Enrico Bertoli

Lipid protein interactions in mitochondria: Spin and fluorescence probe studies on the effect of n-alkanols on phospholipid vesicles and mitochondrial membranes

Abstract The effect of n -butanol on the mobility of phospholipids in phospholipid vesicles and beef heart mitochondrial membranes has been studied using three stearic acid spin labels having a paramagnetic doxyl group in positions 5,12, and 16, respectively, and the fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS). The mobility of the spin labels in the phospholipid aliphatic chains increases from the polar heads toward the methyl groups both in vesicles and in mitochondrial membranes; however, in the latter there is a higher constriction of rotational mobility observed at all levels in the lipid bilayer. Butanol determines a moderate increase in mobility of phospholipids in lipid vesicles, but the effect is more striking in the mitochondrial membranes, where the protein-induced constraint of mobility of the fatty acyl chains is removed at low concentrations of the alcohol. Butanol also enhances the mobility of tightly bound phospholipids residual in lipid-depleted mitochondrial preparations, although higher concentrations of butanol are required for this effect. The effect of the series of aliphatic n -alcohols is related to their hydrophobicity. Alcohols induce a decrease of the fluorescence of ANS bound to both lipid vesicles and mitochondrial membranes. The fluorescence decrease is not the result of a decreased partition of ANS from the aqueous medium to the bilayer, but depends upon a change in the chromophore environment. Since no shift of the emission maximum is observed after alcohol addition, such a change must be ascribed to increased mobility of the probe, in accord with the spin label data. As for the spin label data, the effect of the series of aliphatic n -alcohols is related to their hydrophobicity; at difference with the electron spin resonance results, however, the effects are maximal for pure phospholipid vesicles. It is calculated that alcohols affect both the long-range interactions between phospholipids and proteins in mitochondrial membranes (as detected by spin labels) and the order of phospholipid bilayers near the glycerol region (as detected by ANS). The differences between the two kinds of probes may be related to their differing localization in the lipid bilayer.

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.

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 cofactor function for unsaturated fatty acids in oxidative phosphorylation; studies with a yeast auxotroph.

Abstract Mitochondria from the yeast unsaturated fatty acid auxotroph KD115 when grown with a high unsaturated fatty acid supplement catalyse normal oxidative phosphorylation and dihydrolipate-dependent ATP synthesis in the absence of added cofactors. Mitochondria from unsaturated fatty acid depleted KD115 cells have half the unsaturated fatty acid content of supplemented cells and do not catalyse oxidative phosphorylation and dihydrolipoate dependent ATP synthesis. Dihydrolipoate-dependent ATP synthesis can be restored specifically by addition of cofactor amounts of oleic acid and oleoyl CoA. The results provide further evidence for a cofactor role for an unsaturated fatty acid in oxidative phosphorylation.

A modified subunit of mitochondrial ATPase in mutants of Saccharomyces cerevisiae with decreased sensitivity to dicyclohexylcarbodiimide

Biochemical genetic studies in this laboratory have led to the isolation of mutants of ~~cc~~~o~~ces ce~e~~~~~~ with lowered sensitivity to oligomycin [ 11. The mutations are located on one of three loci on mitochondrial DNA designated OLI, OLII and OLIII [2,3] and may be associated with cross-resistance to venturicidin [3]. It has been shown that the component conferring lowered sensitivity resides in the membrane subunits of the mitochond~al ATPase and not in the catalytic subunits [4]. Dicyclohexylcarbodiimide (DCCD) is a potent inhibitor of oxidative phosphorylation [5] and has been shown to bind to a low molecular weight proteolipid subunit of the ox heart mitochondrial ATPase [6,7]. Although DCCD inhibits yeast mitochondrial ATPase at low concentrations [8], the covalent binding of the inhibitor found in ox heart mitochondria cannot be demonstrated in aerobically grown yeast. Binding of carbodiimides to yeast promitochondria has been shown to be associated with subunit 9 of the oligomycin-sensitive ATPase [S]. We have investigated the physical properties of the carbodiimide binding protein of some oligomycin resistant mutants, and here present data indicating