In Polytomella sp. mitochondria, biogenesis of the heterodimeric COX2 subunit of cytochrome c oxidase requires two different import pathways

In the vast majority of eukaryotic organisms, the mitochondrial cox2 gene encodes subunit II of cytochrome c oxidase (COX2). However, in some lineages including legumes and chlorophycean algae, the cox2 gene migrated to the nucleus. Furthermore, in chlorophycean algae, this gene was split in two different units. Thereby the COX2 subunit is encoded by two independent nuclear genes, cox2a and cox2b, and mitochondria have to import the cytosol-synthesized COX2A and COX2B subunits and assemble them into the cytochrome c oxidase complex. In the chlorophycean algae Chlamydomonas reinhardtii and Polytomella sp., the COX2A precursor exhibits a long (130-140 residues), cleavable mitochondrial targeting sequence (MTS). In contrast, COX2B lacks an MTS, suggesting that mitochondria use different mechanisms to import each subunit. Here, we explored the in vitro import processes of both, the Polytomella sp. COX2A precursor and the COX2B protein. We used isolated, import-competent mitochondria from this colorless alga. Our results suggest that COX2B is imported directly into the intermembrane space, while COX2A seems to follow an energy-dependent import pathway, through which it finally integrates into the inner mitochondrial membrane. In addition, the MTS of the COX2A precursor is eliminated. This is the first time that the in vitro import of split COX2 subunits into mitochondria has been achieved.     

Lineage-specific fragmentation and nuclear relocation of the mitochondrial cox2 gene in chlorophycean green algae (Chlorophyta)

In most eukaryotes the subunit 2 of cytochrome c oxidase (COX2) is encoded in intact mitochondrial genes. Some green algae, however, exhibit split cox2 genes (cox2a and cox2b) encoding two polypeptides (COX2A and COX2B) that form a heterodimeric COX2 subunit. Here, we analyzed the distribution of intact and split cox2 gene sequences in 39 phylogenetically diverse green algae in phylum Chlorophyta obtained from databases (28 sequences from 22 taxa) and from new cox2 data generated in this work (23 sequences from 18 taxa). Our results support previous observations based on a smaller number of taxa, indicating that algae in classes Prasinophyceae, Ulvophyceae, and Trebouxiophyceae contain orthodox, intact mitochondrial cox2 genes. In contrast, all of the algae in Chlorophyceae that we examined exhibited split cox2 genes, and could be separated into two groups: one that has a mitochondrion-localized cox2a gene and a nucleus-localized cox2b gene ("Scenedesmus-like"), and another that has both cox2a and cox2b genes in the nucleus ("Chlamydomonas-like"). The location of the split cox2a and cox2b genes was inferred using five different criteria: differences in amino acid sequences, codon usage (mitochondrial vs. nuclear), codon preference (third position frequencies), presence of nucleotide sequences encoding mitochondrial targeting sequences and presence of spliceosomal introns. Distinct green algae could be grouped according to the form of cox2 gene they contain: intact or fragmented, mitochondrion- or nucleus-localized, and intron-containing or intron-less. We present a model describing the events that led to mitochondrial cox2 gene fragmentation and the independent and sequential migration of cox2a and cox2b genes to the nucleus in chlorophycean green algae. We also suggest that the distribution of the different forms of the cox2 gene provides important insights into the phylogenetic relationships among major groups of Chlorophyceae.     

Unusual location of a mitochondrial gene. Subunit III of cytochrome C oxidase is encoded in the nucleus of Chlamydomonad algae

The algae of the family Chlamydomonadaceae lack the gene cox3 that encodes subunit III of cytochrome c oxidase in their mitochondrial genomes. This observation has raised the question of whether this subunit is present in cytochrome c oxidase or whether the corresponding gene is located in the nucleus. Cytochrome c oxidase was isolated from the colorless chlamydomonad Polytomella spp., and the existence of subunit III was established by immunoblotting analysis with an antibody directed against Saccharomyces cerevisiae subunit III. Based partly upon the N-terminal sequence of this subunit, oligodeoxynucleotides were designed and used for polymerase chain reaction amplification, and the resulting product was used to screen a cDNA library of Chlamydomonas reinhardtii. The complete sequences of the cox3 cDNAs from Polytomella spp. and C. reinhardtii are reported. Evidence is provided that the genes for cox3 are encoded by nuclear DNA, and the predicted polypeptides exhibit diminished physical constraints for import as compared with mitochondrial-DNA encoded homologs. This indicates that transfer of this gene to the nucleus occurred before Polytomella diverged from the photosynthetic Chlamydomonas lineage and that this transfer may have occurred in all chlamydomonad algae.     

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)     

Alteration of a novel dispensable mitochondrial ribosomal small-subunit protein, Rsm28p, allows translation of defective COX2 mRNAs

Mutations affecting the RNA sequence of the first 10 codons of the Saccharomyces cerevisiae mitochondrial gene COX2 strongly reduce translation of the mRNA, which encodes the precursor of cytochrome c oxidase subunit II. A dominant chromosomal mutation that suppresses these defects is an internal in-frame deletion of 67 codons from the gene YDR494w. Wild-type YDR494w encodes a 361-residue polypeptide with no similarity to proteins of known function. The epitope-tagged product of this gene, now named RSM28, is both peripherally associated with the inner surface of the inner mitochondrial membrane and soluble in the matrix. Epitope-tagged Rsm28p from Triton X-100-solubilized mitochondria sedimented with the small subunit of mitochondrial ribosomes in a sucrose gradient containing 500 mM NH4Cl. Complete deletion of RSM28 caused only a modest decrease in growth on nonfermentable carbon sources in otherwise wild-type strains and enhanced the respiratory defect of the suppressible cox2 mutations. The rsm28 null mutation also reduced translation of an ARG8m reporter sequence inserted at the COX1, COX2, and COX3 mitochondrial loci. We tested the ability of RSM28-1 to suppress a variety of cox2 and cox3 mutations and found that initiation codon mutations in both genes were suppressed. We conclude that Rsm28p is a dispensable small-subunit mitochondrial ribosomal protein previously undetected in systematic investigations of these ribosomes, with a positive role in translation of several mitochondrial mRNAs.     

Silent mitochondrial and active nuclear genes for subunit 2 of cytochrome c oxidase (cox2) in soybean: evidence for RNA-mediated gene transfer.

In most plants and other eukaryotes investigated, the mitochondrial genome carries the gene encoding subunit 2 of cytochrome c oxidase (cox2). In this paper, we show that the previously reported mitochondrial cox2 of soybean is actually silent, and that there is an expressed, single-copy, nucleus-encoded cox2. Molecular cloning and sequence analysis of cox2 cDNA and genomic clones show that the soybean nuclear gene encodes an N-terminal extension that resembles a signal sequence for mitochondrial import and whose coding sequence is separated by an intron from that corresponding to mtDNA-encoded cox2. Comparison of soybean mitochondrial and nuclear cox2 sequences clearly indicates that in an ancestor of soybean, cox2 was transferred from the mitochondrion to the nucleus via a C-to-U edited RNA intermediate.     

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.     

Transcription of yeast COX6, the gene for cytochrome c oxidase subunit VI, is dependent on heme and on the HAP2 gene

The COX6 gene encodes subunit VI of cytochrome c oxidase. Previously, this gene and its mRNAs were characterized, and its expression has been shown to be subject to glucose repression/derepression. In this study we have examined the effects of heme and the HAP1 (CYP1) and HAP2 genes on the expression of COX6. By quantitating COX6 RNA levels and assaying beta-galactosidase activity in yeast cells carrying COX6-lacZ fusion genes, we have found that COX6 is regulated positively by heme and HAP2, but is unaffected by HAP1. Through 5' deletion analysis we have also found that the effects of heme and HAP2 on COX6 are mediated by sequences between 135 and 590 base pairs upstream of its initiation codon. These findings identify COX6 as the fourth respiratory protein gene that is known to be regulated positively by heme and HAP2. The other three, CYC1, COX4, and COX5a, encode iso-1-cytochrome c, cytochrome c oxidase subunit IV, and an isolog, Va, of cytochrome c oxidase subunit V, respectively. Thus, it appears that the biogenesis of two interacting proteins, cytochrome c and cytochrome c oxidase, in the mitochondrial respiratory chain, are under the control of common factors.     

The polypeptides COX2A and COX2B are essential components of the mitochondrial cytochrome c oxidase of Toxoplasma gondii

Two genes encoding cytochrome c oxidase subunits, Cox2a and Cox2b, are present in the nuclear genomes of apicomplexan parasites and show sequence similarity to corresponding genes in chlorophycean algae. We explored the presence of COX2A and COX2B subunits in the cytochrome c oxidase of Toxoplasma gondii. Antibodies were raised against a synthetic peptide containing a 14-residue fragment of the COX2A polypeptide and against a hexa-histidine-tagged recombinant COX2B protein. Two distinct immunochemical stainings localized the COX2A and COX2B proteins in the parasite's mitochondria. A mitochondria-enriched fraction exhibited cyanide-sensitive oxygen uptake in the presence of succinate. T. gondii mitochondria were solubilized and subjected to Blue Native Electrophoresis followed by second dimension electrophoresis. Selected protein spots from the 2D gels were subjected to mass spectrometry analysis and polypeptides of mitochondrial complexes III, IV and V were identified. Subunits COX2A and COX2B were detected immunochemically and found to co-migrate with complex IV; therefore, they are subunits of the parasite's cytochrome c oxidase. The apparent molecular mass of the T. gondii mature COX2A subunit differs from that of the chlorophycean alga Polytomella sp. The data suggest that during its biogenesis, the mitochondrial targeting sequence of the apicomplexan COX2A precursor protein may be processed differently than the one from its algal counterpart.     

An archaebacterial terminal oxidase combines core structures of two mitochondrial respiratory complexes.

The operon coding for a respiratory quinol oxidase was cloned from thermoacidophilic archaebacterium Sulfolobus acidocaldarius. It contains three genes, soxA, soxB and soxC. The first two genes code for proteins related to the cytochrome c oxidase subunits II and I, respectively. soxC encodes a protein homologous to cytochrome b, which is a subunit of the mitochondrial and bacterial cytochrome c reductases and the chloroplast cytochrome b6f complex. soxA is preceded by a promoter and the genes are cotranscribed into a 4 kb mRNA. Their protein products form a complex which has been partially purified and has quinol oxidase activity. The reduced minus oxidized absorption spectrum of the complex has two maxima at 586 and 606 nm. The latter is typical of cytochrome c oxidase. The complex contains four haems A. Two haems belong to the 'cytochrome oxidase' part of the complex and two are probably bound to be apocytochrome b (SoxC) and responsible for the 586 nm absorption peak. The homology between the sox gene products and their mitochondrial counterparts suggests that energy conservation coupled to the quinol oxidation catalysed either by the Sulfolobus oxidase or two mitochondrial respiratory enzymes may have a similar mechanism.     

Deletion of the Leader Peptide of the Mitochondrially Encoded Precursor of Saccharomyces Cerevisiae Cytochrome C Oxidase Subunit II

Cytochrome c oxidase subunit II (Cox2p) of Saccharomyces cerevisiae is synthesized within mitochondria as a precursor, pre-Cox2p. The 15-amino acid leader peptide is processed after export to the intermembrane space. Leader peptides are relatively unusual in mitochondrially coded proteins: indeed mammalian Cox2p lacks a leader peptide. We generated two deletions in the S. cerevisiae COX2 gene, removing either the leader peptide (cox2-20) or the leader peptide and processing site (cox2-21) without altering either the promoter or the mRNA-specific translational activation site. When inserted into mtDNA, both deletions substantially reduced the steady-state levels of Cox2p and caused a tight nonrespiratory phenotype. A respiring pseudorevertant of the cox2-20 mutant was heteroplasmic for the original mutant mtDNA and a ρ(-) mtDNA whose deletion fused the first 251 codons of the mitochondrial gene encoding cytochrome b to the cox2-20 sequence. The resulting fusion protein was processed to yield functional Cox2p. Thus, the presence of amino-terminal cytochrome b sequence bypassed the need for the pre-Cox2p leader peptide. We propose that the pre-Cox2p leader peptide contains a targeting signal necessary for membrane insertion, without which it remains in the matrix and is rapidly degraded.     

Differential regulation of the two genes encoding Saccharomyces cerevisiae cytochrome c oxidase subunit V by heme and the HAP2 and REO1 genes.

In Saccharomyces cerevisiae, the COX5a and COX5b genes encode two forms of cytochrome c oxidase subunit V, Va and Vb. We report here that heme increases COX5a expression and decreases COX5b expression and that the HAP2 and REO1 genes are involved in positive regulation of COX5a and negative regulation of COX5b, respectively. Heme regulation of COX5a and COX5b may dictate which subunit V isoform is available for assembly into cytochrome c oxidase under conditions of high- and low-oxygen tension.     

Identification of Reo1, a Gene Involved in Negative Regulation of Cox5b and Anb1 in Aerobically Grown Saccharomyces Cerevisiae

In Saccharomyces cerevisiae, the COX5a and COX5b genes constitute a small gene family that encodes two forms of cytochrome c oxidase subunit V, Va and Vb, either of which can provide a function essential for cytochrome c oxidase activity and respiration. In aerobically grown wild-type yeast cells, Va is the predominant form of subunit V. The COX5b gene alone does not produce enough Vb to support a respiration rate sufficient to allow growth on nonfermentable carbon sources. By selecting for mutations that increase the respiratory capacity of a strain deleted for COX5a, we have identified a gene that is involved in negative regulation of COX5b expression under aerobic growth conditions. Each of four independently isolated reo1 mutations are shown to be recessive, unlinked to COX5b, but dependent on COX5b for phenotypic expression. The mutations define a single complementation and linkage group: designated as REO1 for regulator of expression of oxidase. reo1 mutations increase expression of COX5b in aerobically grown cells, but not in anaerobically grown cells, where expression is already elevated. These mutations have no effect on COX5a, the other member of this small gene family which is positively regulated by heme and oxygen. The REO1 gene does play a role in repression of ANB1, a gene that is normally repressed under aerobic but not anaerobic conditions. Neither rox1 or rox3 mutations, which have previously been shown to increase ANB1 expression, are in the same complementation group as reo1 mutations.     

Pet111 Acts in the 5''-Leader of the Saccharomyces Cerevisiae Mitochondrial Cox2 mRNA to Promote Its Translation

PET111 is a yeast nuclear gene specifically required for the expression of the mitochondrial gene COX2, encoding cytochrome c oxidase subunit II (coxII). Previous studies have shown that PET111 activates translation of the COX2 mRNA. To map the site of PET111 action we have constructed, in vitro, genes coding for chimeric mRNAs, introduced them into mitochondria by transformation and studied their expression. Translation of a chimeric mRNA with the 612-base 5'-untranslated leader of the COX3 mRNA fused precisely to the structural gene for the coxII-precursor protein is independent of PET111, but does require a COX3 mRNA-specific translational activator known to work on the COX3 5'-leader. This result demonstrates that PET111 is not required for any posttranslational step. Translation of a chimeric mRNA with the 54-base 5'-leader of the COX2 mRNA fused precisely to the structural gene for cytochrome c oxidase subunit III was dependent on PET111 activity. These results demonstrate that PET111 acts specifically at a site in the short COX2 5'-leader to activate translation of downstream coding sequences.     

Cox2A/Cox2B subunit interaction in <Emphasis Type="Italic">Polytomella</Emphasis> sp. cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase: role of the Cox2B subunit extension

Subunit II of cytochrome c oxidase (Cox2) is usually encoded in the mitochondrial genome, synthesized in the organelle, inserted co-translationally into the inner mitochondrial membrane, and assembled into the respiratory complex. In chlorophycean algae however, the cox2 gene was split into the cox2a and cox2b genes, and in some algal species like Chlamydomonas reinhardtii and Polytomella sp. both fragmented genes migrated to the nucleus. The corresponding Cox2A and Cox2B subunits are imported into mitochondria forming a heterodimeric Cox2 subunit. When comparing the sequences of chlorophycean Cox2A and Cox2B proteins with orthodox Cox2 subunits, a C-terminal extension in Cox2A and an N-terminal extension in Cox2B were identified. It was proposed that these extensions favor the Cox2A/Cox2B interaction. In vitro studies carried out in this work suggest that the removal of the Cox2B extension only partially affects binding of Cox2B to Cox2A. We conclude that this extension is dispensable, but when present it weakly reinforces the Cox2A/Cox2B interaction.     

Cytochrome oxidase assembly in yeast requires the product of COX11, a homolog of the P. denitrificans protein encoded by ORF3.

The synthesis of cytochrome oxidase in Saccharomyces cerevisiae was recently shown to require a protein encoded by the nuclear gene COX10. This protein was found to be homologous to the putative protein product of the open reading frame ORF1 reported in one of the cytochrome oxidase operons of Paracoccus denitrificans. In the present study we demonstrate the existence in yeast of a second nuclear gene, COX11, whose encoded protein is homologous to another open reading frame (ORF3) present in the same operon of P. denitrificans. Mutations in COX11 elicit a deficiency in cytochrome oxidase. In this and in other respects cox11 and cox10 mutants have very similar phenotypes. An antibody has been obtained against the yeast COX11 protein. The antibody recognizes a 28 kd protein in yeast mitochondria, consistent with the size of the protein predicted from the sequence of COX11. The COX11 protein is tightly associated with the mitochondrial membrane but is not a component of purified cytochrome oxidase. An analysis of cytochrome oxidase subunits in wild type and in a cox11 mutant suggests that the COX11 protein is not required either for synthesis or transport of the subunit polypeptides into mitochondria. It seems more probable that COX11 protein exerts its effect at some terminal stage of enzyme synthesis, perhaps in directing assembly of the subunits.