Timothy R. Schmidt

Adaptive evolution of cytochrome c oxidase subunit VIII in anthropoid primates

Cytochrome c oxidase (COX) is a 13-subunit protein complex that catalyzes the last step in mitochondrial electron transfer in mammals. Of the 10 subunits encoded by nuclear DNA (three are mtDNA products), some are expressed as tissue- and/or development-specific isoforms. For COX subunit VIII, previous work showed that expression of the contractile muscle-specific isoform gene, COX8H, is absent in humans and Old World monkeys, and the other isoform gene, COX8L, is expressed ubiquitously. Here, we show that COX8H is transcribed in most primate clades, but its expression is absent in catarrhines, that is, in Old World monkeys and hominids (apes, including humans), having become a pseudogene in the stem of the catarrhines. The ubiquitously expressed isoform, COX8L, underwent nonsynonymous rate acceleration and elevation in the ratio of nonsynonymous/synonymous changes in the stem of anthropoid primates (New World monkeys and catarrhines), possibly setting the stage for loss of the heart-type (H) isoform. The most rapidly evolving region of VIII-L is one that interacts with COX I, suggesting that the changes are functionally coadaptive. Because accelerated rates of nonsynonymous substitutions in anthropoids such as observed for COX8L are also shown by genes for at least 13 other electron transport chain components, these encoded amino acid replacements may be viewed as part of a series of coadaptive changes that optimized the anthropoid biochemical machinery for aerobic energy metabolism. We argue that these changes were linked to the evolution of an expanded neocortex in anthropoid primates.

A third isoform of cytochrome c oxidase subunit VIII is present in mammals.

The terminal enzyme of the mitochondrial respiratory chain, cytochrome c oxidase (COX), contains three mitochondrial and ten nuclear encoded subunits in mammals. Three of the nuclear subunits (VIa, VIIa, and VIII) have muscle and non-muscle-specific isoforms, subunit IV contains a lung-specific isoform, and subunit VIb contains a testes-specific isoform. For subunit VIII, the smallest nuclear encoded COX polypeptide, we have now found a third gene (COX 8-3), which has been identified in human, lemur, rat, and mouse, suggesting that it is present in a broad range of Eutherian mammals. Sequence similarity and gene structure support the homology of COX8-3 to the other subunit VIII isoforms, indicating that all three are the product of gene duplications. COX VIII-3 protein is mitochondrially-targeted, as shown by a fluorescent COX VIII3/DsRed fusion protein. Both the mitochondrial targeting and its sequence conservation suggest that COXVIII-3 functions as part of the COX holoenzyme and could have a tissue-specific role, as is the case for the other two isoforms. Questions remain about where COX8-3 is predominantly expressed. However, detection of full-length cDNAs, lower levels of sequence divergence at the first and second codon positions compared to the third, and a conserved gene structure indicate that COX VIII-3 is an expressed gene whose origin dates to at least 91 million years ago.

The 5′ region of the COX4 gene contains a novel overlapping gene, NOC4

Abstract. We identified a novel human gene, NOC4 (Neighbor Of COX4), located 5′ to COX4, the gene for cytochrome c oxidase subunit IV, on Chr16q32-ter. Transcripts from this gene were identified among human expressed sequence tags. A full-length, 1.06-kb human retinal NOC4 cDNA encoded a 24-kDa, 210-amino acid hypothetical protein of unknown function. Northern hybridization analysis of human RNAs from various tissues detected NOC4 transcripts of 2.2 and 1.4 kb in all tissues examined, suggesting that NOC4 expression is ubiquitous. Transcription of both the COX4 and NOC4 genes initiates within a 250-bp intergenic promoter and occurs in opposite directions. The bidirectional promoter is G + C-rich, lacks TATA and CCAAT elements, and contains multiple potential binding sites for Sp1 and NRF-2/GABP. Two of the NRF-2/GABP sites are located within 14-bp direct repeats, a conserved feature of mammalian COX4 promoters. The NOC4 and COX4 genes are also linked in the rat, mouse, and bovine genomes. A NOC4-GFP fusion protein is located in both the nucleus and the cytoplasm, including the mitochondria.

Amino acid replacement is rapid in primates for the mature polypeptides of COX subunits, but not for their targeting presequences

We examined inferred amino acid replacements for 16 genes that encode the proteins of the cytochrome c oxidase (COX) holoenzyme in eight vertebrate species. Phylogeny-based analysis revealed that the human lineage (primates) has had an unusually large, statistically significant, number of amino acid replacements in the mature protein coding region of these genes. This finding is similar to earlier observations of an accelerated non-synonymous substitution rate for some lineages of primates for COX1, COX2, COX4, and COX7AH. In contrast, the mitochondrial targeting presequences of these same proteins have not undergone a concomitant rate change. This more comprehensive analysis suggests that COX5A, COX6B, COX6C, COX7C, and COX8L have also undergone an acceleration in amino acid replacement rates in anthropoid primates. Some of these rate accelerations (e.g. in COX5A and COX7C) are so pronounced that non-human mammalian sequences are more similar to sequences from Xenopus or zebrafish than they are to human. Since the functions of the targeting and mature proteins of these polypeptides are different, the mature portions of these genes are likely to have undergone a functionally significant change that is adaptive in nature.

Retention of a Duplicate Gene Through Changes in Subcellular Targeting: An Electron Transport Protein Homologue Localizes to the Golgi

Cytochrome c oxidase (COX), the terminal enzyme complex of the electron transport chain, contains 13 subunits, 3 encoded by mitochondrial DNA and 10 by nuclear. Several of the nuclear subunits, including subunit VIIa, are known to have two tissue- and development-specific isoforms in mammals. A recently identified third member of the gene family, COX7AR, encodes a protein previously thought to function in mitochondria. However, observation of fluorescent pCOX7AR C-terminal fusion proteins in HeLa cells showed that pCOX7AR is localized to the Golgi apparatus. Sequence analyses indicate that the duplication of COX7AR occurred prior to the origin of the Euteleostomi (bony vertebrates) and that pCOX7AR is more highly conserved than the two other isoforms. These results indicate that, after gene duplication and modification of the mitochondrial targeting signal, pCOX7AR was evolutionarily altered to a new and apparently important function in the Golgi. These results also suggest that predictions of function from homology can be misleading and show that specialization and modification of subcellular localization are similar to cis-element subfunctionalization. In cis-element subfunctionalization, complementary null mutations occur to the cis-elements of the descendents of a gene duplication, causing both descendent genes to be obligate. In the process described in this paper, which could be termed subcellular subfunctionalization, complementary null mutations can occur to the subcellular localization signals of the descendants of a gene duplication, causing both descendent genes to be similarly obligate. Noncomplementary null mutations could also uncover an alternate localization, which is the more likely case for pCOX7AR.

Isolation and sequence of the human cytochrome c oxidase subunit VIIaL gene

The gene for human cytochrome c oxidase subunit VIIa liver isoform (COX7AL) was isolated and its sequence determined and analyzed. The three introns of the gene are considerably larger than those of the heart isoform of subunit VIIa (COX7AH), but the position of the introns relative to the cDNA sequences is homologous between the two genes. Comparison with other isolated COX7AL genes suggests that the promoter region binding motifs for transcription factors have evolved along with the coding region. In fibroblasts cultured originally from a Leighs disease patient, a shortened COX7AL cDNA was identified by RT-PCR, consisting of exon I joined to exon IV, omitting exons II and III. No mutation could be identified in COX7AL of the patient, suggesting that the shortened cDNA is due to an alteration of the genome during cell culture. A surprising transcription of COX7AH was observed in cultured fibroblasts, suggesting a potential utility of these cells for study of its gene expression.