Suppression of a nuclear aep2 mutation in Saccharomyces cerevisiae by a base substitution in the 5'-untranslated region of the mitochondrial oli1 gene encoding subunit 9 of ATP synthase.

Mutations in the nuclear AEP2 gene of Saccharomyces generate greatly reduced levels of the mature form of mitochondrial oli1 mRNA, encoding subunit 9 of mitochondrial ATP synthase. A series of mutants was isolated in which the temperature-sensitive phenotype resulting from the aep2-ts1 mutation was suppressed. Three strains were classified as containing a mitochondrial suppressor: these lost the ability to suppress aep2-ts1 when their mitochondrial genome was replaced with wild-type mitochondrial DNA (mtDNA). Many other isolates were classified as containing dominant nuclear suppressors. The three mitochondrion-encoded suppressors were localized to the oli1 region of mtDNA using rho- genetic mapping techniques coupled with PCR analysis; DNA sequencing revealed, in each case, a T-to-C nucleotide transition in mtDNA 16 nucleotides upstream of the oli1 reading frame. It is inferred that the suppressing mutation in the 5' untranslated region of oli1 mRNA restores subunit 9 biosynthesis by accommodating the modified structure of Aep2p generated by the aep2-ts1 mutation (shown here to cause the substitution of proline for leucine at residue 413 of Aep2p). This mode of mitochondrial suppression is contrasted with that mediated by heteroplasmic rearranged rho- mtDNA genomes bypassing the participation of a nuclear gene product in expression of a particular mitochondrial gene. In the present study, direct RNA-protein interactions are likely to form the basis of suppression.

The mature AEP2 gene product of Saccharomyces cerevisiae, required for the expression of subunit 9 of ATP synthase, is a 58 kDa mitochondrial protein.

The nucleotide sequence of the yeast nuclear AEP2 gene, required for the expression of the mitochondrial DNA-encoded subunit 9 of ATP synthase, predicts a primary translation product of 67.5 kDa. The ATP13 gene is allelic to AEP2 but was reported to encode a protein of about 42 kDa in size. We thus investigated genetically and biochemically the size of the AEP2 gene product. Genetic complementation assays using 3' truncated AEP2 genes, here shows that function is abolished by the removal of only 32 amino acids from the C-terminus of the predicted protein product. Cell-free translation of AEP2 produces a 64 kDa polypeptide (consistent with the AEP2 sequence) which is imported into mitochondria and processed to a 58 kDa product by the removal of a presequence of about 50 amino acids.

Suppression of a yeast mitochondrial RNA processing defect by nuclear mutations.

The S. cerevisiae strain h56 is a temperature-sensitive mit- mutant containing a single nucleotide substitution in the region 5' to the reading frame of the mitochondrial var1 gene. The mutation decreases the efficiency of processing of a precursor RNA such that little var1 mRNA is produced at the restrictive temperature, 36 degrees C. This communication reports the isolation and characterization of several strains carrying nuclear mutations which suppress the temperature-sensitivity of h56. Both dominant and recessive suppressor mutations were isolated. One dominant suppressor strain (h56-S4) was characterized biochemically, and the mechanism of suppression shown to involve a restoration of precursor RNA processing at the restrictive temperature, with a concomitant increase in the synthesis of the var1 protein. It appears likely that the suppressing allele encodes a component of an RNA processing endoribonuclease active on var1 transcripts. A genomic library was constructed from the h56-S4 strain, and several plasmids showing suppressed activity were isolated. A preliminary analysis of these plasmids is presented.

Characterization of a second nuclear gene, AEP1, required for expression of the mitochondrial OLI1 gene in Saccharomyces cerevisiae.

Due to mutation in a single nuclear locus, AEP1, the temperature-conditional pet mutant ts1860 of Saccharomyces cerevisiae fails to synthesize mitochondrial ATP synthase subunit 9 at the restrictive temperature of 36 degrees C. The presence at this temperature of near-normal levels of the cognate oli1 mRNA in mutant ts1860 indicates that, as previously shown, the product of the AEP1 gene is required for translation of the mitochondrial oli1 transcript. In this study the AEP1 gene has been cloned from a wild-type yeast genomic library by genetic complementation of a temperature-conditional aep1 strain at the restrictive temperature. A 2,330-bp genomic fragment which restores subunit 9 synthesis in aep1 mutant strains was characterized. This fragment encoded five open reading frames: the longest of these, at 1,554 nucleotides, was identified as the AEP1 gene, since disruption of this reading frame generated a non-conditional pet strain unable to synthesize subunit 9. The predicted product of AEP1 is a basic, hydrophilic protein of 59,571 Da which possesses a putative mitochondrial address sequence. Hybridization studies with AEP1-specific probes indicate that the gene is located on chromosome XIII and produces several poly(A)+ transcripts ranging in size from 0.9 to 2.7 kb. None of the identified reading frames share significant homologies with entries of several data bases.

Characterization of a yeast nuclear gene, AEP2, required for accumulation of mitochondrial mRNA encoding subunit 9 of the ATP synthase.

The temperature-conditional pet mutant, ts379, of Saccharomyces cerevisiae fails to synthesize mitochondrial ATP synthase subunit 9 at the restrictive temperature due to mutation of a single nuclear locus, AEP2. The inability to synthesize subunit 9 correlates with a lowered accumulation of the cognate oli1 mRNA indicating that the AEP2 product is involved in oli1 transcript maturation or stabilization. The AEP2 gene has been isolated in this study from a wild-type yeast genomic library by genetic complementation of ts379 at the restrictive temperature. A 1,740 nucleotide open-reading frame was observed that encodes a basic, hydrophilic protein of 67,534 Da which possesses a putative mitochondrial address signal. Disruption of chromosomal DNA within this reading frame produced a non-conditional respiratory mutant unable to synthesize subunit 9, identifying the AEP2 gene. Hybridization analyses indicate that AEP2 is located on chromosome XIII and produces a 2.1 kb poly(A)+ transcript. Two additional open-reading frames were found in close proximity to that of AEP2. The three open-reading frames shared no significant homology with entries in several data bases.

Properties of two nuclear pet mutants affecting expression of the mitochondrial oli1 gene of Saccharomyces cerevisiae.

This study details the characteristics of two temperature-conditional pet mutants of yeast, strains ts1860 and ts379, which at the non-permissive temperature show deficiencies in the formation of three mitochondrially encoded subunits of the ATP synthase complex. By analysis of mitochondrial translation products, and of mitochondrial transcription in temperature shift experiments from the permissive (22 degrees C) to the non-permissive (36 degrees C) temperature, it was concluded that the nuclear mutations in both mutants primarily inhibit synthesis of ATP synthase subunit 9, and that reductions in subunit 8 and 6 synthesis are secondary pleiotropic effects. Following transfer to 36 degrees C, cells of mutant ts379 display a near complete inhibition of subunit 9 synthesis within 1 h, coincident with a marked reduction in the level of the cognate oli1 mRNA. On the other hand, near complete inhibition of subunit 9 synthesis in strain ts1860 occurs after 3 h at 36 degrees C, at which time there is little change in the level of subunit 9 mRNA. In both mutants the mRNA levels for subunits 6 and 8 are not significantly affected at the time of inhibition of subunit 9 synthesis. Provision of an alternative source of subunit 8, translated extra-mitochondrially for import into the organelle, does not overcome the mutant phenotype of either mutant at 36 degrees C, confirming that subunit 8 is not the sole or primary deficiency in each mutant. The mutants indicate that the products of a least two nuclear genes (designated AEP1 and AEP2) are required for the expression of the mitochondrial oli1 gene and the synthesis of subunit 9. (ABSTRACT TRUNCATED AT 250 WORDS)

A mitochondrial intergenic mutation affecting processing of specific yeast mitochondrial transcripts.

The mutation in the temperature-conditional mit- mutant h56, mapped previously to the var1 gene region of Saccharomyces cerevisiae mitochondrial DNA, results in a specific inhibition of var1 protein synthesis in cells incubated at the non-permissive temperature, 36 degrees C (1). We have now characterized the mutation present in mutant h56 by DNA sequencing and found it to be an A to T transversion located 109 nucleotides upstream of the var1 reading frame. Two spontaneous revertants of mutant h56 restore the parental strain sequence at residue -109, confirming that this single base change within the 5'-untranslated region of the var1 mRNA is responsible for defective synthesis of the var1 protein. A comparison of var1 transcripts in the parental and mutant strains has shown that the mutation specifically blocks formation of var1 mRNA at 36 degrees C and leads to accumulation of precursor transcripts. Expression of the oli1 gene, co-transcribed with the var1 gene in primary transcripts, is not affected. It is concluded that the mutation in mutant h56 alters the secondary structure of the precursor RNA, inhibiting an endonucleolytic cleavage required to generate the 5' end of var1 mRNA.

Biogenesis of mitochondria: a mutation in the 5'-untranslated region of yeast mitochondrial oli1 mRNA leading to impairment in translation of subunit 9 of the mitochondrial ATPase complex.

A temperature-conditional mit- mutant of Saccharomyces cerevisiae has been characterized; the mutant strain h45 cannot grow at 36 degrees C on nonfermentable substrates yet appears to be normal at 28 degrees C. The mutation in strain h45 maps genetically to the oli1 region of the mitochondrial DNA (mtDNA) genome, and prevents the synthesis at 36 degrees C of the oli1 gene product, subunit 9 of the mitochondrial ATPase complex. Since the level of oli1 mRNA in mutant h45 is close to normal at 36 degrees C, it is concluded that there is a specific block in translation of this mRNA at the non-permissive temperature. DNA sequence analysis of mtDNA from strain h45 reveals an additional T residue inserted 88 bp upstream of the oli1 coding region, in the A,T-rich sequence that is transcribed into the 5'-untranslated region of the oli1 mRNA. Sequence data on two revertants show that one returns to wild-type parental (J69-1B) mtDNA sequence, whilst the other contains an inserted A residue adjacent to the T inserted in the original h45 mutant. The results are discussed in terms of the stability of folds in RNA upstream of putative ribosome-binding sites in mitochondrial mRNA, and the potential action of nuclear-coded proteins that might be activators of the translation of specific mitochondrial mRNAs in yeast mitochondria.

Constants and variables of fungal mitochondrial genomes.

Comparative studies of several fungal mitochondrial DNAs have highlighted a conspicuous diversity of organization of a limited, but generally well conserved, information content and a marked variability of gene structure with some conserved features. Attention has also been focused on new and potential genes and phenomena associated with nuclear-mitochondrial interactions.

Biogenesis of Mitochondria: Genetic and molecular analysis of the oli2 region of mitochondrial DNA in Saccharomyces cerevisiae.

A genetic and molecular analysis of the oli2 region in mitochondrial DNA of Saccharomyces cerevisiae has been carried out. The oli2 gene codes for subunit 6 of the mitochondrial ATPase complex. We have isolated a series of 41 mit (-), temperature sensitive and oligomycin resistant mutants containing mutations located in the oli2 region. In addition the sequence in wild type mtDNA of 2820 nucleotides covering the oli2 gene and its flanking regions has been determined. A library of petite mutants which have various deletion end points in the oli2 region has been used to generate marker rescue data that allow the assignment of each mit (-), temperature sensitivity and oligomycin resistance mutation to one of seven uniquely ordered genetic groups. The following physical reference points allow the limits of the physical map positions of a number of the genetic groups to be established: DNA sequence data have been obtained for three of the petites so as to determine unambiguously their relevant deletion end points and the base substitution constituting the mtDNA sequence change in one oligomycin resistant mutant [oli2-23r] has been determined to lie at nucleotide +523 of the oli2 coding region. One group of mit (-) mutations (group N) are known to map within the aap1 gene that is found some 700 bases upstream of the oli2 gene, and which codes for subunit 8 of the mitochondrial ATPase complex. Mutations in the remaining six groups (A-F) map within (or very close to) the coding region of the oli2 gene. Various features of the DNA sequence in and around the oli2 gene are considered, including protein coding regions, sequence divergence in the A,T-rich spacer regions, properties of G,C-rich clusters, and potential ribosome binding sites. Consideration of the transcriptional map of this region of mtDNA suggests that an abundant 4,500 nucleotide transcript may represent the first defined example of a dicistronic messenger RNA in yeast mitochondria carrying distinct coding regions (aap1 and oli2) for two different proteins.

Biogenesis of mitochondria: the mitochondrial gene (aap1) coding for mitochondrial ATPase subunit 8 in Saccharomyces cerevisiae.

A mitochondrial gene (denoted aap1) in Saccharomyces cerevisiae has been characterized by nucleotide sequence analysis of a region of mtDNA between the oxi3 and oli2 genes. The reading frame of the aap1 gene specifies a hydrophobic polypeptide containing 48 amino acids. The functional nature of this reading frame was established by sequence analysis of a series of mit- mutants and revertants. Evidence is presented that the aap1 gene codes for a mitochondrially synthesized polypeptide associated with the mitochondrial ATPase complex. This polypeptide (denoted subunit 8) is a proteolipid whose size has been previously assumed to be 10 kilodaltons based on its mobility on SDS-polyacrylamide gels, but the sequence of the aap1 gene predicts a molecular weight of 5,815 for this protein.

Biogenesis of mitochondria. oli2 Mutations affecting the coupling of oxidation to phosphorylation in Saccharomyces cerevisiae.

1. Two oligomycin-resistant strains of Saccharomyces cerevisiae have been isolated and shown to have mutations in the oli2 region of the mitochondrial DNA. On solid media containing a non-fermentable energy source, the mutant strains were able to grow only slowly at 28 degrees C and not at all at 18 degrees C or 36 degrees C. 2. When grown in a glucose-limited chemostat at 28 degrees C, the mutant strains were almost completely defective in oxidative metabolism. The mutant mitochondria contained significant levels of all respiratory enzymes, and an active, oligomycin-sensitive ATPase, but the ATP-32Pi exchange activity and P : O ratio were very low. 3. The mutations in these strains are genetically closely linked to mit mutations which have been shown to affect a 20 000-dalton ATPase subunit (Roberts, H., Choo, W.M., Murphy, M., Marzuki, S., Lukins, H.B. and Linnane, A.W. (1979) FEBS Lett. 108, 501-504). Since the mitochondrial ATPase in these mutant strains appears to be fully assembled, the defect in the coupling mechanism is probably a result of a small alteration in the structure of the 20 000-dalton ATPase subunit. 4. When the mutant strains were grown at 18 degrees C, the mitochondria had very low cytochrome oxidase activities, and reduced levels of cytochrome aa3. The largest subunit (Mr 40 000) of this enzyme was not synthesized.

The dicyclohexylcarbodiimide-binding protein of the mitochondrial ATPase complex from Neurospora crassa and Saccharomyces cerevisiae. Identification and isolation.

Incubation of mitochondria from Neurospora crassa and Saccharomyces cerevisiae with the radioactive ATPase inhibitor [14C]dicyclohexylcarbodiimide results in the irreversible and rather specific labelling of a low-molecular-weight polypeptide. This dicyclohexylcarbodiimide-binding protein is identical with the smallest subunit (Mr 8000) of the mitochondrial ATPase complex, and it occurs as oligomer, probably as hexamer, in the enzyme protein. The dicyclohexylcarbodiimide-binding protein is extracted from whole mitochondria with neutral chloroform/methanol both in the free and in the inhibitor-modified form. In Neurospora and yeast, this extraction is highly selective and the protein is obtained in homogeneous form when the mitochondria have been prewashed with certain organic solvents. The bound dicyclohexylcarbodiimide label is enriched in the purified protein up to 50-fold compared to whole mitochondria. Based on the amino acid analysis, the dicyclohexylcarbodiimide-binding protein from Neurospora and yeast consists of at least 81 and 76 residues, respectively. The content of hydrophobic residues is extremely high. Histidine and tryptophan are absent. The N-terminal amino acid is tyrosine in Neurospora and formylmethionine in yeast.