMaster 2D Elite version 4.01 software (GE Healthcare) as described previously (50). Spot volumes (intensities), corresponding to pixel intensities integrated over the area of each spot, were then subjected to logarithmic transformation and quantile normalization using R and packages implemented in R (biobase and affyPLM). Differential analysis of the data was performed using limma (linear models for microarray) implemented for the R computing environment (51). For differential analysis, we conducted all pairwise comparisons among the three groups (young rats fed ad libitum vs old rats fed ad libitum, old rats fed ad libitum vs old rats fed a CR diet, and young rats fed ad libitum vs old rats fed a CR diet). The limma function eBayes computes a moderated t statistic for each contrast. The moderated t statistic uses an empirical Bayesian shrinkage estimator for the variance term in the denominator of the statistic, which shrinks the nominal variance for a particular comparison toward the pooled variance across all genes. This computation effectively reduces the dependence of the variance for a particular gene on its mean expression value. We used the false discovery rate method to compute an adjusted p value for each gene (52). Then the differences in the levels of each of the proteins for each of the comparisons were rank ordered according to their log odds of being differentially abundant (B statistics). The proteins showing significant differences with a p value less than.01 for each comparison were selected and Venn diagrams showing the numbers of proteins significant in each comparison were constructed. All of the proteins significant in the three pairwise comparisons showed relatively high B statistics and were well separated from most of the data in a volcano plot.

Identification of Proteins
Spots of interest were excised from the gel using ProteomeWorks Plus Spot Cutter System (Bio-Rad) and were digested in situ with trypsin. The proteins were concentrated and purified further using a Montage In-Gel Digest kit (Millipore, Billerica, MA). The resulting digests were analyzed by mass spectrometry, either a matrix absorption laser desorption ionization-time of flight mass spectrometer (MALDI-TOF/MS; Applied Biosystems Voyager DE-STR; Foster City, CA) a MALDI-TOF/TOF/MS (Applied Biosystems 4700 proteomics analyzer), or a capillary-high-performance liquid chromatography–electrospray ionization tandem mass spectrometer (HPLC-ESI/MS/MS; Thermo Finnigan LCQ; Waltham, MA). Spectra by MALDI-TOF/MS were generated by the summation of 100 laser shots. Internal calibration was achieved by analysis of the peaks obtained from trypsin autolysis. The peptide mass maps produced by MALDI-TOF/MS were searched against the published databases by means of Profound (http://www.unb.br/cbsp/paginiciais/profound.htm) and Mascot (Matrix Science, http://www.matrixscience.com). For the protein search, a mass tolerance of 50 ppm and 1 missing cleavage site for MALDI-TOF/MS were allowed, and the oxidation of methionine residues was considered. The probability score calculated by the software was used as the criterion for correct identification of protein. Proteins that could not be identified by MALDI-TOF/MS were subjected to analysis using MALDI-TOF/TOF-MS/MS and HPLC-ESI/MS. An MS/MS tolerance of 50 ppm for parental ion and 0.3 Da for fragmented ion with one missing cleavage site was allowed, and the oxidation of methionine residues was taken into consideration. Again, the probability scores calculated from software were used as a criterion of identification by MALDI-TOF/TOF/MS. HPLC-electrospray ionization mass spectrometry (HPLC-ESI/MS) was performed on a Thermo Finnigan LCQ, which has been adapted for microspray ionization. Online HPLC separation of the tryptic peptides generated by in situ digestion of each SDS–PAGE spot of interest was accomplished with a Michrom MAGIC 2002 micro HPLC column, PicoFrit (75 µm i.d.; New Objective, Woburn, MA) packed to 10 cm with C18 adsorbent (218MSB5 5 µm, 300 Å; Grace Vydacs; Hesperia, CA) mobile phase A = 0.5% acetic acid/0.005% trifluoroacetic acid (TFA); mobile phase B = 90% acetonitrile/0.5% acetic acid/0.005% TFA; linear gradient of 2%–72% B in 30 minutes; flow rate = 0.4 µL/min. As a part of the data-dependent acquisition protocol, the four most intense ions in each survey scan were sequentially fragmented in the ion trap by collision-induced dissociation (CID) using an isolation width of 2.5 and a relative collision energy of 35%. MS/MS spectra were searched by the SEQUEST component of the LCQ software and by MASCOT (Matrix science) using the mammalian database of Swiss-Prot or The National Center for Biotechnology Information (NCBI). Mass tolerance was set at 100 ppm for the parental ion and 0.5 d for the fragmented ion with one missing cleavage site was allowed, and the oxidation of methionine residues was taken into consideration. Proteins containing at least one significant peptide were selected from the search results. Assignment of the MS/MS fragments was verified by comparison with the predicted ions generated in silico by GPMAW (Lighthouse Data, Odense, Denmark).


   RESULTS
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Using 2-D PAGE, we studied the effects of aging and CR on the mitochondrial proteome. Representative patterns of 2-D PAGE gels of proteins obtained from mitochondria isolated from the liver, heart, and skeletal muscle of Fischer 344 rats are shown in Figure 1 with the protein spots that changed significantly with age and/or CR identified. Figure 2 presents the Venn diagrams showing the proteins that changed in the three groups: young rats fed ad libitum (YAL), old rats fed ad libitum (OAL), and old rats fed a CR diet (OCR). The protein spots that were observed to change significantly (p >.01 level) with age and/or CR were determined by applying the linear model and empirical Bayes method using the limma package implemented in R as described in the Materials and Methods section. Three pairwise comparisons were made: YAL and OAL, OAL and OCR, and YAL and OCR. Thirty of the 795 mitochondrial proteins analyzed in the livers of rats changed with age and/or CR. Twenty-five of these 30 mitochondrial proteins changed with age in rats fed ad libitum, and 18 of these proteins also were significantly different between young rats fed ad libitum and old rats fed the CR diet. Only 5 of the 30 proteins showed changes between young rats fed ad libitum and old rats fed the CR diet. In the heart, 10 mitochondrial proteins of the 757 proteins resolved by 2-D PAGE showed a significant change with age or CR. Three of the 10 protein spots changed between young and old rats fed ad libitum and between young rats fed ad libitum and old rats fed the CR diet. Four of 10 proteins were significantly altered by CR, and two of these proteins also changed with age. In skeletal muscle, 8 of 517 mitochondrial proteins resolved by 2-D PAGE changed with either age or CR. Five of the eight proteins changed with age in ad libitum-fed rats, and three of the eight proteins also showed changes between young rats fed ad libitum and old rats fed the CR diet. One mitochondrial protein changed with CR.


Figure 01
View larger version (114K):
[in this window]
[in a new window]

  		Figure 1. Representative 2D-PAGE gels of mitochondrial proteins. Mitochondria were obtained from liver (A), heart (B), and skeletal muscle (C), and 100 µg of mitochondrial protein was subjected to two-dimensional polyacrylamide gel electrophoreses (2-D PAGE) and stained with Sypro Ruby as described in the Materials and Methods section. The photographs of the resulting gels are shown with the spot identification number of proteins that were observed to change significantly with age or calorie restriction. The gels are oriented with acidic proteins (left) and basic proteins (right). High-molecular-weight proteins are near the top of the gel, whereas low-molecular-weight proteins are near the bottom

 

Figure 02
View larger version (22K):
[in this window]
[in a new window]

  		Figure 2. Venn diagrams showing the mitochondrial proteins that changed with age and/or caloric restriction (CR). Proteins from the mitochondria isolated from liver, heart, and skeletal muscle of young rats fed ad libitum (YAL), old rats fed ad libitum (OAL), or old rats fed a CR diet (OCR) are shown. In the liver, 795 proteins were compared and 30 proteins were observed to change significantly with age and/or CR. In the heart, 757 proteins were compared and 10 proteins were observed to change significantly with age and/or CR. In skeletal muscle, 517 proteins were compared and 8 proteins were observed to change significantly with age and/or CR. Each Venn diagram shows the interactions among the proteins that change with age and/or CR

 
Figure 3 shows the effects of age and CR on the levels of the 30 proteins that changed significantly in the mitochondria isolated from liver. Of the 25 proteins that changed with age, 19 increased and 6 decreased. The increase in expression with age ranged from 45% (#1561) to 300% (#1792), and the decrease in expression ranged from 39% (#351) to 71% (#398). Interestingly, of the proteins that changed with age, only one (#180) was significantly altered by CR. This protein showed a 68% increase with age, which was completely prevented by CR. The five other proteins that increased with age (#1792, #805, #1236, #1345, and #1010) showed a similar trend, an increase with age and reduced expression in the old CR animals. However, the change with CR was not statistically significant for any of these proteins. CR had no effect on the expression of the six proteins that were shown to decrease with age.


Figure 03
View larger version (52K):
[in this window]
[in a new window]

  		Figure 3. Levels of mitochondrial proteins in liver that are differentially regulated with age and/or caloric restriction (CR). Graph shows the 30 liver mitochondrial proteins that changed significantly with age and/or CR. The 25 proteins that changed with age in ad libitum-fed rats are shown by the numbers that are not highlighted with a square or circle. *, 18 proteins that also were significantly different for young ad libitum and old CR rats. The five proteins that were significantly different for only young rats fed ad libitum and old CR rats are circled, and the protein that changed with age and is significantly different in old rats fed ad libitum and given the CR diet are shown with a square. The intensities of the protein spots that were subjected to logarithmic transformation and quantile normalization as described in the Materials and Methods section are plotted showing the mean and standard error of the mean of data collected from nine young rats fed ad libitum (white bars), 10 old rats fed ad libitum (black bars), and 10 old rats fed a CR diet (gray bars)

 
The effects of age and CR on the levels of the 10 proteins that changed significantly in the mitochondria obtained from heart are shown in Figure 4. Of the three proteins that changed with age in rats fed ad libitum, one protein (#536) increased 94% and two decreased [19% (#248) and 44% (#353)] with age. CR resulted in an approximate 40% (#367) to 60% (#345) decrease in the levels of three proteins and a 100% increase in the level of protein (#1259). In skeletal muscle, all five of the mitochondrial proteins altered with age were increased in old rats compared to young rats, ranging from 70% (#1112) to 300% (#1116) (Figure 5).


Figure 04
View larger version (29K):
[in this window]
[in a new window]

  		Figure 4. Levels of mitochondrial proteins in heart that were differentially regulated with age and/or caloric restriction (CR). Graph shows the 10 heart mitochondria proteins that changed significantly with age and/or CR. The three proteins that changed with age in ad libitum-fed rats are shown by the numbers that are not highlighted with a square or circle; all three proteins in this group were significantly different for young ad libitum-fed and old CR rats. The three proteins that changed only between young rats fed ad libitum and old CR rats are circled. The four proteins that are significantly different in old ad libitum-fed and old CR rats are shown with a square, and the two proteins in this group that also are significantly different for young ad libitum-fed and old CR rats are shown with an asterisk in the square. Intensities of protein spots that were subjected to logarithmic transformation and quantile normalization as described in the Materials and Methods section are plotted showing the mean and standard error of the mean of data collected from 8 young rats fed ad libitum (white bars), 9 old rats fed ad libitum (black bars), and 10 old rats fed a CR diet (gray bars)

 

Figure 05
View larger version (26K):
[in this window]
[in a new window]

  		Figure 5. Levels of mitochondrial proteins in skeletal muscle that were differentially regulated with age and/or caloric restriction (CR). Graph shows the seven skeletal muscle mitochondria proteins that changed significantly with age and/or CR. The five proteins that changed significantly with age in ad libitum-fed rats are shown by the numbers that are not highlighted with a square or circle; * three proteins in this group that also are significantly different for young ad libitum-fed and old CR rats. Two proteins that are significantly different for only young rats fed ad libitum and old CR rats are circled, and the protein that shows a significant difference between old ad libitum-fed and old CR rats is shown with a square. Intensities of protein spots that were transformed to logarithm and quantile normalization applied as described in the Materials and Methods section are plotted showing the mean and standard error of the mean of data collected from 10 young rats fed ad libitum (white bars), 10 old rats fed ad libitum (black bars), and 10 old rats fed a CR diet (gray bars)

 
The identities of 36 of the 48 mitochondrial proteins that changed with age and/or CR in the three tissues studied were determined by MS as described in the Material and Methods section, and the names of these proteins are listed in Table 1 for liver, Table 2 for heart, and Table 3 for skeletal muscle. Using MitoProt II as a predictor of mitochondrial proteins, we found that 12 of the proteins listed in Tables 1, 2, and 3 that changed with age and/or CR were proteins annotated as nonmitochondrial proteins; that is, based on sequence of the proteins, there is a low probability (less than 70% using MitoProt II) that the proteins are targeted to the mitochondria. It is not surprising that nonmitochondrial proteins were present in our preparation given that we did not conduct an extensive purification of the mitochondria because we did not want to lose potential mitochondrial proteins that might change with either age or CR during mitochondrial isolation. We found multiple proteins in two spots that were observed to change with age, these spots are shown in Table 1. The one mitochondrial protein, the age-related change of which was reversed by CR, was identified as 10-formyltetrahydrofolate dehydrogenase.


View this table:
[in this window]
[in a new window]

  		Table 1. Proteins Regulated With Age and/or Caloric Restriction in Liver Mitochondria.

 

View this table:
[in this window]
[in a new window]

  		Table 2. Proteins Regulated With Age or Caloric Restriction in Heart.

 

View this table:
[in this window]
[in a new window]

  		Table 3. Proteins Regulated With Age or Caloric Restriction in Skeletal Muscle.

 

   DISCUSSION
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This study represents the first comprehensive analysis of the effect of age on the mitochondrial proteome. Mitochondria isolated from the liver, heart, and skeletal muscle of female Fischer 344 rats were studied to determine whether there are common proteins and pathways affected by aging in the mitochondrial proteome of three different tissues. Mitochondria were isolated from the tissues of female Fischer 344 rats, ages 6–7 months (young) and 24–25 months (old), fed ad libitum or a CR diet. Rats at the age of 6–7 months were used to avoid the effects of developmental changes, and rats at the age of 24–25 months were used because this is the approximate mean life span of Fischer 344 rats (53). To analyze the array of protein intensities on the gels, we applied log transformation and quantile normalization to minimize the variation generated under experimental conditions (e.g., the gel staining process, the washing process, amount of protein applied to the gel, and the systemic errors generated in a specific day) as we have described previously (50). Proteins altered by age and/or CR were then determined significant at an adjusted p value <.01. A total of 2061 mitochondrial proteins from the three tissues (795 for liver, 757 for heart, and 509 for skeletal muscle) were studied. The total proteins analyzed in this study represent approximately 20%–40% of total mitochondrial proteome, based on (i) the estimated number of proteins found in a mitochondrion (47), and (ii) the contamination of nonmitochondrial proteins. Of the three tissues studied, the liver showed the greatest number of mitochondrial proteins that changed significantly with age, e.g., 3.1% of the liver mitochondrial proteins were shown to change with age compared to 0.4% for heart and 1.0% for skeletal muscle. Interestingly, the majority of mitochondrial proteins that changed significantly with age were found to increase. For example, of the total changes observed in all three tissues, 76% of the mitochondrial proteins increased with age, compared to 24% of the proteins that showed a decrease with age. The number of mitochondrial proteins increasing (or decreasing) with age varied considerably from tissue to tissue. In liver, 76% of the proteins analyzed increased with age, compared to 33% for heart, and 100% for skeletal muscle. When the identities of the mitochondrial proteins that changed with age were determined by MS, we found that none of the mitochondrial proteins that changed significantly with age did so in all three tissues studied. In fact, no common mitochondrial proteins were found to change significantly in any two tissues. Thus, it appears that the effect of age on the mitochondrial proteome is tissue specific. However, it is important to note that the statistical approach we used is very conservative (adjusted p value <.01), and was chosen to minimize selecting proteins that did not change with age, that is, false positives. In addition, although we based our sample size for each group on our previous methodological studies on the repeatability, stability, and dose response for mitochondrial proteins using the 2-D PAGE system (50), the variability in the current samples may have exceeded the variability observed in our methodological studies. A post hoc power analysis based on the variability observed in the current samples shows that we had 80% power to detect a significant 2-fold difference for 182/795 liver mitochondrial proteins, 15/757 heart mitochondrial proteins, and 4/517 skeletal muscle mitochondrial proteins. These calculations are based on a Bonferroni adjusted p value, which is far more conservative than the False Discovery Rate (FDR) method used in our analyses. However, the calculations suggest that our comparisons for heart and skeletal muscle may have been underpowered. Plots of the observed log2 fold differences against the average intensity for each comparison, however, revealed that only 2%–5% of the proteins showed a 2-fold or greater difference among the three experimental groups. Taken together, these observations suggest that the statistical approach we used in this study could miss proteins that might change with age or CR that could not meet the statistical rigor we used. Thus, if we relax the criterion for statistical analysis, which would result in an increase in false positives, we may detect common mitochondrial proteins and pathways affected by age in these three tissues. It also should be noted that the 2-D PAGE system used to separate the mitochondrial proteins resulted in our analysis of a protein population enriched in soluble proteins and depleted in membrane-bound proteins because hydrophobic proteins have limited solubility in the aqueous buffers used for isoelectric focusing (54). In addition, as with most global analyses, the more abundant mitochondrial proteins were studied in these experiments.

Because CR has been shown to increase the life span of rodents by retarding aging (39) and because CR has been observed to retard or reverse the age-related changes observed in most pathophysiological processes in rodents (6), including changes in mitochondrial function (14,37,44–46), we were interested in determining the effect of CR on the age-related changes in mitochondrial proteins of rodent liver, heart, and skeletal muscle. Recent studies using gene arrays have shown that CR completely prevented the age-related change in 29%, and partially suppressed 34% of the genes that changed with age in gastrocnemius muscle (54). One of the unexpected findings from our study was that CR had very little effect on the age-related changes in the mitochondrial proteome. For example, of the total of 33 proteins observed to change with age, CR significantly prevented or retarded the age-related change in only one mitochondrial protein, and this occurred in mitochondria isolated from liver. This protein is 10-formyltetrahydrofolate dehydrogenase, which increased 68% with age. CR completely prevented the age-related increase in the level of 10-formyltetrahydrofolate dehydrogenase, that is, the levels of the dehydrogenase were similar in CR rats 24–25 months of age and rats at 6–7 months of age fed ad libitum. 10-Formyltetrahydrofolate dehydrogenase converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate through either an NADP+-dependent dehydrogenase reaction or an NADP+-independent hydrolase reaction (55). The precise metabolic role of 10-formyltetrahydrofolate dehydrogenase in the liver is not completely clear. Because 10-formyl-THF is involved in de novo purine biosynthesis, it is believed that 10-formyltetrahydrofolate dehydrogenase converts excess 10-formyl-THF from the de novo purine pathway back to the THF pool (56). Upregulation of 10-formyltetrahydrofolate dehydrogenase with age may result in diminished levels of 10-formyl-THF, which is a substrate for two reactions of de novo purine biosynthesis. Thus, reduced levels of 10-formyl-THF might lead to diminished DNA and/or RNA biosynthesis and possibly to decreased DNA repair capability, thereby leading to an accumulation of DNA damage.

Although CR significantly reversed the age-related change in only one of the 2061 mitochondrial proteins studied (hepatic 10-formyltetrahydrofolate dehydrogenase), CR significantly affected the expression of several mitochondrial proteins in the heart and skeletal muscle. For example, CR reduced the levels of acyl coenzyme A (CoA) acetyltransferase 2 and L-3-hydroxyacyl CoA dehydrogenase (both involved in fatty acid metabolism), isocitrate dehydrogenase, and creatine kinase, even though these proteins showed no significant change with age. Interestingly, 2 of 4 proteins decreased by CR are involved in fatty acid beta-oxidation. Enoyl-CoA hydratase, another protein involved in fatty acid beta-oxidation, decreased with CR, although the decrease was not statistically significant. Isocitrate dehydrogenase, which is involved in the tricarboxylic acid (TCA) cycle, increased significantly with CR, and another protein identified as isocitrate dehydrogenase, was increased by CR; however, the increase of the latter enzyme was not significant. These enzymatic changes may represent a metabolic shift by CR of an energy substrate preference from fatty acids to glucose. Such a shift may be beneficial, because glucose generates more ATP per oxygen consumed than does hexanoic acid from fatty acids (57). In addition, it has been reported that the contractile performance of the heart is greater at a given rate of oxygen consumption when glucose is used as a metabolic substrate, as opposed to the use of fatty acids (58–60). Recently, impaired carbohydrate oxidation and increased fatty acid metabolism have been associated with heart failure (61), and this observation is supported by a dog study indicating that pharmacological repression of cardiac fatty acid oxidation improved the mechanical efficiency of the left ventricle (62). Therefore, the ability of CR to decrease fatty acid beta-oxidation in the mitochondria may contribute to the prevention of age-associated heart disease.

As noted above, we found that age altered more proteins in liver than in the two other tissues studied. As can be seen from Table 1, 21 of the mitochondrial proteins from liver that changed with age were found to be involved in amino acid metabolism (glutamate dehydrogenase and ornithine transcarboxylase), in the TCA cycle (malate dehydrogenase and a protein similar to succinyl CoA synthase), in respiration (choline dehydrogenase, ATP synthase gamma, which is similar to electron transfer flavoprotein, beta), and in chaperone function (heat shock protein 60 and prohibitin). An increased level of proteins involved in amino acid metabolism, TCA cycle, and respiration may reflect an increased need for energy production in the liver by increasing the flux through amino acid metabolism, the urea cycle, and the TCA cycle to generate energy by the breakdown of amino acids and carbohydrates, and to reallocate available metabolic resources. In addition, we found that heat shock protein 60 was upregulated with age in liver. Heat shock protein 60 is a mitochondrial chaperonin that is essential for protein folding and the import of proteins into the mitochondrion (63), and is proposed to play a protective role against oxidative damage (64,65). The upregulation of heat shock protein 60 in liver may therefore reflect increased oxidative stress in the mitochondria of old animals. Moreover, prohibitin was observed to increase with age. Recently, prohibitin was implicated in regulating assembly of respiratory enzyme complexes and in functioning as a mitochondrial chaperone (66). It is reported that an imbalance in respiratory enzymes by the inhibition of mitochondrial protein synthesis increases the level of prohibitin in human HepG2 hepatocytes (67). In the same study, the authors indicate that prohibitin did not respond to oxidative stress (exposure to hydrogen peroxide); however, the data were not shown in that study. Taken together, the observations of an alteration in the 10-formyltetrahydrofolate dehydrogenase level with age and CR, along with an increased prohibitin level with age, suggest that these changes may be associated with an imbalance of respiratory chain complexes. This imbalance is probably caused by age-related alterations in purine biosynthesis, which ultimately could lead to alteration of mitochondrial respiratory complexes.

The data presented here represent the first comprehensive investigation of the effects of aging and CR on the mitochondrial proteome. A total of 2061 proteins isolated from liver, heart, and skeletal muscle mitochondria were analyzed using a high-resolution 2-D PAGE system. Of the 2061 proteins analyzed, 33 were altered by either age or CR, some increasing and some decreasing. It should be pointed out that very stringent statistical criteria (e.g., p <.01) were used for determination of spot intensity changes to minimize the false detection rate. It is possible that additional proteins, the expressions of which are altered by age and/or CR, may be detected when the data are reanalyzed at a lower degree of statistical stringency (e.g., p <.05). Of the 30 proteins found to be altered by either age or CR, only one protein (hepatic 10-formyltetrahydrofolate dehydrogenase) was altered by age with its age-related decline being reversed by CR. Furthermore, no one mitochondrial protein was found to change with either age or CR in all three tissues examined. Thus, our data suggest that the effect of age appears to be tissue specific and that CR may well affect a relatively limited number of mitochondrial proteins.


   Acknowledgments
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by a Merit Review grant (HVR, AR), an Environmental Hazards Center grant from the Department of Veterans Affairs (HVR, AR) and by National Institutes of Health grants RO1 AG025362-01 (WFW) and RO1 AG23843 (AR).

We thank the University of Texas Health Science Center at San Antonio Institutional Mass Spectrometry Laboratory, supported in part by a San Antonio Cancer Institute grant (CA54174), for assistance in protein identification.


   Footnotes
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Decision Editor: Huber R. Warner, PhD

Received March 15, 2006

Accepted August 23, 2006


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Saraste M. Oxidative phosphorylation at the fin de siecle. Science. 1999;283:1488-1493.[Abstract/Free Full Text]
  2. Green DR, Reed JC. Mitochondria and apoptosis. Science. 1998;281:1309-1312.[Abstract/Free Full Text]
  3. Rizzuto R, Simpson AW, Brini M, Pozzan T. Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature. 1992;358:325-327.[Medline]
  4. Joenje H. Genetic toxicology of oxygen. Mutat Res. 1989;219:193-208.[Medline]
  5. Pryor WA. The free-radical theory of aging revisited: a critique and a suggested disease-specific theory. In: Warner RN, Butler RL, Sprott RL, Schneider EI, eds. Modern Biological Theories of Aging. New York: Raven Press; 1987:89–112.
  6. Van Remmen H, Guo Z, Richardson A. The anti-ageing action of dietary restriction. Novartis Found Symp. 2001;235:221-230.[Medline]
  7. Sastre J, Pallardo FV, Vina J. The role of mitochondrial oxidative stress in aging. Free Radic Biol Med. 2003;35:1-8.[Medline]
  8. Merry BJ. Oxidative stress and mitochondrial function with aging–the effects of calorie restriction. Aging Cell. 2004;3:7-12.[Medline]
  9. Lesnefsky EJ, Hoppel CL. Ischemia-reperfusion injury in the aged heart: role of mitochondria. Arch Biochem Biophys. 2003;420:287-297.[Medline]
  10. Muller-Hocker J, Schafer S, Link TA, Possekel S, Hammer C. Defects of the respiratory chain in various tissues of old monkeys: a cytochemical-immunocytochemical study. Mech Ageing Dev. 1996;86:197-213.[Medline]
  11. Bowling AC, Mutisya EM, Walker LC, Price DL, Cork LC, Beal MF. Age-dependent impairment of mitochondrial function in primate brain. J Neurochem. 1993;60:1964-1967.[Medline]
  12. Torres-Mendoza CE, Albert A, de la Cruz Arriaga MJ. Molecular study of the rat liver NADH: cytochrome c oxidoreductase complex during development and ageing. Mol Cell Biochem. 1999;195:133-142.[Medline]
  13. Lenaz G, Bovina C, Castelluccio C, et al. Mitochondrial complex I defects in aging. Mol Cell Biochem. 1997;174:329-333.[Medline]
  14. Feuers RJ. The effects of dietary restriction on mitochondrial dysfunction in aging. Ann N Y Acad Sci. 1998;854:192-201.[Medline]
  15. Hoppel CL, Moghaddas S, Lesnefsky EJ. Interfibrillar cardiac mitochondrial complex III defects in the aging rat heart. Biogerontology. 2002;3:41-44.[Medline]
  16. Ojaimi J, Masters CL, Opeskin K, McKelvie P, Byrne E. Mitochondrial respiratory chain activity in the human brain as a function of age. Mech Ageing Dev. 1999;111:39-47.[Medline]
  17. Kwong LK, Sohal RS. Age-related changes in activities of mitochondrial electron transport complexes in various tissues of the mouse. Arch Biochem Biophys. 2000;373:16-22.[Medline]
  18. Paradies G, Ruggiero FM, Petrosillo G, Quagliariello E. Age-dependent impairment of mitochondrial function in rat heart tissue. Effect of pharmacological agents. Ann N Y Acad Sci. 1996;786:252-263.[Medline]
  19. Martinez M, Ferrandiz ML, De Juan E, Miquel J. Age-related changes in glutathione and lipid peroxide content in mouse synaptic mitochondria: relationship to cytochrome c oxidase decline. Neurosci Lett. 1994;170:121-124.[Medline]
  20. Boffoli D, Scacco SC, Vergari R, Solarino G, Santacroce G, Papa S. Decline with age of the respiratory chain activity in human skeletal muscle. Biochim Biophys Acta. 1994;1226:73-82.[Medline]
  21. Byrne E, Trounce I, Dennett X. Mitochondrial theory of senescence: respiratory chain protein studies in human skeletal muscle. Mech Ageing Dev. 1991;60:295-302.[Medline]
  22. Drew B, Leeuwenburgh C. Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction. Am J Physiol Regul Integr Comp Physiol. 2003;285:R1259-R1267.[Abstract/Free Full Text]
  23. Kristal BS, Yu BP. Dietary restriction augments protection against induction of the mitochondrial permeability transition. Free Radic Biol Med. 1998;24:1269-1277.[Medline]
  24. Goodell S, Cortopassi G. Analysis of oxygen consumption and mitochondrial permeability with age in mice. Mech Ageing Dev. 1998;101:245-256.[Medline]
  25. Mather M, Rottenberg H. Aging enhances the activation of the permeability transition pore in mitochondria. Biochem Biophys Res Commun. 2000;273:603-608.[Medline]
  26. Gadaleta MN, Petruzzella V, Renis M, Fracasso F, Cantatore P. Reduced transcription of mitochondrial DNA in the senescent rat. Tissue dependence and effect of L-carnitine. Eur J Biochem. 1990;187:501-506.[Medline]
  27. Barazzoni R, Short KR, Nair KS. Effects of aging on mitochondrial DNA copy number and cytochrome c oxidase gene expression in rat skeletal muscle, liver, and heart. J Biol Chem. 2000;275:3343-3347.[Abstract/Free Full Text]
  28. Andreu AL, Arbos MA, Perez-Martos A, et al. Reduced mitochondrial DNA transcription in senescent rat heart. Biochem Biophys Res Commun. 1998;252:577-581.[Medline]
  29. Merlo PM, Raule N, Catani L, et al. Increased transcription of mitochondrial genes for Complex I in human platelets during ageing. FEBS Lett. 2004;558:19-22.[Medline]
  30. Sastre J, Pallardo FV, Pla R, et al. Aging of the liver: age-associated mitochondrial damage in intact hepatocytes. Hepatology. 1996;24:1199-1205.[Medline]
  31. Craig EE, Hood DA. Influence of aging on protein import into cardiac mitochondria. Am J Physiol. 1997;272:H2983-H2988.
  32. Bota DA, Davies KJ. Lon protease preferentially degrades oxidized mitochondrial aconitase by an ATP-stimulated mechanism. Nat Cell Biol. 2002;4:674-680.[Medline]
  33. Marcillat O, Zhang Y, Lin SW, Davies KJ. Mitochondria contain a proteolytic system which can recognize and degrade oxidatively-denatured proteins. Biochem J. 1988;254:677-683.[Medline]
  34. Cortopassi GA, Shibata D, Soong NW, Arnheim N. A pattern of accumulation of a somatic deletion of mitochondrial DNA in aging human tissues. Proc Natl Acad Sci U S A. 1992;89:7370-7374.[Abstract/Free Full Text]
  35. Corral-Debrinski M, Horton T, Lott MT, Shoffner JM, Beal MF, Wallace DC. Mitochondrial DNA deletions in human brain: regional variability and increase with advanced age. Nat Genet. 1992;2:324-329.[Medline]
  36. Farmer KJ, Sohal RS. Relationship between superoxide anion radical generation and aging in the housefly, Musca domestica. Free Radic Biol Med. 1989;7:23-29.[Medline]
  37. Sohal RS, Ku HH, Agarwal S, Forster MJ, Lal H. Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse. Mech Ageing Dev. 1994;74:121-133.[Medline]
  38. Muscari C, Caldarera CM, Guarnieri C. Age-dependent production of mitochondrial hydrogen peroxide, lipid peroxides and fluorescent pigments in the rat heart. Basic Res Cardiol. 1990;85:172-178.[Medline]
  39. Masoro EJ. Food restriction in rodents: an evaluation of its role in the study of aging. J Gerontol. 1988;43:B59-B64.
  40. Merry BJ. Calorie restriction and age-related oxidative stress. Ann N Y Acad Sci. 2000;908:180-198.[Medline]
  41. Weindruch R, Walford RL. The Retardation of Aging and Disease by Dietary Restriction. Springfield, IL: C.C. Thomas; 1988.
  42. Weindruch R, Sohal RS. Seminars in medicine of the Beth Israel Deaconess Medical Center. Caloric intake and aging. N Engl J Med. 1997;337:986-994.[Free Full Text]
  43. Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science. 1996;273:59-63.[Abstract]
  44. Yu BP, Yang R. Critical evaluation of the free radical theory of aging. A proposal for the oxidative stress hypothesis. Ann N Y Acad Sci. 1996;786:1-11.[Medline]
  45. Gredilla R, Barja G, Lopez-Torres M. Effect of short-term caloric restriction on H2O2 production and oxidative DNA damage in rat liver mitochondria and location of the free radical source. J Bioenerg Biomembr. 2001;33:279-287.[Medline]
  46. Gredilla R, Sanz A, Lopez-Torres M, Barja G. Caloric restriction decreases mitochondrial free radical generation at complex I and lowers oxidative damage to mitochondrial DNA in the rat heart. FASEB J. 2001;15:1589-1591.[Free Full Text]
  47. Schatz G. Mitochondria: beyond oxidative phosphorylation. Biochim Biophys Acta. 1995;1271:123-126.[Medline]
  48. Johnson D, Lardy H. Isolation of liver or kidney mitochondria. Methods Enzymol. 1967;10:94-96.
  49. Mela L, Seitz S. Isolation of mitochondria with emphasis on heart mitochondria from small amounts of tissue. Methods Enzymol. 1979;55:39-46.[Medline]
  50. Chang J, Van Remmen H, Cornell J, Richardson A, Ward WF. Comparative proteomics: characterization of a two-dimensional gel electrophoresis system to study the effect of aging on mitochondrial proteins. Mech Ageing Dev. 2003;124:33-41.[Medline]
  51. Smyth GK. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl in Genet and Mol Biol. 2004; 3 Epub 2004 Feb 12.
  52. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc Ser B. 1995;57:289-300.
  53. Thurman JD, Bucci TJ, Hart RW, Turturro A. Survival, body weight, and spontaneous neoplasms in ad libitum-fed and food restricted Fischer 344 rats. Toxicol Pathol. 1994;22:1-9.[Abstract/Free Full Text]
  54. Chevallet M, Santoni V, Poinas A, et al. New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis. Electrophoresis. 1998;19:1901-1909.[Medline]
  55. Lee CK, Klopp RG, Weindruch R, Prolla TA. Gene expression profile of aging and its retardation by caloric restriction. Science. 1999;285:1390-1393.[Abstract/Free Full Text]
  56. Champion KM, Cook RJ, Tollaksen SL, Giometti CS. Identification of a heritable deficiency of the folate-dependent enzyme 10-formyltetrahydrofolate dehydrogenase in mice. Proc Natl Acad Sci U S A. 1994;91:11338-11342.[Abstract/Free Full Text]
  57. Bailey LB. Folate in Health and Disease. In: Bailey LB, ed. New York: Marcel Dekker, Inc.; 1995:23–42.
  58. Wolff AA, Rotmensch HH, Stanley WC, Ferrari R. Metabolic approaches to the treatment of ischemic heart disease: the clinicians' perspective. Heart Fail Rev. 2002;7:187-203.[Medline]
  59. Mjos OD. Effect of free fatty acids on myocardial function and oxygen consumption in intact dogs. J Clin Invest. 1971;50:1386-1389.[Medline]
  60. Simonsen S, Kjekshus JK. The effect of free fatty acids on myocardial oxygen consumption during atrial pacing and catecholamine infusion in man. Circulation. 1978;58:484-491.[Abstract/Free Full Text]
  61. Korvald C, Elvenes OP, Myrmel T. Myocardial substrate metabolism influences left ventricular energetics in vivo. Am J Physiol Heart Circ Physiol. 2000;278:H1345-H1351.[Abstract/Free Full Text]
  62. Stanley WC, Chandler MP. Energy metabolism in the normal and failing heart: potential for therapeutic interventions. Heart Fail Rev. 2002;7:115-130.[Medline]
  63. Sabbah HN, Chandler MP, Suzki G. Ranolazine improves left ventricular mechanical efficiency in dogs with heart failure: comparison with dutamine. J Am Col Cardiol. 2001;37:173A.
  64. Ellis RJ, Hartl FU. Protein folding in the cell: competing models of chaperonin function. FASEB J. 1996;10:20-26.[Abstract]
  65. Cabiscol E, Belli G, Tamarit J, Echave P, Herrero E, Ros J. Mitochondrial Hsp60, resistance to oxidative stress, and the labile iron pool are closely connected in. Saccharomyces cerevisiae. J Biol Chem. 2002;277:44531-44538.
  66. Mitsumoto A, Takeuchi A, Okawa K, Nakagawa Y. A subset of newly synthesized polypeptides in mitochondria from human endothelial cells exposed to hydroperoxide stress. Free Radic Biol Med. 2002;32:22-37.[Medline]
  67. Nijtmans LG, de Jong L, Artal SM, et al. Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins. EMBO J. 2000;19:2444-2451.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation

HOME ARCHIVE SEARCH TABLE OF CONTENTS