The function of mitochondrial genes in Neurospora crassa.

The 18 extranuclear mutants of Neurospora crassa, without exception, have abnormal mitochondrial respiratory systems. On the basis of genetic, phenotypic and physiological criteria, these mutants are divided into four groups: 1) the cytochrome aa3 and b deficient "poky" variants that are defective in mitochondrial ribosomes assembly, 2) the cytochrome aa3 deficient mutants, [mi-3] and [exn-5], that appear to have genetic lesions affecting a component of a regulatory system controlling cytochrome aa3 synthesis, 3) the cytochrome aa3 and b deficient "stopper" mutants with physiological lesions that probably affect mitochondrial protein synthesis, and 4) cni-3, a mutant that is constitutive for an inducible mitochondrial cyanide-insensitive oxidase in spite of having a normal cytochrome mediated electron-transport system. It is proposed that the mitochondrial genophore not only codes for cellular components that are essential for the formation of the mitochondrial protein synthesizing apparatus, but also for components of a regulatory system that coordinates the expression of nuclear and mitochondrial genes during the biogenesis of the mitochondrial electorn-transport system.     

Nuclear mutations affecting mitochondrial structure and function in Chlamydomonas.

Wild type cells of the green alga Chlamydomonas reinhardtii can grow in the in the dark by taking up and respiring exogenously supplied acetate. Obligate photoautotrophic (dark dier, dk) mutants of this alga have been selected which grow at near wild type rates in the light, but rapidly die when transferred to darkness because of defects in mitochondrial structure and function. In crosses of the dk mutants to wild type, the majority of the mutants are inherited in a mendelian fashion, although two have been isolated which are inherited in a clearly nonmendelian fashion. Nine mendelian dk mutants have been analyzed in detail, and belong to eight different complementation groups representing eight gene loci. These mutants have been tentatively grouped into three classes on the basis of the pleiotropic nature of their phenotypic defects. Mutants in Class I have gross alterations in the ultrastructure of their mitochondrial inner membranes together with deficiencies in cytochrome oxidase and antimycin/rotenone-sensitive NADH-cytochrome c reductase activities. Mutants in Class II have a variety of less severe alterations in mitochondrial ultrastructure and deficiencies in cytochrome oxidase activity. Mutants in Class III have normal or near normal mitochondrial ultrastructure and reduced cytochrome oxidase activity. Eight of the nine mutants show corresponding reductions in cyanide-sensitive respiration.     

Synthesis of the mitochondrial inner membrane in cultured Xenopus laevis oocytes.

The purpose of this study was to investigate the contribution of mitochondrial and cytoplasmic protein synthesis to the biogenesis of cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase EC 1.9.3.1) and rutamycin-sensitive adenosine triphosphatase (ATP phosphohydrolase EC 3.6.1.3) in cultured oocytes of the toad, Xenopus laevis. X. laevis cytochrome oxidase was purified over 23-fold with respect to specific activity and over 29-fold with respect to specific heme a content from oocyte submitochondrial particles. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate separated the enzyme into six subunits with molecular weights of 44,000, 33,000, 23,000, 17,000, 12,000 and 9,500. the synthesis of the three larger subunits is sensitive to chloramphenicol (an inhibitor of mitochondrial protein synthesis), indicating that these subunits are made on mitochondrial ribosomes; the synthesis of the three smaller subunits is sensitive to cycloheximide (an inhibitor of cytoplasmic protein synthesis) and therefore occurs on cytoplasmic ribosomes. X. laevis rutamycin-sensitive ATPase, purified over 19-fold from oocyte submitochondrial pparticles, consists of 10 subunits with molecular weights of 56,000, 53,000, 41,000, 32,000, 29,000, 24,000, 21,000, 17,500 (2), and 11,500 on sodium dodecyl sulfate-polyacrylamide gels. The 29,000, 21,000, and one of the 17,500-dalton polypeptides are synthesized in the presence of cycloheximide and are, therefore, products of mitochondrial protein synthesis; the synthesis of the remaining seven subunits occurs in the presence of chloramphenicol, indicating that these subunits are made on cytoplasmic ribosomes. The synthesis of protein by mitochondria in cultured oocytes appears to be dependent upon cytoplasmic protein synthesis. In the presence of cycloheximide, the mitoribosomal synthesis of the subunits of cytochrome oxidase and rutamycin-sensitive ATPase is detectable only after a prior inhibition of mitochondrial protein synthesis by chloramphenicol. Oocyte mitochondrial ribosomes synthesize at least nine polypeptides after chloramphenicol treatment, three of which are components of neither cytochrome oxidase nor rutamycin-sensitive ATPase.     

Androgenic control of hepatic mitochondrial metabolism in an apoda, Gegenophis carnosus (Beddome).

In vivo administration of testosterone significantly stimulated the activities of cytochrome oxidase, alpha-glycerophosphate dehydrogenase (alpha-GPDH), succinate dehydrogenase (SDH) and adenosine triphosphatase (Mg2+ ATPase), in mitochondria isolated from the liver of G. carnosus. Administration of dehydroepiandrosterone and androstenedione while significantly stimulated the activities of cytochrome oxidase and alpha-GPDH, did not change that of SDH and Mg2+ ATPase. Simultaneous injections of testosterone and actinomycin D or chloramphenicol prevented the testosterone-stimulated activities of all the oxidative enzymes studied. The results clearly document the important stimulatory role of androgens in the regulation of hepatic mitochondrial metabolism in G. carnosus.     

Characterization of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatases.

The catalytic properties of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatase have been analysed. The pH profile, effect of inhibitors, cold-stability and substrate specificity are characteristic of mitochondrial adenosine triphosphatases, although a high guanosine triphosphatase activity does appear to be restricted to plant mitochondrial adenosine triphosphatases. The kinetic properties of nucleoside 5'-triphosphate hydrolysis by membrane-bound and soluble enzymes have been studied by means of double-reciprocal plots. These plots were linear in the absence of an activating anion, which may indicate that the catalytic and/or regulatory mechanism of Arum maculatum adenosine triphosphatase is different from that of other enzyme preparations. It is suggested that the differences in subunit composition of plant and mammalian adenosine triphosphatases reported previously [Dunn, Slabas & Moore (1985) Biochem. J. 225, 821-824] are structurally, rather than functionally, significant.     

Rapid method for isolation and screening of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae

We describe here a new method for the specific isolation of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae. One unique feature of the method is the use of tetramethyl-p-phenylenediamine as a cytochrome c oxidase activity stain for yeast colonies. The staining of yeast colonies by tetramethyl-p-phenylenediamine is dependent upon a functional cytochrome c oxidase and is unaffected by other lesions in respiration. Since the tetramethyl-p-phenylenediamine colony staining reaction is rapid and simple, it greatly facilitates both the identification and characterization of cytochrome c oxidase-deficient mutants. Another feature of the method, which is made possible by the tetramethyl-p-phenylenediamine colony stain, is the use of an op1 parent strain for the isolation of nuclear pet or mitochondrial mit mutants in specific protein-coding genes. A parent strain that carries this marker selects against rho0 or rho- classes of pleiotropic respiratory-deficient mutants, since these are lethal in op1 strains. We have used this method to isolate 123 independently derived cytochrome c oxidase-deficient pet mutants and 300 independently derived mit mutants.     

The Largest Mitochondrial Translation Product Copurifying with the Mitochondrial Adenosine Triphosphatase of Saccharomyces cerevisiae Is Not a Subunit of the Enzyme Complex

Mitochondrial adenosine triphosphatase isolated from a double mutant of Saccharomyces cerevisiae lacking cytochrome b apoprotein and subunit II of cytochrome oxidase does not contain the mitochondrial translation product (approximate molecular weight, 32,000) previously suggested to be a subunit of the enzyme complex.     

Localization in yeast mitochondrial DNA of mutations expressed in a deficiency of cytochrome oxidase and/or coenzyme QH2-cytochrome c reductase.

1. Three methods are described for the genetic analysis of yeast cytoplasmic mutants (mit- mutants) lacking cytochrome oxidase or coenzyme QH2-cytochrome c reductase. The procedures permit mutations in mitochondrial DNA to be mapped relative to each other and with respect to drug-resistant markers. The first method is based upon the finding that crosses of mit- mutants with some but not other isonuclear q- mutants lead to the restoration of respiratory functions. Thus a segment of mitochondrial DNA corresponding to a given mit- mutation or to a set of mutations can be delineated. The second method is based on the appearance of wild-type progeny in mit- X mit- crosses. The third one is based on the analysis of various recombinant classes issued from crosses between mit-, drug-sensitive and mit+, drug-resistant mutants. Representative genetic markers of the RIBI, OLII, OLI2 and PAR1 loci were used for this purpose. 2. The three methods when applied to the study of 48 mit- mutants gave coherent results. At least three distinct regions on mitochondrial DNA in which mutations cause loss of functional cytochrome oxidase have been established. A fourth region represented by closely clustered mutants lacking coenzyme QH2-cytochrome c reductase and spectrally detectable cytochrome b has also been studied. 3. The three genetic regions of cytochrome oxidase and the cytochrome b region were localized by the third method on the circular map, in spans of mitochondrial DNA defined by the drug-resistant markers. The results obtained by this method were confirmed by analysis of the crosses between selected mit- mutants and a large number of q- clones whose retained segments of mitochondrial DNA contained various combinations of drug-resistant markers. 4. All the genetic data indicate that the various regions studied are dispersed on the mitochondrial genome and in some instances regions or clusters of closely linked mutations involved in the same respiratory function (cytochrome oxidase) are separated by other regions which code for entirely different functions such as ribosomal RNA.     

Turnover of mitochondrial inner membrane proteins in hepatoma monolayer cultures

The degradation rates of inner mitochondrial membrane proteins were examined in serum-deprived hepatoma cultures. In those nonproliferating cells, the degradation of composite mitochondrial proteins was a first order process with a half-life of 34 h. The half-lives of specific inner mitochondrial membrane polypeptides were determined by examining the 3H/35S of isolated polypeptides from cells given [3H]methionine and [35S]methionine pulses, respectively, before and after a 2-day chase period. The 33 most abundant polypeptides resolved on a bidirectional polyacrylamide gel system showed half-lives ranging from 20 to 100+ h. The 15 polypeptides translated on mitochondrial ribosomes in the presence of inhibitory concentrations of cycloheximide also displayed heterogeneous rates of degradation (t1/2 = 35-100+ h). None of the isolated adenosine triphosphatase (coupling factor F1) or immunoprecipitated cytochrome c oxidase subunits were significantly turned over during the case period. Five of eight cytochrome b-c1 complex subunits, however, were turned over significantly more rapidly (t1/2 = 39-42 h) than the other three (t1/2 = 94+ h). The results demonstrate heterogeneous degradation rates for inner membrane polypeptides, extending in some cases to those within the same respiratory complex.     

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.     

Mosaic organization of a mitochondrial gene: evidence from double mutants in the cytochrome b region of Saccharomyces cerevisiae

The region coding for apocytochrome b in the mitochondrial genome of Saccharomyces cerevisiae is believed to exhibit a mosaic organization, consisting in certain strains of five exons and four introns. This model can be tested by the use of double mutants, each containing two physically, genetically and phenotypically defined mit- lesions in cis, (that is, in the same mitochondrial chromosome). Such mutants have been constructed, and the phenotypes of several examples of each of the four possible classes--exon-exon, exon-intron (downstream), intron (upstream)-exon and intron-intron--have been examined. Our results have shown that upstream mutations are always epistatic to downstream ones for polypeptide products, and that regulation of expression of cytochrome oxidase subunit I by introns is epistatic regardless of position. These findings have provided an independent verification of the mosaic model, and also suggest that at least the majority of novel polypeptides accumulating in intron mutants are hybrid products that contain sequences of the wild-type polypeptide.     

Mitochondrial Complementation and Grain Yield in Hybrid Wheat

ADP/O ratios, cytochrome c oxidase and adenosine triphosphatasehave been measured in mitochondria and mixtures of mitochondriaisolated from two day-old shoots of wheat of known F₁ hybridgrain yield performance. Mixtures of mitochondria from two varieties,Peko and Cappelle-Desprez, which have considerable F₁ hybridyield heterosis, showed a significantly increased ADP/O ratioover the mean value for mitochondria from the varieties assayedindividually, i.e. these varieties showed ‘mitochondrialcomplementation’. No mitochondrial complementation wasdetected for cytochrome c oxidase or adenosine triphosphatase.In other mitochondrial mixtures no complementation in ADP/Oratios were found even when the varieties showed F₁ hybrid yieldheterosis. Mitochondrial ADP/O ratios were studied in six varietiesindividually and in mixtures. In only one mixture was any significantcomplementation detected. However, when all the results wereconsidered together, mitochondrial complementation was significantlycorrelated with F₁ hybrid grain yield heterosis when the plantswere grown at a low seed density but not at a high seed density.New hypotheses are offered to account for mitochondrial complementationand its statistical relationship with yield heterosis.     

Pleiotropic mutations within two yeast mitochondrial cytochrome genes block mRNA processing.

The mRNAs from two yeast mitochondrial genes cob-box (cytochrome b) and oxi-3 (cytochrome oxidase 40,000 dalton subunit) are processed from large (7-10 kb) precursors. Certain mutations in each gene block the maturation of the RNAs from both genes at a variety of specific steps. The pleiotropic cytochrome b mutants seem to lack a functional trans-acting RNA required for the processing of both messengers. In contrast, the oxi-3 mutants may act by producing an activity that inhibits specific steps.     

Integration and regulation of mitochondrial assembly in yeast

Summary The interactions between the mitochondrial and nucleocytoplasmic systems required for mitochondriogenesis have been investigated at several different levels. Those involved in the formation of functional enzyme complexes have been studied using cytochrome oxidase: this multimeric (2 × 7 and 2 × 6 subunits for enzymes from yeast and beef heart respectively) has been resolved, and the mitochondrial contribution has been shown to be dispensible for catalytic function proper. Using novel mutants, with a mitochondrial mode of inheritance, a mitochondrial gene product localized in the oligomycin-sensitive ATPase has been implicated in the assembly not only of this complex, but of cytochrome oxidase as well. Interactions required for the genetic competence of the mitochondrial system have become apparent as a result of studies in the mechanism of action of the highly effective mitochondrial mutagen ethidium bromide. This agent first becomes covalently inserted into mitochondrial DNA and, after its excision, eventually results in extensive degradation of the macromolecule. The excision reaction has now been shown to be performed by a complex between the oligomycin-sensitive ATPase and a DNA-binding protein presumably involved in recognizing the damage. On the level of replication and expression of the mitochondrial genome studies using thermolabile mutants have demonstrated that these processes appear independent of the replication of nuclear DNA but not of its expression.Recipient of Research Career Award K06 5060 from the National Institute of General Medical Sciences, National Institutes of Health; research supported by Research Grant GM 12228 from this Institute.Publication No. 2251.     

Molecular basis for resistance to myxothiazol, mucidin (strobilurin A), and stigmatellin. Cytochrome b inhibitors acting at the center o of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae

The respiratory bc1 complex transfers the electrons from ubiquinol to cytochrome c oxidase. Myxothiazol, strobilurin A (mucidin), and stigmatellin are center o inhibitors preventing electron transfer at the ubiquinone redox site Qo, which is located closer to the outer side of the inner mitochondrial membrane. The cytochrome b gene is carried by the organelle DNA. Yeast mutants resistant to myxothiazol and mucidin have been previously isolated and mapped to specific loci of the cytochrome b gene. In the present work, stigmatellin-resistant mutants were isolated and genetically analyzed. The mutated amino acid residues from seven myxothiazol-, four mucidin-, and six stigmatellin-resistant mutants have been identified by sequencing the relevant segments of the resistant cytochrome b gene. A third myxothiazol-resistant locus and the first stigmatellin-resistant locus were identified. The mutated codons were found to be clustered in two regions of the cytochrome b protein which appeared to be responsible for the resistance to Qo site inhibitors. The first region is within the end of the first, the second, and the beginning of the third exon whereas the second region is within exon five and the beginning of the sixth exon.     

Sporulation of mitochondrial respiratory deficient mit- mutants of Saccharomyces cerevisiae.

Summary The role of mitochondrial protein synthesis, electron transport, and four specific mitochondrial gene products on sporulation were studied in respiratory deficient mit ^- mutants. These mutants were isolated in an op1 strain and localized on the mitochondrial genome by petite deletion mapping. All 153 mutations studied could be assigned to the four mitochondrial regions OXI1, OXI2, OXI3 and COB, known to affect cytochrome c oxidase and cytochrome b. The specific loss of one mitochondrially translated polypeptide was found in some mutants of each locus: OXI1—cytochrome c oxidase subunit 2, OXI2 — subunit 3, OXI3 — subunit 1, and COB — cytochrome b.The ability of diploid mit ^- mutants to sporulate was systematically investigated. About one third of the mutants, representing three loci, were incapable of forming spores. All other cultures produced either respiratory competent mit ⁺ tetrads, both mit ⁺ and mit ^- tetrads, or only mit ^- tetrads. Mutants forming mit ^- tetrads mapped in all four loci. These results demonstrate that in contrast to petite mutants some mit ^- mutants have retained the ability to perform meiosis and sporulation.     

Differential expression of alternative oxidase genes in maize mitochondrial mutants

We have examined the expression of three alternative oxidase (aox) genes in two types of maize mitochondrial mutants. Nonchromosomal stripe (NCS) mutants carry mitochondrial DNA deletions that affect subunits of respiratory complexes and show constitutively defective growth. Cytoplasmic male-sterile (CMS) mutants have mitochondrial DNA rearrangements, but they are impaired for mitochondrial function only during anther development. In contrast to normal plants, which have very low levels of AOX, NCS mutants exhibit high expression of aox genes in all nonphotosynthetic tissues tested. The expression pattern is specific for each type of mitochondrial lesion: the NADH dehydrogenase-defective NCS2 mutant has high expression of aox2, whereas the cytochrome oxidase-defective NCS6 mutant predominantly expresses aox3. Similarly, aox2 and aox3 can be induced differentially in normal maize seedlings by specific inhibitors of these two respiratory complexes. Translation-defective NCS4 plants show induction of both aox2 and aox3. AOX2 and AOX3 proteins differ in their ability to be regulated by reversible dimerization. CMS mutants show relatively high levels of aox2 mRNAs in young tassels but none in ear shoots. Significant expression of aox1 is detected only in NCS and CMS tassels. The induction pattern of maize aox genes could serve as a selective marker for diverse mitochondrial defects.     

Cytoplasmically inherited mutations of a human cell line resulting in deficient mitochondrial protein synthesis.

A large number of mutants deficient in mitochondrial protein synthesis (mtPS-) have been isolated from the human cell line VA2-B by subjecting cells partially depleted of their mtDNA to mutagenic treatments thought to be specific for mtDNA. Each of these mtPS- mutants has less than 10% of the wild-type rate of mitochondrial protein synthesis, exhibits reduced cytochrome oxidase and rutamycin sensitive ATPase activities, requires high concentrations of glucose, and grows indefinitely in the presence of 100 micrograms/ml of chloramphenicol (CAP). Fusion of cytoplasts from seven mtPS- mutants to the nucleated thioguanine-resistant VA2-B derivative TG-6 has yielded numerous cybrid clones which grow in CAP plus thioguanine, whereas almost no clones have resulted from the fusion of nucleated mtPS- cells to TG-6 cells: these results suggest that the gene(s) coding for the phenotype of mtPS- cells is localized in the cytoplasm (mtDNA?).     

Hartmanella culbertsoni: biochemical changes in the brain of the meningoencephalitic mouse.

Meningoencephalitis was produced in albino mice by intranasal inoculation of Hartmanella culbertsoni. In the infected brain phosphatidyl choline (PC), phosphatidyl ethanolamine + phosphatidyl serine (PE + PS), sphingomyelin and cholesterol registered decrease, lysophospholipids and free fatty acids accumulated, and part of the cholesterol was esterified. Phospholipase A (EC 3.1.1.4) and sphingomyelinase (EC 3.1.4.12) were elaborated in the postmitochondrial supernatant fraction. The levels of lipid peroxides, in brain as well as the capacity of brain homogenates to form lipid peroxides in vitro, was higher in the infected animals as compared to the uninfected. The activities of succinate dehydrogenase (EC 1.3.99.1) and cytochrome c oxidase (EC 1.9.3.1) in the mitochondrial fraction of the infected brain decreased while adenosine triphosphatase (EC 3.6.1.3) was stimulated. Addition of cytosol fraction (105,000g supernatant) of infected brain to the mitochondrial fraction of uninfected brain caused inhibition of succinate dehydrogenase and cytochrome c oxidase and stimulation of adenosine triphosphatase. This shows that some toxic substance(s) which inhibits mitochondrial function in brain is produced in the cytosol during infection. This inhibitor was nondialyzable and heat stable.