Localization of mucidin-resistant locus muc3 on mitochondrial DNA with respect to ubiquinol-cytochrome c reductase deficient box loci. Locus muc3 is allelic to box2

Summary Genetic relations between mitochondrial mucidin-resistant locus muc3 and ubiquinol-cytochrome c reductase-deficient box loci have been studied by recombination and petite deletion analysis. It was found that the locus muc3 maps in the segment of mitochondrial DNA corresponding to the locus box2. The results suggest the participation of box2/muc3 locus in the sequences of the structural gene for cytochrome b.     

Crystallization of mitochondrial ubiquinol-cytochrome c reductase.

Ubiquinol-cytochrome c reductase of beef heart mitochondria was crystallized in the presence of decanoyl-N-methylglucamide, heptanetriol, and sodium chloride with poly(ethylene glycol) as precipitant. The largest crystal has dimensions of 4 x 2 x 1 mm. The crystalline enzyme is composed of 10 subunits. It contains 2.5 nmol of ubiquinone, 8.4 nmol of cytochrome b, 4.2 nmol of cytochrome c1, 4.2 nmol of iron-sulfur cluster, and 140 nmol of phospholipid per milligram of protein. Of the last, 36% is with diphosphatidylglycerol. The crystals are very stable in the cold and show full enzymatic activity when redissolved in aqueous solution. Absorption spectra of the redissolved crystals show a Soret to UV ratio of 0.88 and 1.01 in the oxidized and the reduced forms, respectively.     

Genetic localization of diuron- and mucidin-resistant mutants relative to a group of loci of the mitochondrial DNA controlling coenzyme QH2-cytochrome c reductase in Saccharomyces cerevisiae.

Summary Diuron-resistance, DIU (Colson et al., 1977), antimycin-resistance, ANA (Michaelis, 1976; Burger et al., 1976), funiculosin-resistance, FUN (Pratje and Michaelis, 1977; Burger et al., 1977) and mucidin-resistance, MUC (Subik et al., 1977) are each coded by a pair of genetic loci on the mit DNA of S. cerevisiae. In the present paper, these respiratory-competent, drug-resistant loci are localized relative to respiratory-deficient BOX mutants deficient in coenzyme QH₂-cytochrome c reductase (Kotylak and Slonimski, 1976, 1977) using deletion and recombination mapping. Three drug-resistant loci possessing distinct mutated allelic forms are distinguished. DIU1 is allelic or closely linked to ANA2, FUN1 and BOX1; DIU2 is allelic or closely linked to ANA1, MUC1 and BOX4/5; MUC2 is allelic to BOX6. The high recombinant frequencies observed between the three loci (13% on the average for 33 various combinations analyzed) suggest the existence of either three genes coding for three distinct polypeptides or of a single gene coding for a single polypeptide but subdivided into three easily separable segments. The resistance of the respiratory-chain observed in vitro in the drug-resistant mutants and the allelism relationships between respiratory-competent, drug-resistant loci and coQH₂-cyt c reductase deficient, BOX, loci strongly suggest that each of the three drug-resistant loci codes for a structural gene-product which is essential for the normal coQH₂-cyt c reductase activity and is obviously a good candidate for a gene product of the drug-resistant loci mapped in this paper. Polypeptide length modifications of cytochrome b were observed in mutants deficient in the coQH₂-cyt c red and localized at the BOX1, BOX4 and BOX6 genetic loci (Claisse et al., 1977, 1978) which are precisely the loci allelic to drug resistant mutants as shown in the present work. Taken together these two sets of data provide a strong evidence in favor of the idea that there exist three non contiguous segments of the mitochondrial DNA sequence which code for a single polypeptide sequence of cytochrome b. In each segment mutations which modify the polypeptide sequence can occur leading to the loss (BOX mutants) or to a modification (drug resistant mutants) of the enzyme activity.Chercheur qualifié du Fonds National de la Recherche Scientifique     

The nature of the inhibition of 4,7-dioxobenzothiazole derivatives on mitochondrial ubiquinol-cytochrome c reductase

To investigate the inhibitory action and binding site of a quinone-like molecule, 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), a series of 4,7-dioxobenzothiazole derivatives were synthesized and their inhibitory efficiencies studied. Replacing the 6-hydroxyl or 2-hydrogen of UHDBT with a bromo or a methoxy group causes only a slight decrease in inhibitory efficiency, indicating that the 6-hydroxyl or the 2-hydrogen of UHDBT is not a structural requirement for inhibition. 5-Undecyl-6-bromo (or methoxy)-4,7-dioxobenzothiazole shows a pH-dependent inhibition similar to that observed with UHDBT, suggesting that the pH dependence is due to the presence of a dissociable group in the protein complex and not to the deprotonation of the hydroxyl group of the inhibitor. Replacing the 6-hydroxyl group with an azido group causes changes similar to those observed with UHDBT; the inhibition is accompanied by alteration of the epr characteristics of reduced iron-sulfur protein in ubiquinol-cytochrome c reductase. The extent of inhibition is not changed upon illumination of the treated reductase. When the photolyzed, 6-azido-5-(1',2'-[3H] undecyl)-4,7-dioxobenzothiazole [( 3H]6-azido-UDBT)-treated reductase is subjected to organic solvent extraction, no radioactivity is found in the reductase protein. Rather, the radioactivity is located in the phospholipid fraction. A [3H]azido-UDBT-cardiolipin adduct, identified after separation of the phospholipid fraction by high performance liquid chromatography, has 6-azido-UDBT linked to an acyl group, not to the head group of the cardiolipin molecule. These results suggest that inhibition by UHDBT is due to perturbation of specific cardiolipin molecules in ubiquinol-cytochrome c reductase. Since UHDBT and 6-azido-UDBT also inhibit the ubiquinol-cytochrome c reductase activity of delipidated reductase (10% of the original lipid remaining) assayed after reconstitution with ubiquinone and phospholipid, and the [3H]azido-UDBT-cardiolipin adduct is also found in the delipidated reductase, the UHDBT-perturbed cardiolipin molecule is structurally indispensable to reductase and it tightly bound to the reductase protein, most likely the quinone binding proteins.     

Immunochemical study of subunit VI (Mr 13,400) of mitochondrial ubiquinol-cytochrome c reductase.

A preparation containing the Mr 13,400 protein (subunit VI), phospholipid, and ubiquinone was isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase by a procedure involving Triton X-100 and urea solubilization, calcium phosphate-cellulose column chromatography at different pHs, acetone precipitation, and decanoyl-N-methylglucamide-sodium cholate extraction. The protein in this preparation corresponds to subunit VI of ubiquinol-cytochrome c reductase resolved in the sodium dodecyl sulfate-polyacrylamidce gel electrophoresis system of Sch?gger et al. (1987, FEBS Lett. 21, 161-168) and has the same amino acid sequence as that of the Mr 13,400 protein reported by Wakabayashi et al. (1985, J. Biol. Chem. 260, 337-343). The phospholipid and ubiquinone present in the preparation copurify with but are not intrinsic components of, the Mr 13,400 protein. This preparation has a potency and behavior identical to that of a free phospholipid preparation in restoring activity to delipidated ubiquinol-cytochrome c reductase. Antibodies against Mr 13,400 react only with Mr 13,400 protein and complexes which contain it. They do not inhibit intact, lipid-sufficient ubiquinol-cytochrome c reductase. However, when delipidated ubiquinol-cytochrome c reductase is incubated with antibodies prior to reconstitution with phospholipid, a 55% decrease in the restoration activity is observed, indicating that the catalytic site-related epitopes of the Mr 13,400 protein are buried in the phospholipid environment. Antibodies against Mr 13,400 cause an increase of apparent Km for ubiquinol-2 in ubiquinol-cytochrome c reductase. When mitoplasts or submitochondrial particles are exposed to a horseradish peroxidase conjugate of the Fab' fragment of anti-Mr 13,400 antibodies, peroxidase activity is found mainly in the submitochondrial particles preparation; little activity is detected in mitoplasts. This suggests that the Mr 13,400 protein is extruded toward the matrix side of the membrane.     

Interactions of cytochrome c with mitochondrial membranes. Binding to succinate-cytochrome c reductase

Methyl-4-azidobenzoimidate was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c bound to cytochrome c-depleted mitochondria with the same Kd as native cytochrome c and restored oxygen uptake to the same extent. Irradiation of cytochrome c-depleted mitochondrial membranes with 3- to 4-fold excess of photoaffinity-labeled cytochrome c over cytochrome c oxidase resulted in covalent binding of the derivative to the membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on an agarose Bio-Gel-A-5m showed that the labeled cytochrome c was bound covalently to succinate-cytochrome c reductase. The covalently bound cytochrome c was active in mediating electron transfer between its reductase and oxidase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the succinate-cytochrome c reductase containing photoaffinity-labeled 125I-cytochrome c showed that the reductase contained a protein binding site for cytochrome c. It is suggested that cytochrome c1 is the most likely site for the cytochrome c binding in mitochondria in situ.     

Localization of 11q13 loci with respect to regional chromosomal breakpoints

We have employed two strategies to map 13 markers located at 11q13. First, we used pulsed-field gel electrophoresis of DNA fragments obtained with methylation-sensitive restriction enzymes. The markers used in this study were scattered over 8.4 Mb and, for most of them, could not be linked one to another. A second mapping strategy employed hybridization to either DNA of somatic hybrids containing various parts of the long arm of chromosome 11 or metaphase chromosomes of a B-cell line containing the t(11;14)(q13;q32) translocation. We were able to sort out the centromeric from the telomeric probes with respect to translocation breakpoints taken as reference chromosomal landmarks by this approach. BCL1, which corresponds to the region where the t(11;14)(q13;q32) translocation breakpoints are clustered, appears as a boundary between two areas of human/mouse homology present in conserved syntenic regions on mouse chromosomes 7 and 19.     

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.     

Localization of tryptophan residues in NADPH-adrenodoxin reductase with respect to the NADPH-binding center

It was demonstrated that the NADPH-adrenodoxin reductase molecule contains ten tryptophan residues titrated by N-bromosuccinimide. The effectiveness of the non-radiant energy transfer was used to calculate the average distance between the NADPH-binding site of the enzyme and tryptophan residues at different steps of N-bromosuccinimide-induced modification.     

Hematoporphyrin-promoted photoinactivation of mitochondrial ubiquinol-cytochrome c reductase: selective destruction of the histidine ligands of the iron-sulfur cluster and protective effect of ubiquinone.

Purified ubiquinol-cytochrome c reductase of beef heart mitochondria is very stable in aqueous solution; it suffers little damage upon illumination with visible light under aerobic or anaerobic conditions. However, it is rapidly inactivated when the photosensitizer hematoporphyrin is present during illumination. The hematoporphyrin-promoted photoactivation is dependent on sensitizer dose, illumination time, and oxygen. Singlet oxygen is shown to be the destructive agent in this system. The photoinactivation of ubiquinol-cytochrome c reductase is prevented by excess exogenous ubiquinone, regardless of its redox state. This protective effect is not due to protein-ubiquinone interactions but to the singlet oxygen scavenger property of ubiquinone. Ubiquinone also protects against hematoporphyrin-promoted photoinactivation of succinate-ubiquinone reductase and cytochrome c oxidase. The photoinactivation site in ubiquinol-cytochrome c reductase is the iron-sulfur cluster of Rieske's protein. Two histidine residues, presumably serving as two ligands for the iron-sulfur cluster of Rieske's protein, are destroyed. No polypeptide bond cleavage is detected. Photoinactivation has little effect on the spectral properties of cytochromes b and c1 but alters their reduction rates substantially. this photoinactivation also causes the formation of proton-leaking channels in the complex. When the photoinactivated reductase is co-inlaid with intact ubiquinol-cytochrome c reductase or cytochrome c oxidase in a phospholipid vesicle, no proton ejection can be detected during the oxidation of their corresponding substrates.     

Comparative kinetic studies of cytochromes c in reactions with mitochondrial cytochrome c oxidase and reductase.

Kinetic studies of the reactions of selected eukaryotic and prokaryotic cytochromes c with mitochondrial cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase (EC 1.9.3.1) using a standardized complex IV preparation from beef heart are reported. Data on reactions with NADH-linked cytochrome c reductase (complexes I and III) are included. The concentration ranges employed provide a basis for quantitative demonstration of a general rate law applicable to oxidase reactions of cytochrome c of greatly differing reactivities. Results are interpreted on the basis of a modified Minnaert mechanism (Minnaert, K. (1961) Biochim. Biophys. Acta 50, 23), assuming productive complex formation between cytochrome c and free oxidase in addition to further complex binding of a second cytochrome c molecule to the initially formed oxidase complex. Kinetic constants so obtained are consistent with the assumption that binding is the dominant parameter in reactivity, and can be rationalized most simply on this basis.     

The biosynthesis of the ubiquinol-cytochrome c reductase complex in yeast. DNA sequence analysis of the nuclear gene coding for the 14-kDa subunit

The nuclear gene coding for the imported 14-kDa subunit of the ubiquinol-cytochrome c reductase of yeast mitochondria has been sequenced in an attempt to define regulatory and protein topogenic elements. The gene has a length of 381 base pairs and is potentially capable of encoding a polypeptide of 14561 Da. It is transcribed into a single low-abundance RNA of 680 nucleotides whose 5' and 3' termini map, respectively, 30-35 nucleotides upstream and 180-190 nucleotides downstream of the initiator and termination codons. Consistent with the estimated low level of the mRNA, codon usage in the gene is not strongly biased and other features, characteristic of highly expressed genes in yeast, are absent. The 14-kDa protein is predicted to be a predominantly hydrophilic protein, with only a single, short hydrophobic stretch located between positions 19-38. Comparison with other imported mitochondrial proteins so far sequenced has failed to reveal unifying features that might serve as targeting elements. Steady-state levels of the 14-kDa and 11-kDa subunits are reduced in mit- mutants which synthesize truncated forms of apocytochrome b and in these, newly synthesized subunits exhibit a specifically increased turnover rate. We suggest that association of these two subunits with the complex may be mediated or enhanced by interaction with other subunits, in particular cytochrome b.     

Isolation and RNA sequence analysis of cytochrome b mutants resistant to funiculosin, a center i inhibitor of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae

Funiculosin is a well-known inhibitor of the mitochondrial respiratory chain, probably acting at the ubiquinone reducing site or center i of QH2-cytochrome c reductase. We report here the isolation, mapping and RNA sequence analysis of yeast apo-cytochrome b mutants resistant to this inhibitor. Funiculosin-resistance was found to be conferred, in 4 independent isolates, upon replacement of a leucine residue by phenylalanine in position 198 of the cytochrome b polypeptide chain.     

Assembly of the mitochondrial membrane system. Physical map of the Oxi3 locus of yeast mitochondrial DNA

The oxi3 locus of yeast mitochondrial DNA is currently thought to code for Subunit 1 of cytochrome oxidase (Tzagoloff, A., Macino, G., and Sebald, W. (1979) Annu. Rev. Biochem. 48, 419-441). The respiratory competent strain of Saccharomyces cerevisiae D273-10B/A48 was used to obtain cytoplasmic "petite" clones enriched for genetic markers in the oci3 locus. The most complex clone studied (DS6) was ascertained to have a mitochondrial genome with a tandemly repeated segment of mtDNA 16.5 kilobases in length. The oxi3 locus was dissected by mutagenesis of DS6 with ethidium bromide and selection of new clones having less complex genotypes. Six derivative clones with genome sizes ranging from 2.3 to 6.1 kilobases have been extensively analyzed. Most of the restriction sites present in the segments of mtDNA retained by the clones have been mapped, thereby providing a detailed restriction map of the oxi3 gene. Based on the physical locations of the most distal oxi3 mutations, the gene spans approximately 10,000 nucleotides and occupies the region of wild type mtDNA from 44 to 58 map units.     

Mapping of mutation tsm-8 with respect to transfer RNA genes on the mitochondrial DNA of Saccharomyces cerevisiae.

Summary A precise localization of thetsm-8 mutation in relation to the transfer RNA genes has been attempted by rho^- deletion analysis. The data show that thetsm-8 mutation is in close proximity to the isoleucyl transfer RNA gene. However, it is not yet possible to decide whether thetsm-8 mutation is within this transfer RNA gene.     

Localization of a bidirectional DNA replication origin in the native locus and in episomally amplified murine adenosine deaminase loci.

Gene amplification is frequently mediated by the initial production of acentric, autonomously replicating extrachromosomal elements. The 4,000 extrachromosomal copies of the mouse adenosine deaminase (ADA) amplicon in B-1/50 cells initiate their replication remarkably synchronously in early S phase and at approximately the same time as the single-copy chromosomal locus from which they were derived. The abundance of ADA sequences and favorable replication timing characteristics in this system led us to determine whether DNA replication initiates in ADA episomes within a preferred region and whether this region is the same as that used at the corresponding chromosomal locus prior to amplification. This study reports the detection and localization of a discrete set of DNA fragments in the ADA amplicon which label soon after release of synchronized B-1/50 cells into S phase. A switch in template strand complementarity of Okazaki fragments, indicative of the initiation of bidirectional DNA replication, was found to lie within the same region. This putative replication origin is located approximately 28.5 kbp upstream of the 5' end of the ADA gene. The same region initiated DNA replication in the single-copy ADA locus of the parental cells. These analyses provide the first evidence that the replication of episomal intermediates involved in gene amplification initiates within a preferred region and that the same region is used to initiate DNA synthesis within the native locus.