Mutations in the structural gene for cytochrome c result in deficiency of both cytochromes aa 3 and c in Neurospora crassa

The cyt-12-12 mutant of Neurospora crassa is characterized by slow growth and a deficiency of spectrophotometrically-detectable cytochromes aa ₃ and c. Using a sib-selection procedure we have isolated the cyt-12 ⁺ allele from a cosmid library of N. crassa genomic DNA. Characterization of the cyt-12 ⁺ allele reveals that it encodes the structural gene for cytochrome c. DNA sequence analysis of the cyt-12-12 allele revealed a mutation in the cytochrome c coding sequence that results in replacement of a glycine residue, which is invariant in the cytochrome c of other species, with an aspartic acid. Genetic analysis confirms that cyt-12-12 is allelic with the previously-characterized cyc-1-1 mutant, which was also shown to affect the single locus encoding cytochrome c in N. crassa. We suggest that the amount of functional cytochrome c present in mitochondria influences the level of cytochrome aa ₃ .     

Replacement of a conserved glycine residue in subunit II of cytochrome c oxidase interferes with protein function

In this paper we describe the isolation and characterization of a respiration-deficient yeast strain which is defective in the function of subunit II of cytochrome c oxidase. This strain, VC32, carries a mutation in the mitochondrial COX2 gene which converts a conserved glycine residue to arginine. The conserved glycine is in a region implicated as important for ligating the Cu_A redox center and for interaction with cytochrome c. We have also characterized five revertants of VC32 which have recovered respiratory function; all five were mapped to the mitochondrial genome. In three of the five revertants the wild-type glycine codon is restored, while in two of the five the mutant arginine codon is still present. These two strains are likely to possess alterations either in components of the mitochondrial translation machinery or in mitochondrially-encoded gene products that interact directly with subunit II to assemble an active oxidase complex.     

Yeast SCO1 protein is required for a post-translational step in the accumulation of mitochondrial cytochrome c oxidase subunits I and II

Summary Biogenesis of functional cytochrome c oxidase in yeast requires the product of the nuclear gene SCO1. Strains deleted for this gene fail to accumulate the mitochondrially-synthesized cytochrome c oxidase subunits I and II, despite the presence of the respective mRNAs. Here we present data which demonstrate that the observed phenotype does not result from a failure to translate the mRNAs, but from a preferential degradation of the newly synthesized subunits. The SCO1 protein is therefore involved in a post-translational step in the accumulation of cytochrome c oxidase subunits I and II. We propose that the SCO1 protein is required for the correct assembly of both subunits into the cytochrome c oxidase complex.     

Disruption of the gene encoding the 78-kilodalton subunit of the peripheral arm of complex I in Neurospora crassa by repeat induced point mutation (RIP)

We have used the procedure of sheltered RIP to generate mutants of the 78-kDa protein of the peripheral arm of Neurospora crassa complex I. The nuclei containing the mutations were initially isolated as one component of a heterokaryon but subsequent analysis showed that nuclei containing null alleles of the gene could be propagated as homokaryons. This demonstrates that the gene does not serve an essential function. Sequence analysis of one allele shows that 61 transition mutations were created resulting in 39 amino-acid changes including the introduction of four stop codons. Mutant strains grow at a slower rate than wild-type and exhibit a decrease in the production of conidia. Electron paramagnetic spectroscopy of mutant mitochondria suggest that they are deficient in Fe–S clusters N-1, N-3, and N-4.     

Ethanol withdrawal posttranslationally decreases the activity of cytochrome c oxidase in an estrogen reversible manner

Cytochrome c oxidase (COX) is a key mitochondrial enzyme that catalyzes electron transfer at the terminal stage of respiratory chain and is composed of multisubunits. We hypothesize that ethanol withdrawal (EW) impairs the activity of COX and estrogen deprivation exacerbates this problem. Five-month-old ovariectomized rats with or without 17beta-estradiol (E2) replacement received a control dextrin or a liquid ethanol diet (6.5%, 5 weeks). They were then sacrificed either during ethanol exposure or at 24h of EW (EW group). Mitochondria of the cerebellum and cortex were processed to measure the activities of total COX, COX subunit I, and IV. The effects of EW and E2 on the protein levels of these subunits were also assessed using an immunoblotting method. As compared to the control dextrin and ethanol exposure, EW decreased the activities of total COX, COX I, and COX IV. E2 treatment prevented the effects of EW on the activities of total COX and COX IV but not COX I. Neither EW nor E2 altered the protein levels of the subunits. These findings suggest that a counteracting relationship exists between the effects of EW and E2 on the activity of COX in a subunit specific manner.     

Sequence analysis of three deficient mutants of cytochrome oxidase subunit I of Saccharomyces cerevisiae and their revertants

Three respiratory-deficient mutants of cytochrome oxidase subunit I in the yeast mitochondrion have been sequenced. They are located in, or near, transmembrane segment VI, the catalytic core of the enzyme. Respiratory-competent revertants have been selected and studied. The mutant V244M was found to revert at the same site in valine (wild-type), isoleucine or threonine. The revertants of the mutant G251R were of three types: glycine (wild-type), serine and threonine at position 251. A search for second-site mutations was carried out but none were found. Among 60 revertants tested, the mutant K265M was found to revert only to the wild-type allele.     

A new senescence-inducing mitochondrial linear plasmid in field-isolated Neurospora crassa strains from India

Summary Several field-collected strains of Neurospora crassa from the vicinity or Aarey, Bombay, India, are prone to precocious senescence and death. Analysis of one strain, Aarely-1e, demonstrated that the genetic determinants for the predisposition to senescence are maternally inherited. The senescence-prone strains contain a 7-kb, linear, mitochondrial DNA plasmid, maranhar, which is not present in long-lived isolates from the same geographical location. The maranhar plasmid has inverted terminal repeats with protein covalently bound at the 5 termini. Molecular hybridization experiments have demonstrated no substantial DNA sequence homology between the plasmid and the normal mitochondrial (mtDNA) and nuclear genomes of long-lived strains of N. crassa. Integrated maranhar sequences were detected in the mtDNAs of two cultures derived from Aarey-1e, and mtDNAs with the insertion sequences accumulated during subculturing. Nucleotide sequence analysis of cloned fragments of the two insertion sequences demonstrates that that they are flanked by long inverted repeats of mtDNA. The senescence syndrome of the maranhar strains, and the mode of integration of the plasmid, are reminiscent of those seen in the kalilo strains of N. intermedia. Nonetheless, there is no detectable nucleotide sequence homology between the maranhar and kalilo plasmids.     

Age-related decrease in expression of mitochondrial DNA encoded subunits of cytochrome c oxidase in Drosophila melanogaster

A key feature of the aging process is that the mitochondrial respiratory capacity declines and, the production of reactive oxygen species increases in the later part of life span. In previous studies, cytochrome c oxidase (CcO), the terminal component of the mitochondrial electron transport chain, was found to be the only oxidoreductase exhibiting an age-related decrease in activity in Drosophila melanogaster. The present study tested the hypothesis that decreases in the abundance of catalytic subunits of CcO, encoded in mitochondrial DNA, could underlie the age-associated loss of enzyme activity. Protein amounts of subunits I, II and III, which form the catalytic core of CcO, were determined by immunoblot analysis in 15-, 25-, 35-, 47- and 60-day-old flies. Subunits II and III decreased with age by up to 43% and 75%, respectively, whereas the decrease in subunit I was only 15%. The results pinpoint specific changes in a component of the mitochondrial electron transport chain, which could underlie the age-related decrease in mitochondrial respiratory activity and an increase in oxidant production. Apparently, the stoichiometry of CcO holoprotein is dynamically altered during the aging process in D. melanogaster.     

Evidence for the association of yeast mitochondrial ribosomes with Cox11p, a protein required for the CuB site formation of cytochrome c oxidase

Cytochrome c oxidase is the terminal enzyme of the mitochondrial (mt) respiratory chain. It contains copper ions, which are organized in two centres, Cu_A and Cu_B. The Cu_A site of subunit Cox2p is exposed to the mt intermembrane space, while the Cu_B site of subunit Cox1p is buried in the inner mt membrane. Incorporation of copper into the two centres is crucial for the assembly and activity of the enzyme. Formation of the Cu_B site is dependent on Cox11p, a copper-binding protein of the mt inner membrane. Here, we experimentally prove that Cox11p possesses a N_in–C_out topology, with the C-terminal copper-binding domain exposed in the mt intermembrane space. Furthermore, we provide evidence for the association of Cox11p with the mt translation machinery. We propose a model in which the Cu_B site is co-translationally formed by a transient interaction between Cox11p and the nascent Cox1p in the intermembrane space.     

The unusual reversion properties of a mitochondrial mutation in the structural gene of subunit I of cytochrome oxidase of Saccharomyces cerevisiae reveal a probable histidine ligand of the redox center

Summary We have analyzed a mutation in the mitochondrial gene oxi3 coding for subunit I of cytochrome-oxidase in the yeast Saccharomyces cerevisiae. This mutation replaces one of the seven invariant histidines of the polypeptide (position 378) by a tyrosine, and leads to a respiratory deficient phenotype. A total of 157 revertants, which have recovered the ability to grow on a respiratory substrate, have been selected from this mutant (tyrosine 378). The nature of the reversion has been analysed by a rapid screening procedure and 32 of the revertants have been sequenced. They are all true backmutations reintroducing the histidine in position 378. This very exceptional situation suggests that this histidine is a ligand of the redox center of cytochrome oxidase.     

Sequence heterology and gene conversion at his-3 of Neurospora crassa

Although sequence heterology clearly reduces crossing over in yeast, conflicting studies suggest that mismatches may increase or decrease gene conversion. To investigate this issue in an additional species, we measured the effect of local sequence heterology on conversion in his-3 of Neurospora crassa. Mismatches close to the cog recombination initiator or within his-3 reduce conversion to 70% and 30% of the homologous level, respectively, while heterologous insertions between his-3 and cog increase conversion by 20%. We suggest that, in both Neurospora and yeast, mismatches reduce the efficiency of the establishment and resolution stages of recombination, but substantial heterology may increase the progress of already established events by preventing repair synthesis from switching between templates. These data provide additional support that recombination at his-3 (and perhaps at yeast hotspots) proceeds by a synthesis-dependent strand-annealing mechanism, during which synthesis can switch templates, with the process being more tolerant of sequence mismatch in Neurospora.     

A mutation in cytochrome oxidase subunit 2 restores respiration of the mutant pet ts1402

The yeast PET1402/OXA1 gene encoding a 44.8-kDa protein is required for mitochondrial biogenesis. Substitution of Leu240 to serine in the protein results in an accumulation of the precursor form of the mitochondrially encoded subunit 2 of cytochrome oxidase (Cox2) and temperature-sensitive respiration. This temperature sensitivity can be suppressed by a mutation in the cox2 gene changing Ala189 of the Cox2 protein to proline. In the cox2-ts1402 double mutant respiration is restored without removal of the Cox2 pre-sequence. The suppression suggests an interaction of the Pet1402 protein with the cytochrome oxidase complex. Antibodies raised against the predicted C-terminus and the tagged N-terminus of the Pet1402 protein reacted with a 37-kDa polypeptide. This protein, present in the mitochondrial fraction, is localized within the inner membrane. The difference in size can be explained by the removal of the predicted mitochondrial-targeting sequence from the Pet1402 protein. The mitochondrial localization of the protein points to a direct interaction with the cytochrome oxidase complex. Received: 4 December 1996 / 26 January 1997     

Amino-acid substitutions in the zinc finger of NIT2, the nitrogen regulatory protein of Neurospora crassa,alter promoter element recognition

NIT2, the major nitrogen regulatory protein of Neurospora crassa mediates nitrogen catabolite derepression of the structural genes which specify enzymes of nitrogen catabolism. The promoter of the structural gene for L-amino acid oxidase, a nitrogen-regulated enzyme, was found to contain two NIT2 binding sites, each with two copies of a GATA core consensus sequence. Site-directed mutagenesis was employed to create amino-acid substitutions within the single zinc-finger region of NIT2, which serves as the DNA-binding domain. The affect of those mutations upon NIT2 function in vivo in the activation of three separate structural genes was examined by transformation assays and relevant enzyme activities, and DNA-binding activity in vitro was determined by gel band mobility-shift assays. It was shown that specific amino-acid residues within the zinc-finger loop region of NIT2 are important for DNA-binding activity, whereas other residues influence the specificity of DNA binding. Mutant NIT2 proteins were obtained which retain DNA-binding activity and alter the specificity of DNA recognition, thus allowing a distinction between related DNA elements.     

The orientation of gene maps by recombination of flanking markers for the am locus of Neurospora crassa

Fincham (1967), Smyth (1973b) and Rambosek and Kinsey (1983) have each generated fine-structure maps of the am gene of Neurospora crassa. Each map had a consistent linear order of alleles but the assignment of an orientation with respect to other linkage group-V loci differed. Fincham found the end marked by the am ₆ allele to be at the distal end of the locus, Smyth found am ₆ to be at the proximal end while the data of Rambosek and Kinsey did not suggest an orientation. Smyth's orientation has been adopted as the standard, but not unreservedly. We have aligned the genetic and physical maps of the am gene, showing that am ₆ is at the distal end, supporting Fincham's orientation. However, we suggest that an assumption used to orient fine structure genetic maps is flawed and that the conflicting orientation between these three studies follows from the different choice of flanking markers.