Elsevier

Gene

Volume 378, 15 August 2006, Pages 11-18
Gene

Adaptive selection of mitochondrial complex I subunits during primate radiation

https://doi.org/10.1016/j.gene.2006.03.015Get rights and content

Abstract

Mammalian oxidative phosphorylation (OXPHOS) complexes I, III, IV and V are assembled from both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) encoded subunits, with complex I encompassing 39 nDNA and seven mtDNA subunits. Yet the sequence variation of the mtDNA genes is more than ten fold greater than that of the nDNA encoded genes of the OXPHOS complexes and the mtDNA proteins have been found to be influenced by positive (adaptive) selection. To maintain a functional complex I, nDNA and mtDNA subunits must interact, implying that certain nDNA complex I genes may also have been influenced by positive selection. To determine if positive selection has influenced nDNA complex I genes, we analyzed the DNA sequences of all of the nDNA and mtDNA encoded complex I subunits from orangutan, gorilla, chimpanzee, human and all available vertebrate sequences. This revealed that three nDNA complex I genes (NDUFC2, NDUFA1, and NDUFA4) had significantly increased amino acid substitution rates by both PAML and Z-test, suggesting that they have been subjected to adaptive selection during primate radiation. Since all three of these subunits reside in the membrane domain of complex I along with the mtDNA subunits, we compared amino acid changes in these three nDNA genes with those of the mtDNA genes across species. Changes in the nDNA NDUFC2 cysteine 39 were found to correlate with those in the mtDNA ND5 cysteine 330. Therefore, adaptive selection has influenced some nDNA complex I genes and nDNA and mtDNA complex I genes may have co-evolved.

Introduction

Dysfunction of OXPHOS is found in ∼ 1:8000 live births, the majority of which show reduced activity of the first enzyme of the respiratory chain, NADH-ubiquinone oxidoreductase (complex I) (Chinnery, 2003). Altered complex I activity has also been reported in Parkinson disease (Schapira et al., 1989, Schapira et al., 1998, Parker and Swerdlow, 1998, Wallace et al., 2006). However, the inheritance of complex I deficiency is often neither Mendelian nor maternal, raising the possibility that some patients suffer from a faulty interaction between nDNA and mtDNA complex I encoded subunits.

The mitochondrion is unique in that its bioenergetic and biosynthetic systems are assembled from two different information storage and retrieval systems: the nuclear–cytosolic system expressing the nDNA encoded mitochondrial genes and the mitochondrial system expressing the mtDNA encoded genes. The maternally inherited mtDNA encodes the mitochondrial 12S and 16S rRNAs, 22 tRNAs, and 13 polypeptides, all of the latter being essential subunits of the OXPHOS enzyme complexes. These mtDNA subunits include seven (ND 1, 2, 3, 4L, 4, 5, and 6) of the 46 polypeptides of complex I, one (cytochrome b, cytb) of the 11 polypeptides of complex III, three (COI, II, III) of the 13 polypeptides of complex IV, and two (ATP6 and 8) of the 16 subunits of complex V (Wallace, 2005, Wallace et al., 2006).

Mitochondrial OXPHOS generates most of the endogenous reactive oxygen species (ROS) as a toxic by-product of energy production. ROS mutagenizes the mtDNA, partially accounting for the elevated mtDNA sequence variation (Neckelmann et al., 1987, Neckelmann et al., 1989, Wallace et al., 1987). However, the mtDNA sequence variation of indigenous peoples has also been found to correlate dramatically with the geographic and climatic zones they inhabit. Therefore, it has been proposed that on the order of 25% of all mtDNA variation is adaptive, having permitted humans to adjust to varying caloric availability and differing demands on mitochondrial energy production for ATP versus heat generation (Mishmar et al., 2003, Ruiz-Pesini et al., 2004). This adaptive feature of mtDNA variation raises two questions: (1) Do nDNA encoded OXPHOS genes also accumulate adaptive variation and (2) Does variation in interacting nDNA and mtDNA subunits coincide?

Evidence that the nDNA and mtDNA encoded subunits of the same enzyme complex co-evolve has been obtained by the preparation of interspecific cytoplasmic hybrids (cybrids) in which the mtDNA of one species is combined with the nDNA of another. Initial studies demonstrated that the mtDNAs and the nDNAs of mouse and human were genetically incompatible (Wallace and Eisenstadt, 1979, Giles et al., 1980). Combining a mouse nucleus with a rat mtDNA resulted in intact mitochondrial translation, transcription and replication, but with impaired complex I at 46% of control, complex III at 37%, and complex IV at 78% (Yamaoka et al., 2000, McKenzie and Trounce, 2000, McKenzie et al., 2003). Combining the mouse nucleus with the Otomys irroratus mtDNA resulted in complex I activity at 72% of control, complex IV at 44%, and complex III at less than 2% (McKenzie et al., 2003). Similarly, combining a human nucleus with either a chimp or a gorilla mtDNA resulted in a 40% reduction in complex I activity (McKenzie et al., 2003). Combining a Mus musculus domesticus nucleus with a Mus spretus, M. caroli, or M. dunni mtDNA resulted in normal OXPHOS enzymatic activity (Yamaoka et al., 2000, McKenzie et al., 2003). However, introduction of M. pahai mtDNA into a M. musculus domesticus nuclear environment reduced complex IV activity to 59% of control (McKenzie et al., 2003). Hybrid musculusspretus animals showed minor reductions in physical performance (Nagao et al., 1998), but mice in which a M. musculus CBA/H nucleus was combined with a M. musculus NZB mtDNA showed alterations in learning, exploration, sensory development and brain anatomy (Roubertoux et al., 2003).

While these interspecific studies confirm the need for compatible nDNA and mtDNA genes for the OXPHOS complexes, to understand the specifics of the co-evolution of the nDNA and mtDNA subunits requires a direct analysis of the interaction of pairs of nDNA and mtDNA subunits. Such studies have been possible for complexes III and IV in which the high resolution crystal structures are available, permitting directed studies of the interaction of nDNA and mtDNA subunits (Wu et al., 1997, Goldberg et al., 2003, Wildman et al., 2003, Grossman et al., 2004). However, the primary defects seen in primate interspecific cybrid studies are in complex I. While a high resolution structure of the soluble domain of bacteria complex I is available (Sazanov and Hinchliffe, 2006), only low resolution crystal structures are available for mitochondrial complex I from Neurospora crassa to mammals which only reveals an L-shaped structure (Grigorieff, 1998). Some information about the regional localization of subunits within complex I, such as that the mtDNA encoded subunits are integral membrane components, has been deduced from biological, chemical and physical subdivisions of the complex (Hirst et al., 2003, Antonicka et al., 2003, Ugalde et al., 2004, Wallace et al., 2001), but additional chemical analysis of subunit interactions for complex I has been less productive. An alternative approach might be to look for co-evolution of amino acid substitutions within and between individual complex I subunits over evolutionary time.

An evolutionary analysis of complex I subunit variation has recently become vital for understanding human diseases associated with complex I deficiency. While some complex I deficiency cases result from missense mutations in mtDNA complex I subunits (Wallace et al., 2006) such as the mild ND4 missense mutation at nt 11778 (H340R) associated with Leber Hereditary Optic Neuropathy (LHON) (Wallace et al., 1988) and the severe ND6 missense mutation at nt T14487C (M63V) associated with dystonia (Simon et al., 2003, Solano et al., 2003) and other complex I deficiency cases result from severe missense mutations in nDNA complex I subunits (Wallace et al., 2006) such as the NDUFS8 compound heterozygous mutation (P85L and R138H) associated with late-onset Leigh syndrome (Procaccio and Wallace, 2004), over 60% of cases of early-onset human complex I deficiency and the majority of Parkinson diseases cases have not been successfully linked to single complex I gene defect (Triepels et al., 2001, Wallace et al., 2006).

In Eurasian LHON patients, the milder pathogenic mtDNA mutations (ND4 G11778A, ND6 T14484C, and ND3 T10663C) are frequently associated with mtDNA haplogroup J-T (Brown et al., 1997, Brown et al., 2002, Volodko et al., 2006, Torroni et al., 1997, Carelli et al., 2006) indicating the importance to intergenic interactions in complex I deficiency. Moreover, the male bias seen for the milder mtDNA LHON mutations has recently been associated with the inheritance of a region on the X-chromosome around p11.3–11.4, confirming the importance of nDNA–mtDNA interactions (Hudson et al., 2005). Hence some complex I deficient patients could be the result of faulty interactions between polymorphic nDNA and mtDNA complex I subunits.

To determine if faulty nDNA–mtDNA interactions might contribute to idiopathic complex I deficiency, we set out to address two pertinent evolutionary questions: do adaptive missense mutations occur in nDNA-complex I genes, as has been found for mtDNA complex I genes (Mishmar et al., 2003, Ruiz-Pesini et al., 2004), and can we find evidence of coordinate changes in a nDNA encoded and a mtDNA encoded complex I subunit from the same sub-region of the enzyme? Since the nDNA changes at a much slower rate than the mtDNA, we surmised that nDNA variation must be analyzed over a longer time frame than mtDNA variation. Therefore, we searched for adaptive variation in the nDNA complex I genes during the primate and great ape radiation and for nDNA–mtDNA co-evolution throughout animal radiation.

Section snippets

Cell lines

Three Pan troglodytes cell lines were generously provided by Dr. Stephen Warren, Emory University, Atlanta, GA. Gorilla gorilla (Repository number AG05251) and Pongo pygmaeus (Repository number GM04272) cell lines were obtained from Coriell (http://coriell.umdnj.edu/).

Subunit sequences

cDNA sequences were generated for all known nuclear complex I subunits from Chimp (P. troglodytes), Gorilla (G. gorilla) and Orangutan (P. pygmaeus) cell line mRNAs by reverse transcriptase-polymerase chain reaction (RT-PCR) and

Primate complex I gene sequences and phylogenies

To determine if complex I genes functionally change over evolutionary time, we sequenced the orthologues of each of the 46 subunits of complex I: seven mtDNA genes and 39 nDNA genes including the NDUFAF1 (CIA30) assembly factor (Kuffner et al., 1998, Vogel et al., 2005) of chimpanzee (P. troglodytes), gorilla (G. gorilla), and orangutan (P. pygmaeus). The sequences for the nDNA cDNAs were obtained by RT-PCR amplification of mRNAs isolated from cell lines. Gene-specific phylogenetic trees were

Discussion

Certain geographically-correlated human mtDNA amino acid polymorphisms have already been shown to have been enriched by positive selection (Ruiz-Pesini et al., 2004, Mishmar et al., 2003) and positive selection has also been demonstrated for other vertebrate and invertebrate mtDNAs (Rand and Kann, 1996, Reyes et al., 2003, Sackton et al., 2003, Willett and Burton, 2004). Since both mtDNA and nDNA encoded subunits occupy the membrane domain of complex I, it follows that the rapid evolution of

Acknowledgements

This work was supported by NIH grants NS21325, AG13154, and AG24373 awarded to DCW.

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    Current address: Department of Life Sciences, Building 40, room 005, Ben-Gurion University, Beer Sheva, 84105, Israel.

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