Identification of the structural gene for yeast cytochrome c oxidase subunit I on mitochondrial DNA.

Mitochondrial protein synthesis was analyzed in the yeast mit^? mutants of $\text{Saccharomyces}$$\text{cerevisiae}$ which specifically lack cytochrome $\text{c}$ oxidase. [³H]leucine labeled polypeptides synthesized in yeast OXI 3 mutant were analyzed by means of immunoprecipitation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). When compared to control, subunit I was not detectable. This result was substantiated by growing OXI 3 mutant in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Under such conditions SDS-PAGE analysis of [³H]leucine labeled immunoprecipitate shows the absence of subunit I. These data show that the OXI 3 locus contains the structural gene for cytochrome $\text{c}$ oxidase subunit I.     

Cloning and characterization of the yeast nuclear gene for subunit 5 of cytochrome oxidase

The nuclear gene COX5 coding for subunit 5 of cytochrome oxidase has been cloned by transformation of the cox5-1 mutant aE4-238/AL1 with a library of yeast genomic DNA. The recombinant plasmid pG46/ST2 bearing a nuclear DNA insert of 1.17 kilobase pairs restores the ability of cox5 mutants to respire and to synthesize a wild type subunit 5. The COX5 gene has been sequenced and determined to code for a 153-amino acid long protein with a molecular weight of 17,121. The amino-terminal 20 residues comprise the signal peptide. The sequence starting from residue 21 matches the partial sequence reported for the mature subunit 5. The sequence of the subunit 5 gene indicates that the mature protein has a molecular weight of 14,858 which agrees with previous size estimates based on electrophoretic migration. The primary sequence and polarity profile of yeast subunit 5 establishes that it is homologous to subunit 4 of bovine cytochrome oxidase.     

Genetic and physical analysis of the mitochondrial gene for subunit II of yeast cytochrome c oxidase.

The mitochondrial genetic locus oxi 1 contains the structural gene for subunit II of Cytochrome c oxidase. In this study, the oxi 1 locus, or at least a major portion of it, has been localized to a 2·4 kb2 HpaII fragment of mitochondrial DNA, by examining the mtDNA of oxi 1 mutants, and rho^? yeast strains that selectively retained in amplified form, this region of the mitochondria) genome. The 2·4 kb fragment is missing from the mtDNA of an oxi 1 locus deletion mutant, but is present in the mtDNAs retained by two rho^? strains that genetically recombine with all 16 oxi 1 mutants tested, to produce respiring progeny. Two other rho^? strains, that retained different but overlapping portions of the oxi 1 locus as determined genetically, contained mtDNAs consisting of “cloned” segments derived from within the 2·4 kb fragment: these rho^? mtDNAs hybridized only to the 2·4 kb HpaII fragment of wild-type mtDNA and could not be cleaved with HpaII. Furthermore, these two rho^? mtDNAs were found to correspond to sequences from opposite sides of the 2·4 kb fragment that overlap for 100 to 300 base-pairs near the middle of the fragment. Thus, five oxi 1 mutations that recombine with both of these rho^? strains could be further localized to this relatively short region of overlap. One such mutation, of particular interest because it produces an altered form of subunit II, was shown to lie on a 75-base-pair fragment that maps in this region of the overlap. The 75-base-pair fragment from the mutant migrates slightly faster during electrophoresis than the corresponding wild-type fragment. In contrast, the mobility of the fragment from a spontaneous revertant was indistinguishable from wild type.     

Analysis of a yeast nuclear gene involved in the maturation of mitochondrial pre-messenger RNA of the cytochrome oxidase subunit I

We have analyzed the mitochondrial RNA of a yeast nuclear pet mutant with no cytochrome oxidase activity. The product of the gene affected in this mutant appears to be necessary for the correct maturation of the mitochondrial pre-mRNA of the cytochrome oxidase subunit I. It does not affect, however, the overall splicing of cytochrome b pre-mRNA or the intron excision of the 21S ribosomal RNA precursor. This gene has been isolated by genetic complementation in yeast, and its DNA sequence has been determined. It is transcribed, as detected by S1 mapping experiments, and could encode a protein of 436 amino acids.     

PET1402, a nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2 in yeast

The nuclear mutation pet ts1402 prevents proteolytic processing of the precursor of cytochrome oxidase subunit 2 (cox2) in Saccharomyces cerevisiae. The structural gene PET1402 was isolated by genetic complementation of the temperature-sensitive mutation. DNA sequence analysis identified a 1206-bp open reading frame, which is located 215 by upstream of the PET122 gene. The DNA sequence of PET1402 predicts a hydrophobic, integral membrane protein with four transmembrane segments and a typical mitochondrial targeting sequence. Weak sequence similarity was found to two bacterial proteins of unknown function. Haploid cells containing a null allelle of PET1402 are respiratory deficient.     

An unusual mitochondrial import pathway for the precursor to yeast cytochrome c oxidase subunit Va

We have studied the import of the precursor to yeast cytochrome c oxidase subunit Va, a protein of the mitochondrial inner membrane. Like the majority of mitochondrial precursor proteins studied thus far, import of presubunit Va was dependent upon both a membrane potential (delta psi) and the hydrolysis of ATP. However, the levels of ATP necessary for the import of presubunit Va were significantly lower than those required for the import of a different mitochondrial precursor protein, the beta subunit of the F1-ATPase. The rate of import of presubunit Va was found to be unaffected by temperature over the range 0 to 30 degrees C, and was not facilitated by prior denaturation of the protein. These results, in conjunction with those of an earlier study demonstrating that presubunit Va could be efficiently targeted to mitochondria with minimal presequences, suggest that the subunit Va precursor normally exists in a loosely folded conformation. Presubunit Va could also be imported into mitochondria that had been pretreated with high concentrations of trypsin or proteinase K (1 mg/ml and 200 micrograms/ml, respectively). Furthermore, the rate of import into trypsin-treated mitochondria, at both 0 and 30 degrees C, was identical to that observed with the untreated organelles. Thus, import of presubunit Va is not dependent upon the function of a protease-sensitive surface receptor. When taken together, the results of this study suggest that presubunit Va follows an unusual import pathway. While this pathway uses several well-established translocation steps, in its entirety it is distinct from either the receptor-independent pathway used by apocytochrome c, or the more general pathway used by a majority of mitochondrial precursor proteins.     

Nuclear genes for mitochondrial proteins. Identification and isolation of a structural gene for subunit V of yeast cytochrome c oxidase

The gene for yeast cytochrome c oxidase subunit V, COX5, has been isolated from a Saccharomyces cerevisiae DNA library by complementation of a cytochrome c oxidase subunit V mutant, JM28. One complementing plasmid, YEp13-511, with a DNA insert of 4.8 kilobase pairs, has been characterized in detail. This plasmid restores respiratory competency in JM28, results in increased cytochrome c oxidase activity and a new form of subunit V in JM28 mitochondria, and is capable of selecting mRNA for subunit V. These results indicate that YEp13-511 carries the COX5 gene and that the subunit V encoded by this plasmid gene is capable of entering the mitochondrion and assembling into a functional holocytochrome c oxidase.     

Phylogeographic analysis of Pimoidae (Arachnida: Araneae) inferred from mitochondrial cytochrome c oxidase subunit I and nuclear 28S rRNA gene regions

Using mitochondrial DNA cytochrome c oxidase subunit I and nuclear DNA 28S rRNA data, we explored the phylogenetic relationships of the family Pimoidae (Arachnida: Araneae) and tested the North America to Asia dispersal hypothesis. Sequence data were analysed using maximum parsimony and Bayesian inference. A phylogenetic analysis suggested that vicariance, instead of dispersal, better explained the present distribution pattern of Pimoidae. Times of divergence events were estimated using penalized likelihood method. The dating analysis suggested that the emergence time of Pimoidae was approximately 140 million years ago (Ma). The divergence time of the North American and Asian species of Pimoa was approximately 110 Ma. Our phylogenetic hypothesis supports the current morphology‐based taxonomy and suggests that the cave dwelling might have played an important role in the speciation of pimoids in arid areas.     

Paracoccus denitrificans mutants deleted in the gene for subunit II of cytochrome c oxidase also lack subunit I.

As a prerequisite to site-directed mutagenesis on cytochrome c oxidase, two different mutants are constructed by inactivating the cta gene locus encoding subunits II and III (ctaC and ctaE) of the Paracoccus denitrificans oxidase. Either a short fragment encoding part of the putative copper binding site near the C terminus of subunit II, or a substantial fragment, comprising parts of the coding region for both subunits and all of the intervening three open reading frames, are removed and replaced by the kanamycin resistance gene. Each construct, ligated into a suicide vector, is mated into Paracoccus, and mutants originating from double homologous recombination events are selected. We observe complete loss of alpha-type heme and of oxidase subunits, as well as a substantial decrease in the cytochrome c oxidase activity. Upon complementation with the ctaC gene (plus various lengths of downstream sequence extending into the operon), subunit II gets expressed in all cases. Wild-type phenotype, however, is only restored with the whole operon. Using smaller fragments for complementation gives interesting clues on roles of the open reading frames for the assembly process of the oxidase complex; two of the open reading frame genes most likely code for two independent assembly factors. Since homologous genes have been described not only for other bacterial oxidases, but their gene products shown to participate also in the assembly of the yeast enzyme, they seem to constitute a group of evolutionary conserved proteins.     

Species of tetrahymena identical by small subunit rRNA gene sequences are discriminated by mitochondrial cytochrome c oxidase I gene sequences

The mitochondrial cytochrome c oxidase 1 (CO1) genes of two isolates of each of the seven mating types of Tetrahymena thermophila were sequenced and found to differ by < 1% in nucleotide sequence and to be identical by putative protein sequence. As this gene was highly conserved in this species, the CO1 gene sequence was determined for four pairs of Tetrahymena species identical in their small subunit rRNA gene sequences. The following pairs of species showed from 1% to 12% divergence at the nucleotide level, enabling discrimination of all these species: (1) Tetrahymena pyriformis strain T and Tetrahymena setosa strain HZ-1; (2) Tetrahymena canadensis strain UM1215 and Tetrahymena rostrata strain ID-3; (3) Tetrahymena pigmentosa strain UM1285 and Tetrahymena hyperangularis strain EN112; and (4) Tetrahymena tropicalis strain TC-105 and Tetrahymena mobilis. However, because of the synonymous nature of the majority of substitutions, the pairs of species were identical based on the putative protein sequence.     

The sequence of the gene for cytochrome c oxidase subunit I, a frameshift containing gene for cytochrome c oxidase subunit II and seven unassigned reading frames in Trypanosoma brucei mitochrondrial maxi-circle DNA.

A 9.2 kb segment of the maxi-circle of Trypanosoma brucei mitochondrial DNA contains the genes for cytochrome c oxidase subunits I and II (coxI and coxII) and seven Unassigned Reading Frames ("URFs"). The genes for coxI and coxII display considerable homology at the aminoacid level (38 and 25%, respectively) to the corresponding genes in fungal and mammalian mtDNA, the only striking point of divergence being an unusually high cysteine content (about 4.5%). The reading frame coding for cytochrome c oxidase subunit II is discontinuous: the C-terminal portion of about 40 aminoacids, is present in the DNA-sequence in a -1 reading frame with respect to the N-terminal moiety. URF5, 8 and 10, show a low but distinct homology (about 20%) to mammalian mitochondrial URF-1, 4 and 5, respectively. In URF5, the first AUG is found at codon 145, whereas extensive homology to mammalian URF-1 sequences occurs upstream of this position. The possibility exists that UUG can serve as an initiator codon. URF7 and URF9 have a highly unusual aminoacid composition and do not possess AUG or UUG initiator codons. These URFs probably do not have a protein-coding function. The segment does not contain conventional tRNA genes.     

Evolution of the primate cytochrome c oxidase subunit II gene

We examined the nucleotide and amino acid sequence variation of the cytochrome c oxidase subunit II (COII) gene from 25 primates (4 hominoids, 8 Old World monkeys, 2 New World monkeys, 2 tarsiers, 7 lemuriforms, 2 lorisiforms). Marginal support was found for three phylogenetic conclusions: (1) sister-group relationship between tarsiers and a monkey/ape clade, (2) placement of the aye-aye (Daubentonia) sister to all other strepsirhine primates, and (3) rejection of a sister-group relationship of dwarf lemurs (i.e., Cheirogaleus) with lorisiform primates. Stronger support was found for a sister-group relationship between the ring-tail lemur (Lemur catta) and the gentle lemurs (Hapalemur). In congruence with previous studies on COII, we found that the monkeys and apes have undergone a nearly two-fold increase in the rate of amino acid replacement relative to other primates. Although functionally important amino acids are generally conserved among all primates, the acceleration in amino acid replacements in higher primates is associated with increased variation in the amino terminal end of the protein. Additionally, the replacement of two carboxyl-bearing residues (glutamate and aspartate) at positions 114 and 115 may provide a partial explanation for the poor enzyme kinetics in cross-reactions between the cytochromes c and cytochrome c oxidases of higher primates and other mammals. Correspondence to: R.L. Honeycutt