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A M Colson

Publications and source records attributed to A M Colson.

At least 19 recordsLinked to original sources
PubMed1995

Structure-function relationships of the mitochondrial bc1 complex in temperature-sensitive mutants of the cytochrome b gene, impaired in the catalytic center N.

Seven new structures of cytochrome b have been recently identified by isolating and sequencing revertants from cytochrome b respiratory deficient mutants (Coppée, J. Y., Brasseur, G., Brivet-Chevillotte, P., and Colson, A. M. (1994) J. Biol. Chem. 269, 4221-4226). These mutations are located in the center N domain (QN). All the revertants exhibited a modified heme b562 maximum, confirming that part of the NH2-terminal region is in the vicinity of the extramembranous loop between helices IV-V and heme b562. Based on measurements performed on the maximal activities occurring in each segment of the respiratory chain, the decrease observed in the NADH oxidase activities of several revertants was correlated with some bc1 complex activity impairments; this may also explain why a moderate decrease in bc1 complex activity does not limit the succinate oxidase activity. The decrease in the rate of reduction of cytochrome b via the center N pathway is responsible for the impairment of the bc1 complex activity of these revertants. The three double-mutated revertants (S206L/N208K or -Y; S206L/W30C) are temperature-sensitive in vivo, and their mitochondria like that of the original mutant S206L are thermosensitive in vitro. Isolating the W30C mutation does not yield a thermosensitive phenotype: the replacement of serine 206 by leucine is therefore responsible for the thermoinstability of these strains; this temperature sensitivity is reinforced by additional mutations N208K or N208Y, and not by W30C. These data suggest that serine 206 and asparagine 208 are involved in the thermostability of the protein. When bc1 complex activity is lost after incubating mitochondria at a nonpermissive temperature (37 degrees C), heme b is still present, but can no longer be reduced by physiological substrate. The progressive loss of bc1 complex activity seems to be initially linked to a change in the tertiary structure of cytochrome b, which occurs drastically at center N and much more slowly at center P, as shown by kinetic study on the two cytochrome b redox pathways.

Cytochrome b Group↗
PubMed1995

The topology of CuA in relation to the other metal centres in cytochrome-c oxidase of Saccharomyces cerevisiae as determined by analysis of second-site reversions.

Second-site revertants were selected from a respiratory-deficient mutant carrying the mutation D369N located in a loop between helices IX and X close to H376 and H378, the proposed ligands of haem a3 and haem a, respectively. A reversion was observed in subunit II, in the vicinity of the CuA ligands. This same reversion compensates the subunit I deficiency mutation, S140L, assumed to be near H62, the second putative histidine ligand to haem a. These data enable us to propose a three-dimensional topology in which CuA in subunit II is located on top of the Positive-side of subunit I and in proximity to all three of its metal centres.

Binding Sites↗
PubMed1994

Analysis of revertants from respiratory deficient mutants within the center N of cytochrome b in Saccharomyces cerevisiae.

Four modified cytochrome b's carrying mononucleotide substitutions affecting center N residues were analysed. The mutant carrying a G33D change does not incorporate heme into the apocytochrome b and fails to grow on non-fermentable carbon sources. Out of 85 genetically independent revertants derived from this mutant, 82 were true back-mutants restoring the wild type sequence (D33G). The remaining three replaced the aspartic acid by an alanine (D33A) indicating that small size residues are best tolerated at this position which is consistent with the perfect conservation of the G33 during evolution. This glycine may be of crucial importance for helix packing around the hemes. The replacement of methionine at position 221 by lysine (M221K) produced a non-functional cytochrome b [(1993) J. Biol. Chem. 268, 15626-15632]. Non-native revertants replacing the lysine 221 by glutamic acid (K221E) or glutamine (K221Q) expressed a selective resistance to antimycin and antimycin derivatives having a modified dilactone ring moiety. Cytochrome b residues in 33 and in 221 seemed to contribute to the quinone reduction (QN) site of the cytochrome bc1 complex. Possible intramolecular interactions between the N-terminal region and the loop connecting helices IV and V of cytochrome b are proposed.

Antimycin A↗
PubMed1994

Non-native intragenic reversions selected from Saccharomyces cerevisiae cytochrome b-deficient mutants. Structural and functional features of the catalytic center N domain.

A total of 110 revertants have been isolated from two well-characterized cytochrome b deficient (mit-) mutants. The mit- mutations are located in an extramembranous loop linking the transmembrane alpha-helices IV and V of cytochrome b which has been postulated to be part of the catalytic center QN and therefore is assumed to be essential for the functioning of the bc1 complex. The molecular bases of the reversions were identified by sequencing the cytochrome b mRNAs. This allowed us to identify seven new structures of cytochrome b which are more or less compatible with its catalytic activity. The secondary mutations occurred either at the level of the original site mutation or at adjacent positions (region 204-208 of the polypeptide chain), or even at a distance of more than 150 amino acids (position 30) suggesting topological interaction between these two areas. All the revertants recovered cytochrome contents and phosphorylation efficiencies similar to the wild-type ones, albeit differences appeared in their specific growth rates and NADH respirations. The failure in bc1 complex functioning induced by the mutation S206L and its restoration by non native reversions are tentatively explained.

Amino Acid Sequence↗
PubMed1993

Insight into the interactions between subunits I and II of the cytochrome c oxidase of the yeast Saccharomyces cerevisiae by means of extragenic complementation.

In yeast, revertants were selected from two respiratory deficient mutants carrying mutations in the catalytic subunits of cytochrome oxidase. From a mutant carrying a double mutation in the vicinity of the copper binding pocket in subunit II, two genetically independent revertants were obtained in which the same extragenic reversion mutation was observed, A147V, in the putative helix 4 of subunit I. A comparison with revertants derived from the second deficient mutant, carrying the deficiency mutation, S140L, in the loop 3-4 of subunit I, provides additional data in favour of an interaction between helix 4 of subunit I and subunit II.

Electron Transport Complex IV↗
PubMed1993

Insight into topological and functional relationships of cytochrome c oxidase subunit I of Saccharomyces cerevisiae by means of intragenic complementation.

In yeast, revertants were selected from four respiratory deficient mutants carrying mutations in the cytochrome c oxidase subunit I gene. Intragenic second site mutations revealed amino acids which are functionally complementary to the original mutated position and may be in topological interaction with it. The results provide additional data in favour of the model proposed for the structure of the binuclear centre in proton-motive oxidases.

Amino Acid Sequence↗
PubMed1993

Genetic screening in Saccharomyces cerevisiae for large numbers of mitochondrial point mutations which affect structure and function of catalytic subunits of cytochrome-c oxidase.

A new search for mitochondrial respiratory deficient mutants (Mit-) has been undertaken in order to accumulate a large number of point mutations in the coding portions of cytochrome-c-oxidase catalytic subunits and cytochrome b. Therefore, a mitochondrial DNA which retains the exons and lacks all the introns of the cytochrome oxidase subunit I and of the cytochrome-b split genes has been introduced into a strain carrying a nuclear recessive mutation affecting the adenine-nucleotide translocator, the op1 mutation, which is known to prevent the accumulation of large deletion petite mutants and this was used as the parental strain. After a moderate MnCl2 mutagenesis in order to limit multiple mutations, 105 Mit- mutants were isolated from 15,000 mutagenised clones in Saccharomyces cerevisiae. Mutations were mapped to the three catalytic subunits encoding genes (COX1, COX2 and COX3) of the cytochrome-c oxidase (70 mutations) and to the cytochrome-b gene (15 mutations). More than 50% of the mutants tested still exhibited mitochondrial translation products (subunits I, II and III), suggesting that they carry a missense mutation, rather than a nonsense mutation which would normally have led to a truncated protein. Mutations in the COX1 gene were allocated to four different subregions corresponding to exons 4 and 8 or to groups of exons, exons 1, 2, 3 or exons 5, 6, 7. Seven missense monosubstitution mutations and two frameshift mutations were also identified. The amino acid changes of the missense mutations were located in the vicinity of the CuB-heme alpha 3 binuclear centre ligands.

Amino Acid Sequence↗
PubMed1993

Rapid screening of cytochromes of respiratory mutants of Saccharomyces cerevisiae. Application to the selection of strains containing novel forms of cytochrome-c oxidase.

A technique has been developed for the direct analysis by visible spectrophotometry of yeast spots growing on agar plates. This allows rapid semi-quantitative estimations of cytochromes c, b and oxidase and permits the identification of strains with impaired respiratory electron flow. Results of screening of 105 mutants are presented. There appears to be a correlation between the exonic location of the mutation in COX1 of oxidase and the level of optically detectable enzyme. Mutations in cytochrome b of the bc1 complex also affect the level of expression of cytochrome oxidase and can cause either an increased or decreased level of expression of oxidase relative to the wild-type strain. Twelve strains selected by the rapid level-1 screening were grown as lawns on sections of an agar plate and resuspended for a second level of screening. Quantitative estimates have been made of the concentrations of cytochromes, the turnover number of cytochrome oxidase and the kinetics of recombination of carbon monoxide with oxidase after flash photolysis. This confirmed the validity of the rapid screening procedure, and we have identified several strains which contain high levels of a mutant form of cytochrome oxidase with properties worthy of further investigation.

Cytochromes↗
PubMed1993

Random mutant generation and its utility in uncovering structural and functional features of cytochrome b in Saccharomyces cerevisiae.

The generation of random mutations in the mitochondrial cytochrome b gene of Saccharomyces cerevisiae has been used as a most fruitful means of identifying subregions that play a key role in the bc1 complex mechanism, best explained by the protonmotive Q cycle originally proposed by Peter Mitchell. Selection for center i and center o inhibitor resistance mutants, in particular, has yielded much information. The combined approaches of genetics and structural predictions have led to a two-dimensional folding model for cytochrome b that is most compatible with current knowledge of the protonmotive Q cycle. A three-dimensional model is emerging from studies of distant reversions of deficient mutants. Finally, interactions between cytochrome b and the other subunits of the bc1 complex, such as the iron-sulfur protein, can be affected by a single amino acid change.

Amino Acid Sequence↗
PubMed1992

Point mutation in cytochrome b of yeast ubihydroquinone:cytochrome-c oxidoreductase causing myxothiazol resistance and facilitated dissociation of the iron-sulfur subunit.

Cytochrome-c reductase was isolated from Saccharomyces cerevisiae GM50-3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDS/PAGE. Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Q0 center (Q, ubiquinone) of cytochrome-c reductase, were analysed. The single amino acid substitution in the cytochrome-b subunit, N256Y in the mutant Myx-119 and G137R in the mutant Myx-118, caused a general resistance to all methoxyacrylate inhibitors to about fivefold higher concentrations. The kinetic measurements with the substrate analogue nonylbenzohydroquinone revealed a decrease in the Km by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The Km of the G137R mutant was not affected and the Vmax was 50% higher. Cytochrome-c reductase was isolated from mutants to allow determination of the Kd values of methoxyacrylate-stilbene and myxothiazol by means of fluorescence-quench and red-shift titration. Changes in the structure of the multisubunit complex due to a single amino acid exchange became obvious during the purification procedure. SDS/PAGE of the purified enzyme revealed that the substitution N256Y in cytochrome b led to a loss of the iron-sulfur protein and the fifth small subunit with no change in the pattern of the remaining eight subunits. The subunit pattern of the G137R mutant was identical to the wild type. This is the first report of a single amino acid exchange in the catalytic subunit of cytochrome b, greatly affecting the iron-sulfur protein, the second important catalytic subunit of the Q0 center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron-sulfur subunit.

Amino Acid Sequence↗
PubMed1991

Structure/function relationships in mitochondrial cytochrome b revealed by the kinetic and circular dichroic properties of two yeast inhibitor-resistant mutants.

The kinetic and circular dichroic properties of two yeast mutants that are resistant towards specific inhibitors of the mitochondrial cytochrome bc1 complex have been characterized. Both of these mutants have an altered cytochrome b gene in which aromatic residues are exchanged with non-polar residues in a highly conserved region of the protein. The mutant resistant to myxothiazol and mucidin that contains the substitution Phe129----Leu is not greatly affected either in its ubiquinol:cytochrome c reductase or in the spectral properties of cytochrome b. On the other hand, the mutant resistant to stigmatellin that contains the substitution Ile147----Phe shows a large decrease of the catalytic efficiency for ubiquinol and of the maximal turnover of its reductase activity. This stigmatellin mutant also shows an altered circular-dichroic spectrum of the low-potential haem of cytochrome b. This study provides biochemical and biophysical information for identifying a region in mitochondrial cytochrome b that may fulfill a crucial role in the binding of ubiquinol to the bc1 complex. The results are discussed also in terms of the structural model of cytochrome b having a core of four transmembrane helices.

Amino Acid Sequence↗
PubMed1991

Molecular analysis of revertants from a respiratory-deficient mutant affecting the center o domain of cytochrome b in Saccharomyces cerevisiae.

In bc complexes, cytochrome b plays a major role in electron transfer and in proton translocation across the membrane. Several inhibitor-resistant and respiratory-deficient mutants have already been used to study the structure-function relationships of this integral membrane protein. We describe here the selection and the molecular analysis of revertants from a thermo-sensitive mit-mutant of known nucleotide changes. Among 80 independent pseudo-wild type revertants screened by DNA-labelled oligonucleotide hybridization, 33 have been sequenced. Eight suppressor mutations, affecting a region critical for both the function and the binding of center o inhibitors (end of helix C) were identified. Two of them were found to be more resistant to myxothiazol.

Amino Acid Sequence↗
PubMed1990

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.

Alleles↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

Increased diuron resistance in the joint expression of mutations located at the DIU2, DIU3 and DIU4 loci of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, diuron blocks the respiratory pathway at the level of the bc1 complex. Two mitochondrially inherited loci, DIU1 and DIU2, located in the cytochrome b gene, and two nuclearly inherited loci, DIU3 and DIU4, have previously been identified. The present work genetically characterizes two double mutants. One mutant, Diu-217, carries two nuclearly inherited mutations, diu3-217a and diu-217b; the second mutant, Diu-783, carries the previously described nuclear mutation diu3-783 and a mitochondrial mutation diu2-783. Each mutation, independent of its location, exhibits a weak diuron resistance. The joint expression of two or three mutations leads to a cumulative or a cooperative enhanced diuron-resistant phenotype.

Alleles↗
PubMed1989

Two nuclearly inherited loci conferring increased diuron resistance to NADH oxidase in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, diuron blocks the respiration pathway at the level of the bc1 complex. Nuclear diuron-resistant mutations which confer in vitro resistance to mitochondrial NADH oxidase have been identified. Five mutations were found to be clustered at two distinct nuclear loci DIU3 and DIU4. The distance between the two loci was estimated to be about 36.7 cM. These loci do not appear to be centromere-linked and did not show a linkage to any of the genes coding for bc1 complex subunits. DIU3 and DIU4 loci might, therefore code for other components of the respiratory chain.

Alleles↗