Anne-Marie Colson

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 cytochromeb gene ofSaccharomyces cerevisiae has been used as a most fruitful means of identifying subregions that play a key role in thebc1 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 cytochromeb 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 cytochromeb and the other subunits of thebc1 complex, such as the iron-sulfur protein, can be affected by a single amino acid change.

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

SummaryDiuron-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.

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

Four modified cytochrome bs 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 (Q N ) site of the cytochrome bc 1 complex. Possible intramolecular interactions between the N‐tenninal region and the loop connecting helices IV and V of cytochrome b are proposed.

Allelism relationships between diuron-resistant, antimycin-resistant and funiculosin-resistant loci of the mitochondrial map in Saccharomyces cerevisiae

SummaryUsing allelism tests, two diuron (DIU1, DIU2), one funiculosin (FUN1), and two antimycin (ANA1, ANA2) resistance loci are resolved into two mitochondrial drug-resistant genetic loci. DIU1 is allelic to ANA2 and FUN1. DIU2 is allelic to ANA1.

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.

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

Inbc complexes, cytochromeb plays a major role in electron transfer and in proton translocation accross 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.

A cytoplasmic gene for partial suppression of a nuclear pleiotropic respiratory deficient mutant in the petite negative yeast Schizosaccharomyces pombe

SummaryThe nuclear pleiotropic respiratory-deficient mutant pet1 (previously M126) exhibits cytochromes aa3 and b deficiencies accompanied by loss of the oligomycin-sensitivity of the mitochondrial ATPase. The mutant pet1, unable to grow on glycerol, exhibits in addition sensitivity of Antimycin A of the growth on glucose. The latter phenotypic trait symbolized by ANAS-D, exhibits a high frequency (2 to 4×10-5) of spontaneous suppression into Antimycin A-resistant strains. Mutagenesis with MnCl2 increases by a factor of 102 the frequency of ANAR-D derivatives. This suppression is partial since none of the suppressed strains is able to grow on glycerol even when respiratory functions and cytochromes activities are restored as in the pet1 [SUP2] strain. In the latter strain it is concluded that the extralocus suppressor gene [SUP2] is responsible for the ANAR-D trait. Tetrad analysis in a cross homozygous for pet1 demonstrates a non-Mendelian segregation pattern for the SUP2 suppressor gene. In stable diploids, homozygous for pet1, the [SUP2] suppressor exhibits a mitotic segregation pattern. Furthermore the transmission of the [SUP2] gene is decreased by ethidium bromide treatment. Therefore, the [SUP2] suppressor gene responsible for partial suppression of the nuclear pleiotropic phenotype in mutant pet1 is of cytoplasmic heredity.

Fructose-bisphosphatase-deficient mutants of the yeast Schizosaccharomyces pombe

We showed that in the yeast Schizosaccharomyces pombe, fructose-bisphosphatase is not subject to catabolite inactivation as it was observed in Saccharomyces cerevisiae. However, this enzyme activity is sensitive to catabolite repression in both yeasts. Two mutants lacking completely fructose-bisphosphatase activity were found. They were unable to grow on glycerol medium. They were still respiratory competent and exhibited the ability to derepress partially malate dehydrogenase activity. In glucose exponential phase culture, the parental strain lacks completely the fructosebisphosphatase activity due to catabolite repression. In these conditions, the growth is slowed down only in the mutants eventhough both mutants and their parental strain lack this enzyme activity. Normal sporulation and poor spore germination were observed for one mutant whereas, only in the presence of glucose, normal sporulation and normal spore germination were observed for the second mutant. Mendelian segregation of glycerol growth was found for the well germinating mutant. It is of nuclear heredity. The two mutations appeared to be closely linked.

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.

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

SummaryIn 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.