Assembly of the mitochondrial membrane system. DNA sequence and organization of the cytochrome b gene in Saccharomyces cerevisiae D273-10B

The mitochondrial genomes of cytoplasmic "petite" (rho-) mutants of Saccharomyces cerevisiae have been used to sequence the cytochrome b gene. A continuous sequence of 6.2 kilobase pairs has been obtained from 71.4 to 80.2 units of the wild type map. This region contains all the cytochrome b mutations previously assigned to the cob1 and cob2 genetic loci. Analysis of the DNA sequence has revealed that in the strain D273-10B, the cytochrome b gene is composed of three exons. The longest exon (b1) codes for the first 252 to 253 amino acids from the NH2-terminal end of the protein. The next two exons (b2 and b3) code for 16 to 18 and 115 to 116 amino acids, respectively. The complete cytochrome b polypeptide chain consists of 385 amino acids. Based on the amino acid composition, the yeast protein has a molecular weight of 44,000. The three exon regions of the cytochrome b gene are separated by two introns. The intron between b1 and b2 is 1414 nucleotides long and contains a reading frame that is continuous with the reading frame of exon b1. This intron sequence is potentially capable of coding for another protein of 384 amino acid residues. The second intron is 733 nucleotides long. This sequence is rich in A + T and includes a G + C cluster that may be involved in processing of the cytochrome b messenger. The organization of the cytochrome b region in S. cerevisiae D273-10B is somewhat less complex than has been reported for other yeast strains i which exon b1 appears to be further fragmented into three smaller exons.     

Assembly of the mitochondrial membrane system. Sequence of the oxi 2 gene of yeast mitochondrial DNA

The region of mitochondrial DNA (mtDNA) containing the oxi 2 locus has been sequenced in a rho- clone (DS40) derived from the respiratory competent strain D273-10B/A48 of Saccharomyces cerevisiae. The DS40 clone was established to have retained only genetic markers in the oxi 2 locus and to have a segment of mtDNA extending from 18.6 to 24.3 units of the wild type map. The mitochondrial genome of DS40 includes a sequence that has been tentatively identified as the structural gene of Subunit 3 of cytochrome oxidase. The coding sequence is 810 nucleotides long and generates a protein with a molecular weight of 30,340. The amino acid composition of the oxi 2 gene product deduced from the nucleotide sequence is in agreement with the composition of the purified Subunit 3 of yeast cytochrome oxidase. The orientation of the DS40 mtDNA segment relative to wild type mtDNA indicates that the oxi 2 gene is transcribed from the same DNA strand as the oxi 1 and several other mitochondrial genes.     

Assembly of the mitochondrial membrane system. Characterization of nuclear mutants of Saccharomyces cerevisiae with defects in mitochondrial ATPase and respiratory enzymes.

Mutants of Saccharomyces cereviaiae showing defects in cytochrome oxidase, coenzyme QH2-cytochrome c reductase, and rutamycin-sensitive ATPase are described. The mutations have been established to be nuclear, based on complementation with a cytoplasmic petite tester strain and 2:2 segregation of tetrads. Genetic analysis indicate the coenzyme QH2-cytochrome c reductase and cytochrome oxidase mutants fall into 9 and 10 different complementation groups, respectively. The mutants also form distinct classes based on absorption spectra of the mitochondrial cytochromes. Two of the ATPase mutants lack detectable F1 ATPase, while the third synthesizes F1 but does not integrate it into a membrane complex. The latter mutant is missing one of the mitochondrially synthesized subunits of the rutamycin-sensitive ATPase complex.     

Petite deletion map of the mitochondrial oxi3 region in Saccharomyces cerevisiae.

Summary Fifty eight mitochondrial mutants (p ⁺ mit^- mutants), all deficient in cytochrome oxidase activity and previously assigned to the genetic region oxi3 on the mitochondrial DNA, were mapped by the method of petite deletion mapping.This procedure resulted in the identification of at least twenty one different classes of oxi3 mutants, which could be arranged in a linear order.Moreover, it provided a set of twenty three p ^- petite mutants, each containing a differentially deleted mit DNA segment included in the oxi3 region. The two sets of mutants, p ⁺ oxi3 ^- and p ^- oxi3 ⁺, will be of interest for a further genetic and physical analysis of this mitochondrial DNA segment which spans over about ten thousand base pairs and controls the subunit I of cytochrome oxidase.     

Assembly of the mitochondrial membrane system: mutations in the pho2 locus of the mitochondrial genome of Saccharomyces cerevisiae.

Two mutants of Saccharomyces cerevisiae which show a loss of mitochondrial rutamycin-sensitive ATPase activity are described. Although phenotypically similar to mutants of the mitochondrial locus pho1 [F. Foury and A. Tzagoloff (1976) Eur. J. Biochem. 68, 113-119], these mutants define a second ATPase locus on the mitochondrial DNA (designated pho2), which is genetically unlinked to pho1. Analysis of recombination in crosses involving multiple antibiotic resistance markers indicates that the locus is in the segment of the genome between ery1 and oli2, very close to oli1. In fact it is proposed that the oli1 and pho2 mutations are in the same gene. Supporting evidence for this proposal includes: 1. The analysis of marker retention in petite mutants shows that the oli1 and pho2 loci were either retained or lost together in all cases. 2. Recombination frequencies of 0.05% or less are observed in crosses between the oli1 and pho2 loci. 3. When rho+ revertants are isolated from the pho2 mutants they frequently are oligomycin resistant. 4. pho2 mutants have an altered subunit 9 of the ATPase complex.     

Assembly of the mitochondrial membrane system. Genetic complementation of mit- mutations in mitochondrial DNA of Saccharomyces cerevisiae.

A method has been devised to test intergenic complementation of mutations in the mitochondrial DNA of Saccharomyces cerevisiae. The test is based on the observation that diploids issued from pairwise crosses of certain mit- mutants with deficiencies in cytochrome oxidase, or coenzyme QH2-cytochrome c reductase, acquire high levels of respiratory activity shortly after zygote formation. Under our experimental conditions neither biochemical complementation, interallelic complementation, nor recombination has been found to contribute to any significant extent toward the respiration measured in the diploids at early times. The test has been used to study the number of complementation groups represented by a large number of mit- mutants. Results of pairwise crosses of mutants in the oxi 1, oxi 2, oxi 3, cob 1, and cob 2 loci indicate that complementation occurs between the oxi and cob loci between different oxi loci but not between the two cob loci. The five loci have, therefore, been assigned to four different complementation groups.     

Assembly of the mitochondrial membrane system: two separate genes coding for threonyl-tRNA in the mitochondrial DNA of Saccharomyces cerevisiae.

1. Mitochondria of Saccharomyces cerevisiae contain two tRNA's that are acylated with threonine. The two isoaccepting species (tRNA1Thr and tRNA2Thr) can be separated by reversed-phase chromatography on RPC-5. 2. A cytoplasmic mutant has been isolated which lacks tRNA1Thr but has normal levels of tRNA2Thr. This mutation was previously shown to map between the oxi 1 and oxi 2 loci on mitochondrial DNA. 3. tRNA1Thr and tRNA2Thr hybridize to wild type mitochondrial but not nuclear DNA and are capable of partially competing with each other. Hybridization of each species to different segments of mitochondrial DNA isolated from p- clones indicate that there are two threonyl tRNA genes. One gene is located between oxi 1 and oxi 2 and codes for tRNA1Thr. The second gene codes for tRNA2Thr and is near the cap locus. 4. Binding assays to E. coli ribosomes indicate that tRNA2Thr recognizes the threonine triplet ACA and may also recognize the other three triplets but with a much lower efficiency. None of the four codons for threonine stimulate the binding of tRNA1Thr to the ribosomes.     

Assembly of the mitochondrial membrane system: isolation of nuclear and cytoplasmic mutants of Saccharomyces cerevisiae with specific defects in mitochondrial functions.

A selection procedure is described which permits a large number of Saccharomyces cerevisiae mutants to be screened for specific lesions in mitochondrial respiratory enzymes and the adenosine triphosphatase. The method has been used to isolate nuclear mutant strains with specific lesions in coenzyme QH2-cytochrome c reductase, cytochrome oxidase, and adenosine triphosphatase. In addition, two cytoplasmic mutants have been found whose primary defect is in cytochrome oxidase, and others have been found that show variable degrees of abnormalities in their mitochondrial translation products.     

Assembly of the mitochondrial membrane system. Cytoplasmic mutants of Saccharomyces cerevisiae with lesions in enzymes of the respiratory chain and in the mitochondrial ATPase.

Mutants of Saccharomyces cervisiae with defects in enzymes of the electron transfer chain and in the rutamycin-sensitive ATPase have been isolated. Some of the mutants are specifically affected in either cytochrome oxidase, coenzyme QH2-cytochrome c reductase or ATPase. Other strains are deficient in both cytochrome oxidase and coenzyme QH2-cytochrome c reductase but still have rutamycin-sensitive ATPase. All the mutants reported in this study fail to be complemented by a rho0 tester derived from a respiratory competent strain. The meiotic spore progeny obtained by mating the mutants to a respiratory competent haploid yeast, when scored for growth on glycerol, show a non-Mendelian segregation of the phenotype. These two genetic tests indicate the mutations to be cytoplasmically inherited.     

Characterization of a mitochondrial inorganic pyrophosphatase in Saccharomyces cerevisiae.

We have studied a mitochondrial inorganic pyrophosphatase (PPase) in the yeast Saccharomyces cerevisiae. The uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and the ionophores valinomycin and nigericin stimulate the PPase activity of repeatedly washed yeast mitochondria 2-3-fold. We have previously cloned a yeast gene, PPA2, encoding the catalytic subunit of a mitochondrial PPase. Uncouplers stimulate the PPase activity several-fold in mitochondria from both cells that overexpress PPA2 from a high copy number plasmid and cells with normal expression. These results indicate that the PPA2 polypeptide functions as an energy linked and membrane associated PPase. The stimulation of mitochondrial PPase activity by FCCP, but not by valinomycin and nigericin, was greatly enhanced by the presence of DTT. The antibiotics Dio-9, equisetin and the F0F1-ATPase inhibitor oligomycin also increase mitochondrial PPase activity several fold. This stimulation is much higher, whereas basal PPase activity is lower, in isotonic than in hypotonic solution, which indicates that intact membranes are a prerequisite for maximal effects.     

Assembly of the mitochondrial membrane system: isolation of mitochondrial transfer ribonucleic acid mutants and characterization of transfer ribonucleic acid genes of Saccharomyces cerevisiae

A method is described for isolating cytoplasmic mutants of Saccharomyces cerevisiae with lesions in mitochondrial transfer ribonucleic acids (tRNA's). The mutants were selected for slow growth on glycerol and for restoration of wild-type growth by cytoplasmic "petite" testers that contain regions of mitochondrial deoxyribonucleic acid (DNA) with tRNA genes. The aminoacylated mitochondrial tRNA's of several presumptive tRNA mutants were analyzed by reverse-phase chromatography on RPC-5. Two mutant strains, G76-26 and G76-35, were determined to carry mutations in the cysteine and histidine tRNA genes, respectively. The cysteine tRNA mutant was used to isolate cytoplasmic petite mutants whose retained segments of mitochondrial DNA contain the cysteine tRNA gene. The segment of one such mutant (DS504) was sequenced and shown to have the cysteine, histidine, and threonine tRNA genes. The structures of the three mitochondrial tRNA's were deduced from the DNA sequence.     

Characterization and localization of the sporulation glucoamylase of Saccharomyces cerevisiae

Glucoamylase (SGA) was purified approximately 250-fold from sporulating Saccharomyces cerevisiae cells. The partially purified enzyme was active against glycogen, starch, maltotriose and maltose. It exhibited maximum catalytic activity against glycogen at pH 5.5. The enzyme appears to be glycosylated, because it bound to lentil-lectin Sepharose. SGA was expressed in vegetatively growing cells under the control of the GAL1 promoter, and the cellular location of the enzymatic activity determined by fractionation techniques. SGA was preferentially recovered in fractions which were enriched for the vacuolar hydrolases, carboxypeptidase Y and alpha-mannosidase.