Mitochondrial membrane biogenesis: characterization and use of pet mutants to clone the nuclear gene coding for subunit V of yeast cytochrome c oxidase

A nuclear pet mutant of Saccharomyces cerevisiae that is defective in the structural gene for subunit V of cytochrome c oxidase has been identified and used to clone the subunit V gene (COX5) by complementation. This mutant, E4-238 [24], and its revertant, JM110, produce variant forms of subunit V. In comparison to the wild-type polypeptide (Mr = 12,500), the polypeptides from E4-238 and JM110 have apparent molecular weights of 9,500 and 13,500, respectively. These mutations directly alter the subunit V structural gene rather than a gene required for posttranslational processing or modification of subunit V because they are cis-acting in diploid cells; that is, both parental forms of subunit V are produced in heteroallelic diploids formed from crosses between the mutant, revertant, and wild type. Several plasmids containing the COX5 gene were isolated by transformation of JM28, a derivative of E4-238, with DNA from a yeast nuclear DNA library in the vector YEp13. One plasmid, YEp13-511, with a DNA insert of 4.8 kilobases, was characterized in detail. It restores respiratory competency and cytochrome oxidase activity in JM28, encodes a new form of subunit V that is functionally assembled into mitochondria, and is capable of selecting mRNA for subunit V. The availability of mutants altered in the structural gene for subunit V (COX5) and of the COX5 gene on a plasmid, together with the demonstration that plasmid-encoded subunit V is able to assemble into a functional holocytochrome c oxidase, enables molecular genetic studies of subunit V assembly into mitochondria and holocytochrome c oxidase.     

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.     

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.     

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.     

Mitochondrial import of cytochrome c oxidase subunit VIIa in Saccharomyces cerevisiae. Identification of sequences required for mitochondrial localization in vivo

Subunit VIIa of yeast cytochrome c oxidase is a small (59 amino acids) protein of the inner mitochondrial membrane that lacks a cleavable amino-terminal presequence. To identify regions within this polypeptide that are essential for its import, gene fusions were constructed using a leader peptide substitution vector (pLPS) developed in this laboratory (Glaser, S. M., Trueblood, C. E., Dircks, L. K., Poyton, R. O., and Cumsky, M. G. (1988) J. Cell. Biochem. 36, 275-287). In this vector, oligonucleotide sequences encoding all or part of subunit VIIa were fused in-frame with the coding region of mature cytochrome c oxidase subunit Va. The plasmid pLPS is ideal for assaying protein sequences for their ability to direct mitochondrial import in vivo since subunit Va's leader peptide is essential for import and because subunit V is required for cytochrome c oxidase activity and respiration. Strains containing these fusions but lacking both subunit V genes (COX5a and COX5b) were analyzed to determine whether the chimeric protein is directed to mitochondria. Our findings indicate that the amino-terminal 17 amino acids of subunit VIIa are sufficient to localize subunit Va to the mitochondrion and that a 6-amino acid-long region within the amino terminus (Gly8-Arg13) is essential. In addition, some import (approximately 10% of wild type) is observed with the highly charged carboxyl terminus of subunit VIIa, suggesting that the subunit may contain redundancy in its import information.     

Yeast cytochrome c oxidase subunit VII is essential for assembly of an active enzyme. Cloning, sequencing, and characterization of the nuclear-encoded gene

The gene COX VII coding for yeast cytochrome c oxidase subunit VII has been cloned by a two-step procedure. Two degenerate oligonucleotides corresponding to amino- and carboxyl-terminal protein segments were used in a polymerase chain reaction for the amplification of a major portion of subunit VII (residues 1-52), which was then used for the cloning of complete COX VII. From the nucleotide sequence, an additional amino-terminal and two additional carboxyl-terminal amino acids are predicted as compared with the described primary sequence (Power, S. D., Lochrie, M. A., and Poyton, R. O. (1986) J. Biol. Chem. 261, 9206-9209). Beside subunit VIIa the subunit described here is the only nuclear encoded subunit of cytochrome c oxidase in yeast without a leader sequence. COX VII exists as a single copy per haploid genome as shown by Southern blot and gene disruption. Null mutants produced by gene disruption at the COX VII locus were respiratory-deficient. No cytochrome c oxidase activity was detectable nor was there an assembly of the oxidase complex.     

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.     

The isoforms of yeast cytochrome c oxidase subunit V alter the in vivo kinetic properties of the holoenzyme.

One of the nuclear-coded subunits of yeast cytochrome c oxidase is specified by a gene family composed of two genes, COX5a and COX5b. These genes are regulated differentially by oxygen and encode isoforms of subunit V, designated Va and Vb, which have only 66% primary sequence identity. Yeast cells require one or the other isoform for a functional cytochrome c oxidase (Trueblood, C. E., and Poyton, R. O. (1987) Mol. Cell Biol. 7, 3520-3526). To determine if these isoforms of subunit V alter the catalytic properties of holocytochrome c oxidase, we have analyzed various aspects of cytochrome c oxidase function in intact yeast cells that produce only one type of isoform. From measurements of room temperature turnover numbers and low temperature rates of ligand binding, single turnover cytochrome c oxidation, and internal electron transfer (heme a oxidation), we have found that isozymes which incorporate the Vb isoform have both higher turnover rates and higher rates of heme a oxidation than isozymes which incorporate Va. These findings support the conclusion that the isoforms of subunit V modulate cytochrome c oxidase activity in vivo and suggest that they do so by altering the rates of one or more intramolecular electron transfer reactions.     

The untranslated leader of nuclear COX4 gene of Saccharomyces cerevisiae contains an intron.

The nuclear gene for subunit IV of cytochrome oxidase (COX4) in Saccharomyces cerevisiae contains a 342 bp intron which is contained entirely within the 5' leader of the message. Splicing of the intron results in removal of several small open reading frames; subsequently, the COX4 AUG becomes the 5' proximal initiation codon. A strain with an rna2- mutation fails to splice mRNA efficiently at restrictive temperature and was used to map the intron splice junctions by RNase protection. Two major mRNA initiation sites were mapped by primer extension of synthetic oligodeoxynucleotides. The splice junctions and internal TACTAAC box conform to consensus sequences previously determined from other yeast introns. One gene for subunit V of cytochrome oxidase (COX5b) has also been shown to contain an intron. The significance of introns in two nuclear genes encoding subunits of cytochrome oxidase is discussed.     

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.     

Structure of the human cytochrome c oxidase subunit Vb gene and chromosomal mapping of the coding gene and of seven pseudogenes.

Subunit Vb of mammalian cytochrome c oxidase (COX; EC 1.9.3.1) is encoded by a nuclear gene and assembled with the other 12 COX subunits encoded in both mitochondrial and nuclear DNA. We have cloned the gene for human COX subunit Vb (COX5B) and determined the exon-intron structure by both hybridization analysis and DNA sequencing. The gene contains five exons and four introns; the four coding exons span a region of approximately 2.4 kb. The 5' end of the COX5B gene is GC-rich and contains many HpaII sites. Genomic Southern blot analysis of human DNA probed with the human COX Vb cDNA identified eight restriction fragments containing COX Vb-related sequences that were mapped to different chromosomes with panels of human x Chinese hamster somatic cell hybrids. Because only one of these fragments hybridized with a 210-bp probe from intron 4, we conclude that there is a single expressed gene for COX subunit Vb in the human genome. We have mapped this gene to chromosome 2, region cen-q13.     

Cytochrome c oxidase subunit IV is essential for assembly and respiratory function of the enzyme complex

Cytochrome c oxidase or complex IV, catalyzes the final step in mitochondrial electron transfer chain, and is regarded as one of the major regulation sites for oxidative phosphorylation. This enzyme is controlled by both nuclear and mitochondrial genomes. Among its 13 subunits, three are encoded by mitochondrial DNA and ten by nuclear DNA. In this work, an RNA interference approach was taken which led to the generation of mouse A9 cell derivatives with suppressed expression of nuclear-encoded subunit IV (COX IV) of this complex. The amounts of this subunit are decrease by 86% to 94% of normal level. A detail biosynthetic and functional analysis of several cell lines with suppressed COX IV expression revealed a loss of assembly of cytochrome c oxidase complex and, correspondingly, a reduction in cytochrome c oxidase-dependent respiration and total respiration. Furthermore, dysfunctional cytochrome c oxidase in the cells leads to a compromised mitochondrial membrane potential, a decreased ATP level, and failure to grow in galactose medium. Interestingly, suppression of COX IV expression also sensitizes the cells to apoptosis. These observations provide the evidence of the essential role of the COX IV subunit for a functional cytochrome c oxidase complex and also demonstrate a tight control of cytochrome c oxidase over oxidative phosphorylation. Finally, our results further shed some insights into the pathogenic mechanism of the diseases caused by dysfunctional cytochrome c oxidase complex.     

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)     

COX8, the structural gene for yeast cytochrome c oxidase subunit VIII. DNA sequence and gene disruption indicate that subunit VIII is required for maximal levels of cellular respiration and is derived from a precursor which is extended at both its NH2 and COOH termini

From the amino acid sequence of yeast cytochrome c oxidase subunit VIII published previously (Power, S. D., Lochrie, M.A., Patterson, T.E., and Poyton, R.C. (1984) J. Biol. Chem. 259, 6571-6574), we have synthesized a pair of oligonucleotide probes and used them to identify COX8, its structural gene. By genomic Southern blot analysis and disruption of the COX8 chromosomal locus, we have shown that this gene is present in one copy per haploid genome and that its product, subunit VIII, is essential for maximal levels of cellular respiration and cytochrome c oxidase activity. Alignment of the amino acid sequence predicted from the DNA sequence of COX8 with the determined amino acid sequence of subunit VIII indicates that mature subunit VIII is derived from a larger precursor that extends from both the NH2 and COOH termini of the mature polypeptide. Thus, like many other nuclear coded mitochondrial proteins, subunit VIII is derived from a precursor which carries a leader peptide. In addition, this precursor, like that for yeast cytochrome c oxidase subunit VIIa, appears to carry a four-amino acid "trailer peptide" at its COOH terminus.     

Assembly of the mitochondrial membrane system. Characterization of a yeast nuclear gene involved in the processing of the cytochrome b pre-mRNA

The cytochrome b gene of Saccharomyces cerevisiae D273-10B was previously shown to be composed of three exons and two introns (Nobrega, F.G., and Tzagoloff, A. (1980) J. Biol. Chem. 255, 9828-9837). In the present study nuclear respiratory deficient mutants of this strain have been screened for defects in processing of the cytochrome b pre-mRNA. Fifteen independently isolated mutants lacking cytochrome b have been assigned to a single genetic complementation group (G36). Members of this complementation group are blocked in the excision of the second intervening sequence of cytochrome b and consequently are unable to produce the mature mRNA. The wild type gene defined by this class of mutants has been named CBP2. A recombinant plasmid with the CBP2 gene has been selected from a library of wild type nuclear DNA and further subcloned by transformation of a cbp2 mutant to respiratory competency. The smallest plasmid (pG36/T5) capable of complementing cbp2 mutants and of restoring their ability to complete processing of the cytochrome b pre-mRNA has a nuclear DNA fragment of 2.6 kilobase pairs inserted at the BamHI site of the yeast vector YEp13. The sequence of the cloned DNA fragment has revealed an 1890-nucleotide-long reading frame encoding a basic protein with a molecular weight of 74,000. Deletion analysis confirms that the entire reading frame is required for complementation of cbp2 mutants. This reading frame is proposed to code for the CBP2 gene product.