* Department of Neurology, University of Miami School of Medicine; Institute for Anthropology and Human Genetics, Department Biology II, Ludwig-Maximilians University, Munich, Germany; and
Department of Cell Biology and Anatomy, University of Miami School of Medicine
Correspondence: E-mail: cmoraes{at}med.miami.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: anthropoid primates coevolution cybrids oxidative phosphorylation mitochondrial DNA
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adaptive coevolution of OXPHOS components has been proposed to have an important role in the adaptation to larger brains (Goldberg et al. 2003) or to colder climates (Ruiz-Pesini et al. 2004). Natural selection will favor evolutionary coadaptation of interacting proteins that maintain or improve physiological functions. One example of the biological importance of intergenomic coadaptation is the evidence that mtDNA interaction with the nuclear genome modifies cognition in mice (Roubertoux et al. 2003).
To study the functional importance of mtDNA and nuclear DNA coevolution, several mammalian models have been generated by transferring mtDNA of different species to a human or murine nuclear environments. Human xenomitochondrial cybrids were made by fusing human cells devoid of mtDNA (termed ° cells) with enucleated cells (cytoplasts) from different species. Human cells harboring mtDNA from chimpanzee and gorilla were viable and had a functional OXPHOS (Kenyon and Moraes 1997). These human xenomitochondrial cybrids had a specific partial defect in complex I because of nuclear-mitochondrial incompatibilities (Barrientos, Kenyon, and Moraes 1998). Orangutan mtDNA, which diverged earlier than chimpanzee and gorilla from humans was not able to functionally replace human mtDNA (Kenyon and Moraes 1997). Mus musculus domesticus cybrids with several species of mouse mtDNA or rat (Rattus norvegicus) mtDNA have been described (Dey, Barrientos, and Moraes 2000; McKenzie et al. 2003). Mouse xenomitochondrial cybrids harboring rat mtDNA had a defect in oxidative phosphorylation, with reduced activities of multiple OXPHOS complexes (Dey, Barrientos, and Moraes 2000; McKenzie and Trounce 2000; McKenzie et al. 2003).
In this study, we have developed several novel primate ° lines and attempted to repopulate them with mtDNA from various other primate species. The results were surprising, indicating that there is some directionality in specific nuclear-mitochondrial interactions. These studies also unveiled a fast adaptive coevolution of complex V subunits in orangutan.
![]() |
Material and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells were cultured in high-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 µg/ml sodium pyruvate, and 50 µg/ml of uridine. Galactose medium contained 5.5 mM galactose, in no-glucose Dulbecco's modified Eagle's medium supplemented with 5% dialyzed fetal bovine serum and 100 µg/ml sodium pyruvate. Selective medium without uridine was prepared by supplementing high-glucose Dulbecco's modified Eagle's medium with 10% dialyzed fetal bovine serum and 100 µg/ml sodium pyruvate.
Generation of Primate ° Cell Lines
To generate derivative ° cell lines (devoid of mtDNA), chimpanzee fibroblasts were grown in the presence of 150 ng/ml ethidium bromide for 4 months. Gorilla and baboon fibroblasts were grown for 3 months in the same conditions. Orangutan and rhesus macaque cell lines were grown in the presence of 500 ng/ml ethidium bromide for 3 and 4 months, respectively. Total depletion of mtDNA after treatment was assessed by Southern blot and the inability of the cell lines to grow in media lacking uridine (Moraes, Dey, and Barrientos 2001) or where glucose was replaced by galactose (Robinson et al. 1992).
Cybrid Fusions
To generate xenomitochondrial cybrids, mitochondrial donor cell lines were chemically enucleated by actinomycin D treatment and fused with the correspondent ° cell line using polyethylene glycol (PEG) as described (Moraes, Dey, and Barrientos 2001). Briefly, 0.3 x 106 cells were plated in 35 mm dishes, treated with actimomycin D for 15 hours (Human TEX, 0.5 µg/ml; orangutan, 1 µg/ml; rhesus macaque 1.5 µg/ml), and fused with
° cells with PEG 1,500 (60 s). Fused cells were grown 24 hours in complete medium and then trypsinized and plated at low density on 10 mm dishes with selective medium (without uridine). Individual clones were isolated by the cloning ring method.
Southern Blots
The mitochondrial DNA haplotype of xenomitochondrial cell lines was determined by Southern blots. Total DNA was digested with PvuII in the cases of chimpanzee/human and gorilla/human cybrids and the respective parental cell lines. MtDNA was detected in the blots using a probe corresponding to human mtDNA positions 14747 to 15000. In the case of orangutan/human cybrids, total DNA was digested with KpnI, and mtDNA was detected in the blots using a probe corresponding to human mtDNA positions 4120 to 4530. The probe was labeled with 5 µCi of [32P]dCTP by the random primer method (Roche, Burlington, NC).
Nuclear Markers
Human microsatellite markers have been used for analysis of genetic variation in apes (Coote and Bruford 1996). Nuclear markers were obtained from Research Genetics/Invitrogen (Carlsbad, Calif.). The loci with polymorphic tetranucleotide repeats used were: D3S2427, D14S587, D17S1290, D9S934, and DXS6797-F. Total DNA was PCR amplified with a [32P]-labeled oligonucleotide. PCR products were denatured at 80°C and resolved by electrophoresis in a 6% PAGE, 7 M urea sequencing gels. Gels were dried and exposed to an X-ray film at 80°C.
Interspecies Comparative Genomic Hybridization
Interspecies comparative genomic hybridization (iCGH) was employed to differentiate orangutan from human chromosomes (Barrientos et al. 2000). Approximately 400 ng each of genomic orangutan DNA (digoxigenin-dUTP labeled) and human genomic DNA (biotin-dUTP labeled) were ethanol precipitated. Hybridization in situ to metaphase preparations of human, orangutan, or xenomitochondrial cybrids cell lines was performed for 72 hours at 37°C. Posthybridization washes included 3 x 5 minutes in 0.1xSSC, 60°C. Biotinylated probe was detected by avidin-Cy3, digoxigenin-labeled probe by sheep antidigoxigenin FITC. Twenty-fourcolor M-FISH karyotyping for chromosome identification and microscopy was performed as described (Muller, Neusser, and Wienberg 2002).
Growth Curves
To determine rate of cell growth in glucose or galactose containing medium, 20,000 cells (when grown in glucose), or 30,000 cells (when grown in galactose) were plated in triplicates on 24-well plates. Cells were trypsinized and counted every 24 hours on a Z1 Coulter Cell Counter (Beckman Coulter, Fullerton, Calif.). Medium was replaced every other day during the experiment.
Oxygen Consumption
Oxygen consumption was measured polarographically in digitonin-permeabilized cells with a Clark oxygen electrode (Hansatech Instruments, Norfolk, UK), as described (Barrientos, Kenyon, and Moraes 1998). Cells were resuspended at 4 x 106 cells/ml in 500 µl of respiration buffer (20 mM HEPES, pH 7.1, 250 mM sucrose, 10 mM MgCl2, 2 mM potassium phosphate, and 1 mM ADP). Digitonin was added from a 1% solution to 8 µg/106 cells, in the oxygen electrode chamber. After 10 to 15 minutes of equilibration, the oxygen consumption was recorded while substrates and inhibitors were added for each respiratory complex (Villani and Attardi 1997). Sodium malate and glutamate 5 mM were used as complex I substrates, rotenone (100 nM) was used as a specific inhibitor of complex I. Sodium succinate and glycerol 3-phosphate (5 mM) were used as complex II+III substrates, and antimycin A (20 nM) was used as an inhibitor of complex III. Ascorbate (10 mM), and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) (0.2 mM) were added as complex IV substrates, and potassium cyanide (1 mM) was used to inhibit complex IV.
Mitochondrial Protein Synthesis
Mitochondrial protein synthesis was determined by pulse-labeling cell cultures in the presence of emetine as described by Chomyn (1996). Cells were grown to confluence in 60-mm dishes treated with 100 µg/ml emetine for 4 minutes, pulse labeled with 300 µCi of [35S] methionine/[35S] cysteine (Easy Tag EXPRESS, NEN/PerkinElmer, Boston, Mass.) for 60 minutes, and immediately harvested. Aliquots of 45 µg of total protein were resolved by electrophoresis on a 15% polyacrylamide gel. The gel was stained with Coomassie brilliant blue, fixed in methanol/acetic acid/water solution (30%, 10%, 60%) and treated with Enhance (NEN/PerkinElmer), dried, and exposed to an X-ray film at 80°C.
Blue Native-PAGE
Blue native electrophoresis of respiratory complexes was performed as described (Nijtmans, Henderson, and Holt 2002). Cell pellets were resuspended in PBS to a final concentration of 5.0 mg/ml, permeabilized by incubation with 1 volume of 8 mg/ml digitonin, 10 minutes on ice. Samples were diluted seven times and centrifuged 10 minutes at 10,000 x g, and the pellets were resuspended in buffer (1.5 M aminocaproic acid, 50 mM Bis-Tris, pH 7.0). Respiratory complexes were solubilized adding 10% lauryl maltoside (to a final concentration 10 mg/ml) and incubating 30 minutes on ice. Insolubilized material was removed by centrifugation at 20,000 xg for 30 minutes at 4°C. Five percent Coomassie brilliant blue G in 100 mM Bis-Tris, 500 mM aminocaproic acid, pH 7.0, was added to the supernatants to a final concentration of 2.5 mg/ml. Eighty micrograms of protein were separated in 5% to 13% blue native gradient gels (Nijtmans, Henderson, and Holt 2002). Respiratory complexes were detected in the blots, with specific antibodies obtained from Molecular Probes (Eugene, Ore.) directed against complex I, NDFS3; complex II, SDH (Fp); complex III, core 2 and iron sulfur protein; complex IV, COXI; and complex V, ATPaseß.
Phylogenetic Analysis
Phylogenetic comparison of mitochondrial proteins in different primates was performed by ClustalW using MegAlign version 5.5 (DNASTAR Inc.).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MtDNA-less cells, also know as ° cells, were used as nuclear donors for fusion experiments. To introduce mtDNA from different species, we used a novel procedure that takes advantage of the DNA replication-toxic effect of actinomycin D as a chemical enucleator (Bayona-Bafaluy, Manfredi, and Moraes 2003). Actinomycin Dtreated cells were PEG-fused to
° cells, as described in Materials and Methods. Fusion products were selected in the absence of uridine. Table 1 summarizes the results of the selection. As expected, we were able to isolate uridine-independent clones having chimpanzee nucleus and human mtDNA as well as gorilla nucleus and human mtDNA. However, contrary to our expectations, we also obtained cells with orangutan nucleus and human mtDNA, albeit at a lower number than with the former two nuclear donors (table 1). We also had the chance to test whether the mtDNA from Old World monkeys (rhesus macaque or baboon) could be replaced by an ape mtDNA (human or orangutan) or by each other's mtDNA. We were unable to rescue clones in the selective medium for OXPHOS function when either rhesus macaque or baboon were used as nuclear donors and any of the catarrhine's mtDNA was used.
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous work showed that the maintenance of mtDNA in xenomitochondrial cybrids did not depend on OXPHOS function, but rather on competing molecular recognition cues (Moraes, Kenyon, and Hao 1999). However, technical limitations require that we apply some form of selection to obtain xenomitochondrial cybrids. Without selection, most of the cells after fusion would be the unfused parental ° nuclear donor cells. The little OXPHOS function retained by the orangutan/human xenomitochondrial cybrids was enough to keep them growing, albeit poorly, in no-uridine medium. Subsequently, we showed that their growth in high-glucose medium was only slightly decreased, and they were unable to survive in galactose medium.
By studying hybrids of human and orangutan cells (i.e., with mixed nuclear backgrounds from both species) containing only orangutan mtDNA, previously we have shown a dominant negative effect specifically in complex IV assembly and activity (Barrientos et al. 2000). Based on what we know from the present work, the incompatibilities in complexes I and V were probably high enough to allow only orangutan nuclear-coded factors to assemble with the orangutan mtDNA-coded factors in these hybrids, whereas human nuclear-coded complex IV subunits could assemble with the orangutan mtDNA-coded counter parts and trigger a dominant negative defect in catalysis of COX. This effect on complex IV was smaller when the assembled enzyme was made of orangutan nuclear-coded subunits and human mtDNA-coded subunits, showing a directionality in the severity of misinteractions.
Complex I was affected by nuclear-mitochondrial incompatibilities in the orangutan/human xenomitochondrial cybrids. There are seven mtDNA-coded subunits and close to 40 nuclear-coded subunits in complex I, which would make it more susceptible to evolutionary changes. This sensitivity of complex I to small evolutionary variations has been observed even in xenomitochondrial cybrids with closely related mtDNA, such as human, chimpanzee, and gorilla (Barrientos, Kenyon, and Moraes 1998). Still, there is some complex I assembly in orangutan/human cybrids, even though its activity was impaired. It is likely that less-than-optimal nuclear-mitochondrial interactions are affecting electron transfer in the assembled complex I of orangutan/human xenomitochondrial cybrids.
The undetectable levels of assembled complex V in the human/orangutan xenomitochondrial cybrids suggest a major disruption of interactions between nuclear-coded and mtDNA-coded components of the F1/F0 ATP synthetase. The reason for this is probably related to the fact that in orangutan, the two mtDNA-coded subunits of complex V had an accelerated rate in nonsynonym amino acids substitutions (fig. 4). The small subunit (A8) has only 67% identity between orangutan and other apes A8 polypeptides. This is an extraordinarily high divergence value for what is considered a conserved polypeptide among closely related primate species. It is also far higher than what is observed for subunits of other complexes (fig. 4B). A6 of orangutan and human have 82% identity, but this is still smaller than the identity of the remaining polypeptides coded by mtDNA. It is possible that the undetectable band in mitochondrial protein synthesis of orangutan/human cybrids was A6, which could be extremely unstable if not assembled.
With such a high degree of divergence observed in complex V subunits between orangutan and human mtDNA, it is expected that nuclear-coded protein partners had to adapt accordingly. Nuclear-mitochondrial coadaptive evolution is a well-known phenomenon, and it was confirmed by our functional approach. A typical example of adaptive coevolution can be seen in cytochrome oxidase genes of some primates (Grossman et al. 2001). COXII was a rapidly evolving gene in primates compared with rodents, a phenomenon that occurred during the radiation of the anthropoid primates (Adkins and Honeycutt 1994). The rate of evolution of COX II in anthropoid primates is similar to the rate observed for cytochrome c, indicating a possible coevolutionary pressure (Cann, Brown, and Wilson 1984). Phylogenetic analysis of COX IV and COXVIII also showed accelerated nonsynonym substitution rates in catarrhine and anthropoid evolution (Wu et al. 1997; Wildman et al. 2002; Goldberg et al. 2003). In complex III, subunits that interact with the rapidly evolving cytochrome c also underwent a faster rate of amino acid substitutions (Grossman et al. 2001). Although the reason for a faster evolving rate of some OXPHOS complex in primates is unclear, it has been suggested to be necessary for adapting to the energy-demanding neocortex of catarrhine (Goldberg et al. 2003).
We now show that adaptive coevolution of rapidly evolving mtDNA genes also occurred in recently diverged branches of anthropoid primates. Why would complex V undergo a faster evolution than other OXPHOS complexes in orangutan when compared with other apes? It has been recently suggested that variations in human mtDNA haplotypes helped in the adaptation of certain populations to cold weather (Ruiz-Pesini et al. 2004). It is possible that changes in complex V helped the orangutan adapt to climate conditions of its present habitat, the Indo-Malayan region. Even more intriguing, nuclear-mitochondrial interactions were shown to affect cognition in mice (Roubertoux et al. 2003). This observation, together with fast evolution of specific complexes, opens up tantalizing hypotheseis related to adaptation to life styles (e.g., solitary versus groups and arboreal versus terrestrial). Whatever the selective pressure was, it is clear that to maintain an optimized OXPHOS function, the nuclear-coded and mtDNA-coded subunits of complex V had to coevolve fast in the Pongo genus.
Our findings expand previous observations made for OXPHOS complexes III and IV (Grossman et al. 2001) by showing adaptive coevolution of complexes I and V subunits in primates by a functional approach.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adkins, R. M., and R. L. Honeycutt. 1994. Evolution of the primate cytochrome c oxidase subunit II gene. J. Mol. Evol. 38:215231.[ISI][Medline]
Arnason, U., A. Gullberg, A. Janke, and X. Xu. 1996. Pattern and timing of evolutionary divergences among hominoids based on analyses of complete mtDNAs. J. Mol. Evol. 43:650661.[ISI][Medline]
Barrientos, A., L. Kenyon, and C. T. Moraes. 1998. Human xenomitochondrial cybrids: cellular models of mitochondrial complex I deficiency. J. Biol. Chem. 273:1421014217.
Barrientos, A., S. Muller, R. Dey, J. Wienberg, and C. T. Moraes. 2000. Cytochrome c oxidase assembly in primates is sensitive to small evolutionary variations in amino acid sequence. Mol. Biol. Evol. 17:15081519.
Bayona-Bafaluy, M. P., G. Manfredi, and C. T. Moraes. 2003. A chemical enucleation method for the transfer of mitochondrial DNA to rho(o) cells. Nucleic Acids Res. 31:e98.
Cann, R. L., W. M. Brown, and A. C. Wilson. 1984. Polymorphic sites and the mechanism of evolution in human mitochondrial DNA. Genetics 106:479499.
Chomyn, A. 1996. In vivo labeling and analysis of human mitochondrial translation products. Methods Enzymol. 264:197211.[Medline]
Coote, T., and M. W. Bruford. 1996. Human microsatellites applicable for analysis of genetic variation in apes and Old World monkeys. J. Hered. 87:406410.[Abstract]
Dey, R., A. Barrientos, and C. T. Moraes. 2000. Functional constraints of nuclear-mitochondrial DNA interactions in xenomitochondrial rodent cell lines. J. Biol. Chem. 275:3152031527.
Goldberg, A., D. E. Wildman, T. R. Schmidt, M. Huttemann, M. Goodman, M. L. Weiss, and L. I. Grossman. 2003. Adaptive evolution of cytochrome c oxidase subunit VIII in anthropoid primates. Proc. Natl. Acad. Sci. USA 100:58735878.
Grossman, L. I., T. R. Schmidt, D. E. Wildman, and M. Goodman. 2001. Molecular evolution of aerobic energy metabolism in primates. Mol. Phylogenet. Evol. 18:2636.[CrossRef][ISI][Medline]
Hao, H., and C. T. Moraes. 1996. Functional and molecular mitochondrial abnormalities associated with a C T transition at position 3256 of the human mitochondrial genome: the effects of a pathogenic mitochondrial tRNA point mutation in organelle translation and RNA processing. J. Biol. Chem. 271:23472352.
Harris, E. E. 2000. Molecular systematics of the Old World monkey tribe papionini: analysis of the total available genetic sequences. J. Hum. Evol. 38:235256.[CrossRef][ISI][Medline]
Kenyon, L., and C. T. Moraes. 1997. Expanding the functional human mitochondrial DNA database by the establishment of primate xenomitochondrial cybrids. Proc. Natl. Acad. Sci. USA 94:91319135.
Lochmuller, H., T. Johns, and E. A. Shoubridge. 1999. Expression of the E6 and E7 genes of human papillomavirus (HPV16) extends the life span of human myoblasts. Exp. Cell Res. 248:186193.[CrossRef][ISI][Medline]
McKenzie, M., M. Chiotis, C. A. Pinkert, and I. A. Trounce. 2003. Functional respiratory chain analyses in murid xenomitochondrial cybrids expose coevolutionary constraints of cytochrome b and nuclear subunits of complex III. Mol. Biol. Evol. 20:11171124.
McKenzie, M., and I. Trounce. 2000. Expression of Rattus norvegicus mtDNA in Mus musculus cells results in multiple respiratory chain defects. J. Biol. Chem. 275:3151431519.
Moraes, C. T., R. Dey, and A. Barrientos. 2001. Transmitochondrial technology in animal cells. Methods Cell Biol. 65:397412.[ISI][Medline]
Moraes, C. T., L. Kenyon, and H. Hao. 1999. Mechanisms of human mitochondrial DNA maintenance: the determining role of primary sequence and length over function. Mol. Biol. Cell. 10:33453356.
Muller, S., M. Neusser, and J. Wienberg. 2002. Towards unlimited colors for fluorescence in-situ hybridization (FISH). Chromosome Res. 10:223232.[CrossRef][ISI][Medline]
Nijtmans, L. G., N. S. Henderson, and I. J. Holt. 2002. Blue native electrophoresis to study mitochondrial and other protein complexes. Methods 26:327334.[CrossRef][ISI][Medline]
Robinson, B. H., R. Petrova-Benedict, J. R. Buncic, and D. C. Wallace. 1992. Nonviability of cells with oxidative defects in galactose medium: a screening test for affected patient fibroblasts. Biochem. Med. Metab. Biol. 48:122126.[ISI][Medline]
Roubertoux, P. L., F. Sluyter, M. Carlier, et al. (12 co-authors).2003. Mitochondrial DNA modifies cognition in interaction with the nuclear genome and age in mice. Nat. Genet. 35:6569.[CrossRef][ISI][Medline]
Ruiz-Pesini, E., D. Mishmar, M. Brandon, V. Procaccio, and D. C. Wallace. 2004. Effects of purifying and adaptive selection on regional variation in human mtDNA. Science 303:223226.
Villani, G., and G. Attardi. 1997. In vivo control of respiration by cytochrome c oxidase in wild-type and mitochondrial DNA mutation-carrying human cells. Proc. Natl. Acad. Sci. USA 94:11661171.
Wildman, D. E., W. Wu, M. Goodman, and L. I. Grossman. 2002. Episodic positive selection in ape cytochrome c oxidase subunit IV. Mol. Biol. Evol. 19:18121815.
Wu, W., M. Goodman, M. I. Lomax, and L. I. Grossman. 1997. Molecular evolution of cytochrome c oxidase subunit IV: evidence for positive selection in simian primates. J. Mol. Evol. 44:477491.[ISI][Medline]
Xu, X., and U. Arnason. 1996. The mitochondrial DNA molecule of Sumatran orangutan and a molecular proposal for two (Bornean and Sumatran) species of orangutan. J. Mol. Evol. 43:431437.[ISI][Medline]
|