Nuclear cytochrome-deficient mutants of Neurospora crassa: isolation, characterization, and genetic mapping.

Summary We have isolated twenty-six nuclear, singlegene cytochrome-deficient mutants of Neurospora crassa as an initial step toward the study of the structural components and regulatory mechanisms involved in the biogenesis of the mitochondrial cytochrome system. These mutants, together with two previously described mutants, cyt-1 and cyt-2, have been classified into six distinct groups on the basis of cytochrome phenotype: a) cytochrome aa ₃ deficiency (due to mutations affecting loci designated cya); b) cytochrome b deficiency (cyb-1 locus); c) cytochrome b deficiency with a partial deficiency of cytochrome aa ₃ (cyb-2 locus); d) deficiency of both cytochromes aa ₃ and b (cyt loci); e) deficiency of both cytochromes aa ₃ and c (cyt-2 locus); and f) partial deficiency of cytochromes aa ₃ and c (cyt-12 locus).Four of seven mutations affecting cya loci have been mapped and are located on linkage groups I, II, V, and VI. It is not yet known whether these genes code for structural components of cytochrome oxidase or have a regulatory function that affects synthesis or assembly of the enzyme. The cyb-1 and cyb-2 genes are located on linkage groups V and VI, respectively, and appear to code for regulatory elements that control the biogenesis of cytochromes b and aa ₃ . The positions of the cyt mutations that cause a simultaneous deficiency of cytochromes aa ₃ and b are dispersed throughout the genome, except for two gene clusters on the left arm of linkage group I. Some of these mutants may be deficient in mitochondrial protein synthesis. Two mutations, cyt-2 and cyt-12, are located on linkage groups VI and II, respectively, and appear to affect genes that code for components of a regulatory system that controls the biogenesis of cytochromes aa ₃ and c.     

Nuclear suppressors of the [poky] cytoplasmic mutant in Neurospora crassa. III. Effects on other cytoplasmic mutants and on mitochondrial ribosome assembly in [poky].

Summary We have previously isolated six non-allelic, nuclear mutations (su ^I loci) that partially suppress the growth, respiratory and cytochrome abnormalities of the extranuclear [poky] mutant.A comparison of the mitochondrial ribosome profiles of suppressed and unsuppressed [poky] strains revealed that five of the six suppressors alleviate at least partially the deficiency of mitochondrial small ribosomal subunits that is associated with the [poky] genotype.Six independently isolated Group I extranuclear mutants, namely [exn-1], [exn-2], [exn-4], [stp-B ₁], [SG-1] and [SG-3], which have growth and cytochrome phenotypes similar to [poky], also were found to be deficient in small subunits of mitochondrial ribosomes. Using cytochrome aa ₃ and b production as a criterion for mitochondrial protein synthesis, it could be shown that the nuclear su ^I suppressors of [poky] also suppress the other six Group I extranuclear mutants. However, differences in the efficiencies of suppression by su ^I suppressors suggest that at least some of Group I extrachromosomal mutants are not simply re-isolates of [poky], but represent distinct extranuclear mutations.     

Isolation and Classification of Extranuclear Mutants of NEUROSPORA CRASSA

Four extranuclear mutants, [exn-1], [exn-2],[exn-4], and [stp-C], were obtained from N-methyl-N'-nitro-N-nitrosoguanidine-treated conidia and mycelium of Neurospora crassa. The three exn mutants grow with a pronounced lag from conidia and ascospores and are female fertile, whereas [stp-C] has a stop-start growth phenotype and is female sterile. The mitochondria from all four mutants are deficient in cytochromes a+a(3) and b, but contain an excess of cytochrome c. On the basis of growth and fertility, nuclear suppressors and complementation in heteroplasmons, 16 of the extranuclear mutants now available in Neurospora can be divided into three groups. Group I consists of 8 female-fertile variants with both poky-like growth and cytochrome defects. Their slow growth is suppressed by the nuclear factor, f, but not by a second nuclear suppressor, su-1([mi-3]). They complement with group III mutants in mixed cytoplasmons. Group II is represented by a single variant, [mi-3]. It is phenotypically modified by the su-1([mi-3]) factor, but not by f. Its unique cytochrome spectrum shows a deficiency of cytochrome a, but c and b are present. It complements in heteroplasmons with group I and III mutants. Group III included 7 female-sterile variants with stopper growth phenotypes and the same cytochrome defects as group I. Group III mutants complement both with group I and II isolates, but they are unaffected by either f or su-1.     

Growth,respiration and cytology of acetate-negative mutants ofCandida albicans

A number of acriflavine-induced mutants ofCandida albicans, characterized by their inability to grow on acetate as a source of energy, were screened for their cytochrome absorption spectra. Three mutants with different spectra, along with their parent, were selected for comparative studies, of their growth, respiratory activities and cellular structure. The spectrum of one of the mutants was the same as that of the wild-type, but the growth rate and yield of cells on glucose medium were only about 60% of the wild-type's; those of a second mutant deficient in cytochromes aa₃ were 50%, and those of a third mutant deficient in cytochromes aa₃ and b were less than 5% of those of the wild-type. The cytochrome-complete mutant and the wild-type showed respiratory activity both on glucose and ethanol well above the endogenous, the cytochrome aa₃-deficient mutant showed only endogenous respiration, and the cytochrome aa₃, b-deficient mutant no respiration at all. Electron microscopy of the wild-type cells revealed discrete, regular ovoidal, cristate mitochondria spaced near the periphery of the protoplasm; the cytochrome-complete mutant showed an abundance of large, cristate, but morphologically irregular mitochondria; the cytochrome aa₃-deficient mutant had fewer but still large, cristate, somewhat irregular mitochondria; and the cytochrome aa₃, b-deficient mutant only a few simple vesicles without discernible cristae.     

A temperature-sensitive mutant of Neurospora crassa deficient in cytochrome b.

Summary A nuclear gene mutant of Neurospora crassa designated cyb-3 is deficient in cytochrome b and coenzyme QH₂-cytochrome c reductase. Nearly normal when grown at 25°C, the strain expresses a mutant phenotype at 38°C. Mitochondria from cybr-3 mycelium, which has undergone 3–4 mass doublings at the elevated temperature, possess 3-fold less cytochrome b, 2-fold more cytochrome, c, 5-fold less coenzyme QH₂-cytochrome c reductase activity, and require 3-fold less antimycin A per milligram of protein to inhibit NADH oxidation than do wild type mitochondria. The activity of coenzyme QH₂-cytochrome c reductase declines rather slowly in cultures of cyb-3 transferred to 38° C, and the in vitro thermostability of the enzyme is very similar in wild type and mutant mitochondria. Therefore, the mutation may decrease synthesis or impair integration into the membrane of cytochrome b and perhaps other proteins of the enzyme complex.Contribution No. 1294-j, Division of Biology, Agricultural Experiment Station, Kansas State University, Manhattan, Kansas.     

Genetic interactions in the control of mitochondrial functions in Paramecium

Summary The genetic and physiological properties of two nuclear mutants of Parameccium tetraurelia affecting mitochondrial properties, and first screened as resistant to tetrazolium (TTC) are described. The mutant TTC _64-1 ^R is strongly deficient in cytochrome c and the mutant TTC _66p ^R is partially deficient in cytochrome aa₃; both mutants display cyanide insensitive respiration in exponential growth phase. In the double mutant TTC _64-1 ^R -TTC _66p ^R /TTC _64-1 ^R -TTC _66p ^R the deficiency in cytochrome aa₃ due to the TTC _64-1 ^R mutation is suppressed. The mutation TTC _64-1 ^R does not suppress cytochrome aa₃ deficiencies due to mitochondrial mutations, but does interact with another nuclear mutation, cl ₁, (compatible only with mitochondria deficient in cytochrome oxidase) in such a way that the double mutant TTC _64-1 ^R -cl ₁/TTC _64-1 ^R -cl ₁ displays a normal amount of cytochrome aa₃. The possible mechanisms and physiological significance of these suppressive effects are discussed.Abbreviations TTC^R/TTC^S resistant/sensitive to tetrazolium - KCN^R/KCN^S cyanide insensitive/sensitive respiration - aa ₃ ^- /aa ₃ ⁺ deficient/normal amount of cytochrome aa₃ - c^-/c⁺ deficient/normal amount of cytochrome c     

Evidence that energy conserving electron transport pathways to nitrate and cytochrome o branch at ubiquinone in Paracoccus denitrificans

The organisation and function of electron transport pathways in Paracoccus denitrificans has been studied with both inhibitors and electrode probes for O₂ or N₂O respiration and membrane potential. Myxothiazol completely inhibits electron flow through the cytochrome bc₁ region of the electron transport chain, as judged by its effect on nitrous oxide respiration. Electron flow to oxygen via the cytochrome o oxidase was shown to be insensitive to myxothiazol in a mutant, HUUG 25, that lacks cytochrome c and in which the aa₃ oxidase is therefore inactive. Myxothiazol did not inhibit nitrate reduction. It is concluded that myxothiazol is a specific inhibitor of electron flow through the cytochrome bc₁ region and more potent than antimycin which does not give complete inhibition.As neither antimycin nor myxothiazol, nor a combination of the two, inhibits electron transport to either nitrate reductase or cytochrome o it is concluded that electron transport pathways to these enzymes do not involve the cytochrome bc₁ region but rather branch at the level of ubiquinone. Although the cytochrome o pathway branches at ubiquinone, it is associated with the generation of a protonmotive force; this is shown by measurements of membrane potential in vesicle preparations from the mutant HUUG 25.In contrast to antimycin and myxothiazol, the ubiquinone analogues 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and 2-n-undecyl-3-hydroxy-1,4-naphthoquinone (UHNQ) inhibit electron flow through both the cytochrome bc₁ complex and the cytochrome o pathway, although a higher titre is required in the latter case. These two inhibitors were without effect on the nitrate reductase pathway. Thus myxothiazol is the inhibitor of choice for selective and complete inhibition of cytochrome bc₁.Recently published schemes for electron transport in P. denitrificans are analysed.Non standard abbreviations S-13 2,5-dichloro-3-t-butyl-4-nitrososalicylanilide - UHNQ 2-n-undecyl-3-hydroxy-1,4-naphthoquinone - UHDBT 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole     

Nuclear mutants of Neurospora crassa temperature-sensitive for the synthesis of cytochrome aa3. I. Isolation and preliminary characterization.

Summary Three nuclear mutants of Neurospora crassa, temperature-sensitive for the synthesis of cytochrome aa ₃ have been isolated. When grown at 41°C the mutants have large amounts of KCN-insensitive respiration, reduced amounts of cytochrome aa ₃ and cytochrome c oxidase activity, and grow more slowly than wild-type cultures grown at the same temperature. When the mutants are grown at 23°C, they are virtually indistinguishable from wild-type strains.The mutants were selected on the basis of their slow growth at 41°C in medium containing salicylhydroxamic acid, and by their inability to reduce 2,3,5-triphenyltetrazolium chloride at 41°C. The selection technique was designed to eliminate mutants that did not carry thermolabile electron transport chain components. However, studies on the thermolability of the cytochrome oxidase activity in isolated mitochondria indicate that the enzyme of the mutants is no more susceptible to heat denaturation than is the enzyme in wild-type mitochondria. This suggests that the synthesis or assembly of cytochrome aa ₃ may be altered in the mutants at the restrictive temperature.Supported by National Research Council of Canada Grant Number A-6351Recipient of a National Research Council of Canada Postgraduate Scholarship     

An alternate respiratory pathway in Candida albicans

Usual concentrations of antimycin A, rotenone and EDTA, individally or in combination, reduced aerobic growth rate and cell yield of Candida albicans to about half its normal level and to about the levels of previously-described acetate-negative, cytochrome-complete and aa₃-deficient variants which were little affected by the inhibitors. Anaerobic conditions (not affected by antimycin A) reduced growth rate and cell yield of all cultures-including that of a nonrespiring aa₃, b-deficient mutant-to low, equal levels. Antimycin A but not rotenone prevented growth of the normal strain on ethanol medium. Cyanide and antimycin A blocked most of the respiration of the normal strain and cytochrome-complete variant, but did not affect that of the cytochrome aa₃-deficient mutant. Rotenone and EDTA did not affect respiration of any of the cultures. SHAM blocked cyanide- and antimycin A-insensitive respiration and prolonged the lag phases of the three respiring cultures, especially in the presence of antimycin A, but alone increased oxygen-uptake rate of the cytochromecomplete cultures while curtailing that of the cytochrome aa₃-deficient mutant. Resting cells, especially wild-type, grown in medium containing antimycin A exhibited lowered oxygen-uptake rate, which was increased upon the addition of cyanide or antimycin A. Antimycin A stimulated, but cyanide inhibited, respiration of cytochrome-complete cultures grown in the presence of rotenone but did not affect that of the cytochrome aa₃-deficient mutant. SHAM inhibited respiration of all antimycin A- or rotenone-grown cultures. The high rate of respiration of C. albicans in the presence of inhibitors for three sites of electron transport in the conventional oxidative pathway, the inhibition of this respiration by SHAM and its loss by the absence of cytochrome b, indicate an alternate oxidative pathway in this organism which crosses the conventional one at cytochrome b.This work was supported by Public Health Service Graduate Dental Training Grant DE 00144 and the Graduate School and the Department of Microbiology, Southern Illinois University.     

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.     

Oxidative phosphorylation in yeast X. Phosphorylation ability of mutants deficient in cytochromes a and b

1. Mitochondria from three non-allelic mutants of Saccharomyces cerevisiae, each lacking cytochrome aa₃ as a consequence of single nuclear gene mutation, exhibited oxidative phosphorylation with ferricyanide as electron acceptor with the same efficiency as wild-type yeast mitochondria.     

Functional mitochondria in snake Bothrops alternatus erythrocytes and modulation of HbO2 affinity by mitochondrial ATP

Digitonin was applied to permeabilize the plasma membrane of Bothrops alternatus erythrocytes to study respiration, oxidative phosphorylation and Ca²⁺ transport by mitochondria in situ. These mitochondria oxidized added NAD-linked substrates, succinate and N,N,N, N-tetramethyl-p-phenylenediamine. Respiration was sensitive to rotenone and cyanide but not to antimycin A. This indicates that Bothrops mitochondria possess the respiratory complexes NADH-ubiquinone, succinate-ubiquinone, and ferrocytochrome c-oxygen oxidoreductases, although the lack of sensitivity to antimycin A raises doubt about the composition of the ubiquinol cytochrome c-reductase complex. An ability to build up and sustain a membrane potential was documented by their capacity to accumulate tetraphenylphosphonium and Ca²⁺ through an uncoupler-sensitive mechanism. Addition of ADP caused a transient decrease in the membrane potential, indicating that this is the predominant driving force for ATP synthesis as in most types of mitochondria. Uncoupling of phosphorylation from the oxidative process increased hemoglobin O₂ affinity, which suggests that ATP production by mitochondria may participate in modulation of O₂ transport by hemoglobin.Abbreviations membrane potential - BAE Bothrops alternatus erythrocytes - DNP 2,4-dinitrophenol - DPG 2,3-diphosphoglycerate - EGTA ethyleneglycol tetra-acetic acid - FCCP carbonylcyanide p-trifloromethoxyphenylhydrazone - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - TPP⁺ tetraphenylphosphonium - TRIS tris-(hydroxymethyl)aminomethane     

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1 I. Spectral properties and redox potentials

1. Purified mitochondria have been prepared from wild type Paramecium tetraurelia and from the mutant Cl₁ which lacks cytochrome aa₃. Both mitochondrial preparations are characterized by cyanide insensitivity. Their spectral properties and their redox potentials have been studied.2. Difference spectra (dithionite reduced minus oxidized) of mitochondria from wild type P. tetraurelia at 77 K revealed the α peaks of b-type cytochrome(s) at 553 and 557 nm, of c-type cytochrome at 549 nm and a-type cytochrome at 608 nm. Two α peaks at 549 and 545 nm could be distinguished in the isolated cytochrome c at 77 K. After cytochrome c extraction from wild type mitochondria, a new peak at 551 nm was unmasked, probably belonging to cytochrome c₁. The a-type cytochrome was characterized by a split Soret band with maxima at 441 and 450 nm. The mitochondria of the mutant Cl₁ in exponential phase of growth differed from the wild type mitochondria in that cytochrome aa₃ was absent while twice the quantity of cytochrome b was present. In stationary phase, mitochondria of the mutant were characterized by a new absorption peak at 590 nm.3. Cytochrome aa₃ was present at a concentration of 0.3 nmol/mg protein in wild type mitochondria and ubiquinone at a concentration of 8 nmol/mg protein both in mitochondria of the wild type and the mutant Cl₁. Cytochrome aa₃ was more susceptible to heat than cytochromes b and c,c₁.4. CO difference spectra at 77 K revealed two different Co-cytochrome complexes. The first, found only in wild type mitochondria, was a typical CO-cytochrome a₃ complex characterized by peaks at 596 and 435 nm and troughs at 613 and 450 nm. The second, found both in mitochondria of the wild type and the mutant, was a CO-cytochrome b complex with peaks at 567, 539 and 420 nm and a trough at 558-549 nm. Both complexes are photo-dissociable.5. Spectral evidence was obtained for interaction of cyanide with the a-type cytochrome (shift of the α peak at 77 K from 608 to 605 nm), but not with the b-type cytochrome.6. The mid-point potentials of the different cytochromes at neutral pH are as follows: cytochrome aa₃ 235 and 395 mV, cytochrome c,c₁ 233 mV, cytochromes b 120 mV.     

Biochemical and biophysical studies on cytochrome aa3. VI. Reaction of cyanide with oxidized and reduced enzyme

1.

1. The reaction of cyanide with cytochrome aa₃ in the fully oxidized and reduced states has been studied. In both cases a single molecule of cyanide is bound reversibly per molecule of aa₃ (that is, 1 mole cyanide per 2 equivalents of haem a).     

Cytochrome aa 3 from Methylococcus capsulatus (Bath)

A cytochrome aa ₃-type oxidase was isolated with and without a c-type cytochrome (cytochrome c-557) from Methylococcus capsulatus Bath by ion-exchange and hydrophobic chromatography in the presence of Triton X-100. Although cytochrome c-557 was not a constitutive component of the terminal oxidase, the cytochrome c ascorbate-TMPD oxidase activity of the enzyme decreased dramatically when the ratio of cytochrome c-557 to heme a dropped below 1:3. On denaturing gels, the purified enzyme dissociated into three subunits with molecular weights of 46,000, 28,000 and 20,000. The enzyme contains two heme groups (a and a ₃), absorption maximum at 422 nm in the resting state, at 445 and 601 nm in the dithionite reduced form and at 434 and 598 nm in the dithionite reduced plus CO form. Denaturing gels of the cytochrome aa ₃-cytochrome c-557 complex showed the polypeptides associated with cytochrome aa ₃ plus a heme c-staining subunit with a molecular weight of 37,000. The complex contains approximately two heme a, one heme c, absorption maximum at 420 nm in the resting state and at 421, 445, 522, 557 and 601 nm in the dithionite reduced form. The specific activity of the purified enzyme was 130 mol O₂/min · mol heme a compared to 753 mol O₂/min · mol heme a when isolated with cytochrome c-557.Abbreviations MMO methan monooxygenase - sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - TMPD N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride - Na₂EDTA disodium ethylenediamine-tetraacetic acid     

Properties of the menaquinol oxidase (Qox) and of qox deletion mutants of Bacillus subtilis

Menaquinol oxidase isolated from the membrane of Bacillus subtilis W23 was found to consist of four polypeptides (QoxA, B, C, and D) that were predicted by the sequence of the qox operon of B. subtilis 168 (Santana et al. 1992). The preparation contained 7 mol cytochrome aa ₃ per g protein, which corresponds to 2mol heme A per mol enzyme of 144 kDa molecular mass. Respiration with dimethylnaphthoquinol catalyzed by the enzyme was ten times faster than that with menadiol. Activities with more electropositive quinols were negligible. The activity of the enzyme was inhibited by equimolar amounts of HQNO, while antimycin, myxothiazol, and stigmatellin were more than tenfold less effective. When cells of both strains of B. subtilis (W23 and 168) were grown with glucose, quinol respiration was an order of magnitude more active than respiration with N,N,N,N-tetramethyl-1,4-phenylenediamine plus ascorbate. Surprisingly, the same result was obtained with mutant strains lacking qoxB. As cytochromes a and d were virtually absent, a second quinol oxidase, possibly of the cytochrome o-type, was apparently formed by the mutants.Abbreviations cat Chloramphenicol resistance gene - cta Cytochrome oxidase genes - DMN 2,3-Dimethyl-1,4-naphthoquinone - DMNH ₂ Reduced DMN - HQNO 2-(n-Heptyl)-4-hydroxyquinoline-N-oxide - qox Quinol oxidase genes - TMPD N,N,N,N-tetramethyl-1,4-phenylenediamine