Isolation and sequence of the structural gene for cytochrome c oxidase subunit VI from Saccharomyces cerevisiae

Using synthetic oligodeoxyribonucleic acid probes we have identified and isolated COX6, the structural gene for subunit VI of cytochrome c oxidase from Saccharomyces cerevisiae. The nucleotide sequence of COX6 predicts an amino acid sequence, for the mature subunit VI polypeptide, which is in perfect agreement with that determined previously. The nucleotide sequence of COX6 also predicts that subunit VI is derived from a precursor with a highly basic 40-amino acid NH2-terminal presequence. This precursor has been observed after in vitro translations programmed by yeast poly(A+)RNA. Northern blot analysis of poly(A+) RNA from strain D273-10B reveals that COX6 is homologous to three RNAs of 1800, 900, and 700 bases in length. By means of Southern blot analysis, the cloned gene was shown to be co-linear with yeast chromosomal DNA and to exist in a single copy in the yeast genome. An additional open reading frame, consisting of 82 codons, terminates 22 codons upstream from COX6. It is "in frame" with the COX6 coding region.     

A gene in Paracoccus for subunit III of cytochrome oxidase

The region of Paracoccus denitrificans chromosome where the genes coding for cytochrome oxidase (cytochrome aa3) subunits are located has been cloned. DNA sequencing revealed an open reading frame that codes for a protein homologous to the subunit III of the eukaryotic, mitochondrial enzyme. This subunit is absent from the isolated Paracoccus oxidase. It now seems that it is part of the native enzyme in the bacterial cytoplasmic membrane. This may explain the observed discrepancies in the function of the isolated enzyme.     

Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria

Three mitochondrial DNA–encoded proteins, Cox1, Cox2, and Cox3, comprise the core of the cytochrome c oxidase complex. Gene-specific translational activators ensure that these respiratory chain subunits are synthesized at the correct location and in stoichiometric ratios to prevent unassembled protein products from generating free oxygen radicals. In the yeast Saccharomyces cerevisiae, the nuclear-encoded proteins Mss51 and Pet309 specifically activate mitochondrial translation of the largest subunit, Cox1. Here we report that Mam33 is a third COX1 translational activator in yeast mitochondria. Mam33 is required for cells to adapt efficiently from fermentation to respiration. In the absence of Mam33, Cox1 translation is impaired, and cells poorly adapt to respiratory conditions because they lack basal fermentative levels of Cox1.     

The amino acid sequence of cytochrome c oxidase subunit VI from Saccharomyces cerevisiae

The complete amino acid sequence of the nuclearly coded cytochrome c oxidase subunit VI was determined for a genetically defined haploid strain of Saccharomyces cerevisiae. The subunit contains 108 amino acids, has Mr = 12,627, is acidic (net charge of -9.7 at pH 7) and is quite polar (polarity index, 50.9%). Distribution of charges within the polypeptide chain is highly non-random. The NH2- and COOH-terminal regions are predominantly acidic whereas an apolar and a basic region are found in the interior, Subunit VI shows between 28 and 40% sequence homology (depending on the method of alignment) with subunit V of bovine cytochrome c oxidase; since the yeast subunit VI lacks methionine and contains only a single histidine residue very close to the NH2 terminus, it is unlikely that either of the two subunits carries heme alpha in the native enzyme.     

Isolation and characterization of COX12, the nuclear gene for a previously unrecognized subunit of Saccharomyces cerevisiae cytochrome c oxidase.

We have cloned and sequenced COX12, the nuclear gene for subunit VIb of Saccharomyces cerevisiae cytochrome c oxidase. This subunit, which was previously not found in cytochrome c oxidase purified from S. cerevisiae, has a deduced amino acid sequence which is 41% identical to the sequences of subunits VIb of bovine and human cytochrome c oxidases. The chromosomal copy of COX12 was replaced with a plasmid-derived copy of COX12, in which the coding region for the suspected cytochrome oxidase subunit was replaced with the yeast URA3 gene. The resulting Ura+ deletion strain grew poorly at room temperature and was unable to grow at 37 degrees C on ethanol/glycerol medium, whereas growth was normal at both temperatures on dextrose. This temperature-dependent, petite phenotype of the deletion strain was complemented to wild-type growth with a single copy plasmid carrying COX12. Cytochrome c oxidase activity in mitochondrial membranes from the cox12 deletion strain is decreased to 5-15% of that in membranes from the wild-type parent, and this activity is restored to normal when the cox12 deletion strain is complemented by the plasmid-borne COX12. Optical spectra of mitochondrial membranes from the cox12 deletion strain revealed that optically detectable cytochrome c oxidase is assembled at room temperature and at 37 degrees C, although the heme a + a3 absorption is diminished approximately 50%. The N-terminal amino acid sequence of the protein encoded by COX12 is identical to the N-terminal sequence of a subunit found in yeast cytochrome c oxidase purified by a new procedure (Taanman, J.-W., and Capaldi, R. A. (1992) J. Biol. Chem. 267, 22481-22485). We conclude that COX12 encodes a subunit of yeast cytochrome c oxidase which is essential during assembly for full cytochrome c oxidase activity but apparently can be removed after the oxidase is assembled, with retention of oxidase activity. This is the first instance in which deletion of a subunit of cytochrome c oxidase results in assembly of optically detectable cytochrome c oxidase but having markedly diminished activity.     

Deletion of the COX7 gene in Saccharomyces cerevisiae reveals a role for cytochrome c oxidase subunit VII In assembly of remaining subunits

Cytochrome c oxidase from Saccharomyces cerevisiae is composed of nine subunits. Subunits I, II and III are products of mitochondrial genes, while subunits IV, V, VI, VII, VIIa and VIII are products of nuclear genes. To investigate the role of cytochrome c oxidase subunit VII in biogenesis or functioning of the active enzyme complex, a null mutation in the COX7 gene, which encodes subunit VII, was generated, and the resulting cox7 mutant strain was characterized. The strain lacked cytochrome c oxidase activity and haem a/a3 spectra. The strain also lacked subunit VII, which should not be synthesized owing to the nature of the cox7 mutation generated in this strain. The amounts of remaining cytochrome c oxidase subunits in the cox7 mutant were examined. Accumulation of subunit I, which is the product of the mitochondrial COX1 gene, was found to be decreased relative to other mitochondrial translation products. Results of pulse-chase analysis of mitochondrial translation products are consistent with either a decreased rate of translation of COX1 mRNA or a very rapid rate of degradation of nascent subunit I. The synthesis, stability or mitochondrial localization of the remaining nuclear-encoded cytochrome c oxidase subunits were not substantially affected by the absence of subunit VII. To investigate whether assembly of any of the remaining cytochrome c oxidase subunits is impaired in the mutant strain, the association of the mitochondrial-encoded subunits I, II and III with the nuclear-encoded subunit IV was investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cytochrome oxidase subunit I split gene in Saccharomyces cerevisiae: genetic and physical studies of the mtDNA segment encompassing the ''cytochrome b-homologous'' intron

Summary We have constructed a refined genetic and physical map of 38 oxi3 mutations. With the help of the rho ^- clones derived from short and long genes, pairwise crosses between mutants, estimations of their reversion frequencies and analyses of mitochondrially synthesized proteins, we have characterized and localized several mutants in the exon A4 and in the intron aI4. We present genetic and physical evidence that in the long gene the exon A5 is split into at least three quite distinct exons, A5-1, A5-2 and A5-3 where numerous mutations are localized. We suggest that a novel 56 Kd polypeptide, which accumulates in some cis-dominant oxi3 ^- mutants results from the translation of the upstream exons and the downstream aI4 intron.     

Species-specific expression of cytochrome c oxidase subunit III mRNA

1. A cDNA probe encoding cytochrome c oxidase subunit III cloned from rat liver mitochondria was used to quantify mRNA levels in rat, mouse and rabbit tissues. This was compared to its phenotypic expression, using enzyme activity. 2. Enzyme activities were highest in mouse, intermediate in rat, and lowest in rabbit tissues. 3. Subunit III mRNA levels were easily quantified in rat, but could not be accurately measured in rabbit or mouse tissues despite high cytochrome c oxidase activities. 4. Significant subunit III sequence divergence must exist, among these species. Caution should be exercised in quantifying the expression of this mitochondrial gene.     

Deletion of the gene for subunit III leads to defective assembly of bacterial cytochrome oxidase.

COIII is one of the major subunits in the mitochondrial and a bacterial cytochrome c oxidase, cytochrome aa3. It does not contain any of the enzyme's redox-active metal centres and can be removed from the enzyme without major changes in its established functions. We have deleted the COIII gene from Paracoccus denitrificans. The mutant still expresses spectroscopically detectable enzyme almost as the wild-type, but its cytochrome c oxidase activity is much lower. From 50 to 80% of cytochrome a is reduced and its absorption maximum is 2-3 nm blue-shifted. The EPR signal of ferric cytochrome a is heterogeneous indicating the presence of multiple cytochrome a species. Proteolysis of the membrane-bound oxidase shows new cleavage sites both in COI and COII. DEAE-chromatography of solubilized enzyme yields fractions that contain a COI + COII complex and in addition haem-binding, free COI as well as free COII. The mutant phenotype can be complemented by introducing the COIII gene back to cells in a plasmid vector. We conclude that cytochrome oxidase assembles inefficiently in the absence of COIII and that this subunit may facilitate a late step in the assembly. The different oxidase species in the mutant represent either accumulating intermediates of the assembly pathway or dissociation products of a labile COI + COII complex and its conformational variants.     

Aberrant translation of cytochrome c oxidase subunit 1 mRNA species in the absence of Mss51p in the yeast Saccharomyces cerevisiae

Expression of yeast mitochondrial genes depends on specific translational activators acting on the 5'-untranslated region of their target mRNAs. Mss51p is a translational factor for cytochrome c oxidase subunit 1 (COX1) mRNA and a key player in down-regulating Cox1p expression when subunits with which it normally interacts are not available. Mss51p probably acts on the 5'-untranslated region of COX1 mRNA to initiate translation and on the coding sequence itself to facilitate elongation. Mss51p binds newly synthesized Cox1p, an interaction that could be necessary for translation. To gain insight into the different roles of Mss51p on Cox1p biogenesis, we have analyzed the properties of a new mitochondrial protein, mp15, which is synthesized in mss51 mutants and in cytochrome oxidase mutants in which Cox1p translation is suppressed. The mp15 polypeptide is not detected in cox14 mutants that express Cox1p normally. We show that mp15 is a truncated translation product of COX1 mRNA whose synthesis requires the COX1 mRNA-specific translational activator Pet309p. These results support a key role for Mss51p in translationally regulating Cox1p synthesis by the status of cytochrome oxidase assembly.     

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.     

Properties and reconstitution of a cytochrome oxidase deficient in subunit III

Three different preparations of beef heart cytochrome oxidase (EC 1.9.3.1) were reconstituted into the membranes of artificial liposomes, and the electrical charge/electron ratios were determined for charge translocation coupled to enzymic activity. Our previously characterised subunit-III-deficient preparation, which apparently lacks H+ translocation capacity [Saraste et al. (1981) Eur. J. Biochem. 115, 261-268] has a decreased charge/electron ratio (0.9-1.0) as determined from the uptake of potassium in the presence of valinomycin, in contrast to the intact reconstituted cytochrome oxidase (1.9-2.0). A third preparation that was depleted of three minor polypeptides by trypsin treatment (these polypeptides are also removed together with subunit III using the present method), but which retains subunit III, had a K+/e- ratio of 1.5 but also a relatively low respiratory control index. The pH-dependence of the Em of cytochrome a determined in the presence of cyanide is abolished in the subunit-III-deficient enzyme. Electron transfer activities are nearly identical for the original and subunit-III-depleted enzymes at an infinite concentration of cytochrome c in a polarographic assay with supplemented phospholipids. The optical spectral properties are very similar for both preparations, but with a small shift to the blue of the alpha-peak in the modified enzyme. These results support the hypothesis that the removal of subunit III abolishes the H+-translocating function of cytochrome oxidase. This occurs by an intrinsic decoupling of H+ transport from electron transfer, and yields a preparation with only half-maximal efficiency of energy conservation.     

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     

Pet191 is a cytochrome c oxidase assembly factor in Saccharomyces cerevisiae

The twin-Cx(9)C motif protein Pet191 is essential for cytochrome c oxidase maturation. The motif Cys residues are functionally important and appear to be present in disulfide linkages within a large oligomeric complex associated with the mitochondrial inner membrane. The import of Pet191 differs from that of other twin-Cx(9)C motif class of proteins in being independent of the Mia40 pathway.