David E. Griffiths

Studies on energy-linked reactions: Inhibition of oxidative phosphorylation and energy-linked reactions by dibutyltin dichloride

Recent studies in this laboratory have demonstrated that a new trialkyltin analogue, dibutylchloromethyltin chloride (DBCT), inhibits oxidative phosphorylation in yeast mitochondria by reacting covalently with a non-protein lipid cofactor associated with the mitochondrial inner membrane and the ATP synthase (oligomycin-sensitive ATPase) complex and which has been provisionally identified by thin-layer chromatography (t.1.c.) and bioassay as lipoic acid [l] . In addition dihydrolipoate will specifically reverse the inhibitory effects of DBCT [l] . Other dithiols including dihydrolipoamide are ineffective, indicating that reversal of inhibition is due to replacement of the DBCT-titrated cofactor by dihydrolipoate with consequent restoration of ATP synthesis and ATPdriven energy-linked reactions. During the course of these studies it was observed that dibutyltin dichloride (DBT), the precursor used for DBCT synthesis, was also a potent inhibitor of the yeast oligomycin-sensitive ATPase [l] . Previous studies of Aldridge and Cremer [2] had indicated that the inhibition of oxidative phosphorylation by dialkyltin compounds was due to the inhibitor reacting with the dihydrolipoate cofactor in the pyruvate and a-ketoglutarate dehydrogenase complexes. This paper describes studies which demonstrate that DBT not only titrates the dihydrolipoate found in the pyru-

Oligomycin resistant mutants in yeast.

The increasing amount of information available relating to the structure and activity of yeast mitochondria has made feasible the use of a genetic approach to the problem of the mechanisms of respiration and oxidative phosphorylation by isolating mutants with lesions in mitochondrial metabolism [l] . Such mutants are usually detected by either their inability to grow on non-fermentable carbon sources, their staining reaction to dyes feeding into the electron transport chain, their spectral characteristics or their resistance to drugs which affect oxidative phosphorylation or electron transport [2,3] . Some of the mutants so far isolated, such as the cytoplasmic petites and a number of nuclear gene mutants with a similar phenotype are unsuitable for biochemical analysis of energy coupling reactions or respiration due to the large extent of the biochemical lesions. However, other more specific mutants [4-71 have been reported. The purpose of the investigation was to attempt to define a series of mutants in which mitochondrial function relating to oxidative phosphorylation had been altered, and to carry out a systematic genetic and biochemical study on them. The results presented relate to a series of 50 mutants of Saccharomyces cerevisiae which were resistant to concentrations of oligomycin 4-20 fold greater than that inhibiting the nonresistant strain. The cross resistance to other inhibitors of oxidative phosphorylation such asN, N1 -dicyclohexylcarbodiimide (DCCD) and aurovertin was investigated. 2. Materials and methods

Evidence for an extra-chromosomal element involved in mitochondrial function: A mitochondrial episome?

Biochemical genetic studies of cytoplasmic mutants of Saccharomyces cerevisiae resistant to inhibitors of aerobic (mitochondrial) metabolism such as oligomycin and venturicidin have shown that the resistance determinants are located on mitochondrial DNA [ 1,2]. Three separate loci, OL 1, OL II and OL III which probably specify two different subunits of the mitochondrial ATPase complex have been identified [2-41. Other mutants resistant to inhibitors and uncouplers with a specific mode of action on aerobic energy conservation reactions are under investigation including triethyltin, ‘1799’, bongkrekic acid, rhodamine 6G and valinomycin. Detailed studies of cytoplasmic triethyltin mutants and another class of venturicidin resistant mutants [2,5] indicate that these resistance determinants are unlinked to other loci on mt-DNA suggesting that the VENRTETR determinants are located on a cytoplasmic DNA species different from mt-DNA [2]. However, there are several features of these mutants which suggest a close association with mitochondrial function, e.g. the loss of the VENRTETR phenotype on ethidium bromide treatment always leads to the pstate and a significant proportion of spontaneous petites co-) have lost resistance determinants. In this paper we present evidence that the VENR TETR resistance determinants are located on a cytoplasmic DNA species other than mt-DNA. Our studies

Plasma Membrane Perturbation Induced by Organotins on Erythrocytes from Salmo irideus Trout

Tributyltin chloride and its degradation products monobutyltin and dibutyltin act as water pollutants, owing to the use of tributyltin chloride as a biocide in marine paint formulations. These compounds are lipid-soluble and undergo bioaccumulation and bioconcentration. Salmo irideus trout erythrocytes were studied to evaluate the possible effects of these compounds on freshwater fish, which could be exposed to long-term effects due to bioaccumulation of organotins. Data showed that tributyltin increases the haemolysis rate, starting at 10 μM, while dibutyltin has a scant protective effect at each concentration tested. Similar studies were performed in the presence of carbon monoxide (CO), which is protective against membrane oxidative stress due to haemoglobin (Hb) auto-oxidation. In these conditions all the organotins tested induced an increase in the haemolysis rate. These results suggest that the consequence of auto-oxidation of Hb could condition the effects of some organotin compounds. Steady-state fluorescence of probes embedded in the lipidic part of the membrane was used to evaluate the modifications induced by organotins to the physico-chemical state of phospholipids.

The role of lipid in regulation of mitochondrial adenosine triphosphatase

Interactions between lipid and protein within the mitochondrial membrane are important in the regulation of many membrane-bound functions [l-3]. Such membrane activities show characteristic discontinuities in the Arrhenius kinetics, which probably reflect the influence on the enzymes of lipid phase transitions [4-61. However, in some cases discrepancies have been observed between activity transition temperatures and the membrane lipid phase transition temperatures. As shown by Lenaz et al. [7], several mitochondrial enzymes exhibit breaks in Arrhenius plots at temperatures which are well above the observed lipid phase transition temperature of the whole membrane [8]. Disparities between the transition temperature of membrane lipids and the breaks in Arrhenius plots of proline uptake and succinate-dehydrogenase were also observed by Esfahani et al. [9] in membranes of Escherichia coli, and recently in our laboratory for the membrane-bound mitochondrial ATPase [lo]. These observations might indicate that the distribution of the lipids within the membrane is heterogeneous. One direct approach to resolving this problem is to study the physical and functional properties of the lipid micro environment surrounding the enzyme. In this paper we provide direct evidence that the change in activation energy of oligomycin sensitive adenosine triphosphatase is a consequence of a liquid-crystalline to a gel-phase transition in the lipid molecules which form the immediate environment of the enzyme.

The isolation and genetic characterization of extrachromosomal chloramphenicol and oligomycin-resistant mutants from the petite-negative yeast Kluyveromyces lactis.

SummarySpontaneous mutants of the petite-negative yeast Kluyveromyces lactis, resistant to the antibiotics chloramphenicol and oligomycin, were isolated and genetically characterized.Three chloramphenicol-resistant mutants showed non-Mendelian inheritance when crossed to sensitive parents.Of 5 oligomycin-resistant strains studied, three exhibited resistance due to the presence of an extrachromosomal mutation. The resistance of the other two deriving from a nuclear and recessive mutation.When two factor crosses in trans configuration were performed between two of the chloramphenicol and the five oligomycin-resistant mutants a polarity in recombination was observed with a predominance of sensitive (OSCS) over resistant (ORCR) reciprocal recombinants.Allelism tests carried out among the oligomycin-resistant mutants indicated the presence of at least two distinct extrachromosomal regions responsible for the resistance.

DNA sequence analysis of the Olir2-76 and Ossr1-92 alleles of the Oli-2 region of the yeast Saccharomyces cerevisiae. Analysis of related amino-acid substitutions and protein-antibiotic interaction☆

Petite deletion mapping helped to generate a fine-structure genetic map of the Oli-2 region of the mitochondrial genome of Saccharomyces cerevisiae. Here we report the DNA sequence analysis of the Oli-2 region from two drug-resistant alleles (Olir2-76 and Ossr1-92) which are located in the gene for subunit-6 of mitochondrial ATPase, in agreement with their genetic locations on the mitochondrial genome. An analysis of the corresponding amino-acid substitutions is also presented in the context of protein-antibiotic interactions.

Interaction of the dibutylchloromethyltin chloride binding site with the carbodiimide binding site in mitochondria

Abstract An examination of the effect of dibutylchloromethyltin/chloride on the carbodiimide binding proteolipid of mitrochondrial ATPase has revealed that in the presence of the alkyltin, (1) binding of dicyclohexycarbodiimide is decreased (2) the electron spin resonance spectrum of a nitroxide analogue of dicyclohexylcarbodiimide exhibits line broadening characteristic of either an increase of polarity or a decrease in viscosity of the carbodiimide binding site (3) the rate of reduction of the nitroxide probe by ascorbate is increased threefold. These phenomena suggest a possible mode of action for the inhibition of ATP synthesis by alkyltins.

Studies of energy-linked reactions: Dihydrolipoate- and oleate- dependent ATP synthesis in yeast promitochondria

A role for lipoic acid residues in oxidative phosphorylation has been demonstrated in yeast and heart mitochondrial preparations and a requirement for a unsaturated fatty acid as a cofactor has been demonstrated in ATP synthase preparations [l-4] . It is proposed that the terminal reactions of oxidative phosphorylation are analogous to substrate-level phosphorylation, with lipoic acid residues providing a functional link (energy-transfer system) between the respiratory chain and the ATP synthase complex. In these experiments the respiratory chain was inhibited by rotenone and antimycin A which apparently inhibit the contribution of the respiratory chain. However, in a complex membrane preparation it may not be possible to inhibit all the functional capacity of the respiratory chain and thus evaluate the role of all respiratory chain components. Yeast promitochondria from anaerobically grown cells do not possess a functional respiratory chain and lack the cytochrome system and ubiquinone as shown by Criddle and Schatz [5]. However, promitochondria still retain a functional energy-transfer system as evidenced by an oligomycin sensitive ATPase and an uncoupler-sensitive Pi-ATP exchange reaction [6] and thus provides an experimental system for evaluating the role of lipoic acid in the energy-transfer system which is still present in these organelles. In addition, the unsaturated fatty acid

Studies of energy-linked reactions: a lipoic acid requirement for oxidative phosphorylation in Escherichia coli.

A role for lipoic acid (or a lipoic acid conjugate) in oxidative phosphorylation catalysed by yeast and heart mitochondria has been demonstrated and net synthesis of ATP by ATP-synthase preparations has been shown to require dihydrolipoate and an unsaturated fatty acid as cofactors [ 1,2]. It is proposed that lipoic acid residues provide a link between the respiratory chain and the ATP-synthase complex and that the terminal reactions of oxidative phosphorylation involve a cycle of transacylation and transphosphorylation reactions, analogous to those involved in substrate level phosphorylation [ 11. The availability of lipoic acid auxotrophs of Escherichia coli [3] provides an experimental system for evaluating the role of lipoic acid in the oxidative phosphorylation system present in the plasma membrane of E.coZi. This paper describes studies of oxidative phosphorylation in membrane vesicles from a lipoic acid requiring mutant of E.coli and demonstrates a requirement for lipoic acid in oxidative phosphorylation with NADH, succinate and D-lactate as substrates.