Preparation of highly phosphorylating mitochondria from the yeast Schizosaccharomyces pombe.

Schizosaccharomyces pombe yeast cells grown on either fermentable or respiratory media were efficiently converted to stable spheroplasts by the alpha-(1-->3)-glucanase Novozym 234 in the presence of 1.2 M sorbitol. Lysis of spheroplasts by gentle homogenization in dilute sorbitol resulted in the preparation of mitochondria with a structure similar to that observed within the starting yeast cells.

Functional nucleotide-binding domain in the F0F1-ATPsynthase alpha subunit from the yeast Schizosaccharomyces pombe.

The segment R165-T330 of the alpha subunit of Schizosaccharomyces pombe F1-ATPase, corresponding to a putative nucleotide-binding domain by comparison with related nucleotide-binding proteins, has been overexpressed in Escherichia coli. Produced as a nonsoluble material, it was purified in a nonnative form, using a rapid procedure that includes one reversed-phase chromatography step. Refolding of the domain, called DN alpha 19, was achieved quantitatively by using a high-dilution step and monitored by circular dichroism and intrinsic fluorescence.

Structural mapping of catalytic site with respect to alpha-subunit and noncatalytic site in yeast mitochondrial F1-ATPase using fluorescence resonance energy transfer.

The intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1 is a very sensitive probe to differentiate nucleotide binding to catalytic and noncatalytic sites (Divita, G., Di Pietro, A., Roux, B.

Chemical modification of alpha-subunit tryptophan residues in Schizosaccharomyces pombe mitochondrial F1 adenosine 5'-triphosphatase: differential reactivity and role in activity.

Chemical modification of mitochondrial F1-ATPase from Schizosaccharomyces pombe by the tryptophan-specific reagent N-bromosuccinimide (NBS) at pH 5.0 in the presence of 20% glycerol produced a characteristic lowering in both enzyme absorbance at 280 nm and intrinsic fluorescence at 332 nm that varied with NBS/F1 molar ratio up to a value of 130. Fluorometric titration of tryptophans and correlation to residual ATPase activity showed that modification of three reactive residues among the seven present on alpha- and epsilon-subunits did not markedly modify the enzyme activity but efficiently released endogenous ATP and abolished the fluorescence quenching related to GDP or ATP binding to the catalytic site.

Differential nucleotide binding to catalytic and noncatalytic sites and related conformational changes involving alpha/beta-subunit interactions as monitored by sensitive intrinsic fluorescence in Schizosaccharomyces pombe mitochondrial F1.

Mitochondrial F1 from the yeast Schizosaccharomyces pombe exhibits an intrinsic tryptophan fluorescence sensitive to adenine nucleotides and inorganic phosphate [Divita, G., Di Pietro, A., Deléage, G.

Large-scale purification and characterization of the five subunits of F1-ATPase from pig heart mitochondria.

A large-scale purification procedure was developed to isolate the five subunits of F1-ATPase from pig heart mitochondria. The previously described procedure (Williams, N. and Pedersen, P.

Subunit delta of chloroplast F0F1-ATPase and OSCP of mitochondrial F0F1-ATPase: a comparison by CD-spectroscopy.

CD spectra have been recorded with subunit delta from chloroplast CF0CF1 and with OSCP from mitochondrial MF0MF1. These subunits are supposed to act similarly at the interface between proton transport through the F0-portion and ATP-synthesis in the F1-portion of their respective F0F1-ATPase. Evaluation of the data for both proteins revealed a very high alpha-helix content of approximately 85% and practically no beta-sheets.

Alteration of apparent negative cooperativity of ATPase activity by alpha-subunit glutamine 173 mutation in yeast mitochondrial F1. Correlation with impaired nucleotide interaction at a regulatory site.

The first described alpha-subunit mutation of yeast mitochondrial F1 has been recently identified as a single Gln173----Leu substitution in a strongly conserved sequence (Falson, P., Maffey, L., Conrath, K.

Intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1-ATPase. A powerful probe for phosphate and nucleotide interactions.

Mitochondrial F1 from the yeast Schizosaccharomyces pombe, in contrast to the mammalian enzyme, exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 291 nm and a maximal emission at 332 nm. Low values of Stern-Volmer quenching constants, 4.0 M-1 or 1.

A 28 kDa mitochondrial protein is radiolabelled by crosslinking with a 125I-labelled presequence.

A 13-residue peptide containing the first 12 amino acids of the N-terminal part of the signal sequence of yeast cytochrome c oxidase subunit IV is shown by chemical crosslinking to interact with a mitochondrial protein. This result is obtained with mitochondria from four different origins. Submitochondrial localization experiments suggest that the 28 kDa labelled component is present on the outer face of the inner membrane.

Interaction between delta and epsilon subunits of F1-ATPase from pig heart mitochondria. Circular dichroism and intrinsic fluorescence of purified and reconstituted delta epsilon complex.

A delta epsilon complex has been purified as a molecular entity from pig heart mitochondrial F1-ATPase. This delta epsilon complex has also been reconstituted from purified delta and epsilon subunits. Both isolated and reconstituted delta epsilon complexes have delta 1 epsilon 1 stoichiometry and are indistinguishable by their chromatographic behavior, their circular dichroism spectra (CD spectra), and their intrinsic fluorescence features.

Structure-function relationships of mitochondrial ATPase-ATPsynthase using Schizosaccharomyces pombe yeast mutants with altered F1 subunits.

Phenotypic revertants have been selected from mutants of the yeast Schizosaccharomyces pombe devoid of either alpha or beta subunits of mitochondrial ATPase-ATPsynthase. In contrast to parental mutants, phenotypic revertants are able to grow on glycerol respiratory medium and show immunodetectable alpha and beta subunits. However, growth and cellular respiration are only partially restored as compared to the wild strain, indicating that the recovered subunits are mutated.

Purification from a yeast mutant of mitochondrial F1 with modified beta-subunit. High affinity for nucleotides and high negative cooperativity of ATPase activity.

Mitochondrial F1 containing genetically modified beta-subunit was purified for the first time from a mutant of the yeast Schizosaccharomyces pombe. Precipitation by poly(ethylene glycol) allowed us to obtain a very stable and pure enzyme from either mutant or wild-type strain. In the presence of EDTA, purified F1 retained high amounts of endogenous nucleotides: 4.

A yeast strain with mutated beta-subunits of mitochondrial ATPase-ATPsynthase: high azide and bicarbonate sensitivity of the ATPase activity.

A phenotypic revertant with modified beta-subunits of mitochondrial ATPase-ATP synthase has been obtained for the first time by selection from a beta-less mutant of the yeast Schizosaccharomyces pombe. Contrary to the parental mutant, the phenotypic revertant grows on glycerol, has normal respiratory activity and shows immunodetectable beta-subunits. However the kinetic properties of its submitochondrial particles ATPase activity differ markedly from those of the wild strain.

Fate of nucleotides bound to reconstituted Fo-F1 during adenosine 5'-triphosphate synthesis activation or hydrolysis: role of protein inhibitor and hysteretic inhibition.

The protein ATPase inhibitor entraps about five nucleotides in pig heart mitochondrial F1, one at least being a triphosphate [Di Pietro, A., Penin, F., Julliard, J.

Cyclic GMP, cyclic AMP, glucose at birth, and maturation of rat liver mitochondria.

The improvement in mitochondrial functions which normally occurs in newborn rat liver in vivo during the few hours following delivery is inhibited by a glucose injection at birth (Meister, R., Comte, J., Baggetto, L.

[Comparative effects of pentoxifylline and of HWA 285 on mitochondrial maturation and biosynthesis of proteins in rats in the perinatal period].

To improve our understanding of the beneficial effects of alkylxanthines in various disorders, two animal models were used. The biochemical modifications due to pentoxifylline and HWA 285 on mitochondrial maturation and protein biosynthesis during the neonatal period were determined simultaneously.

ANTHEPROT: a package for protein sequence analysis using a microcomputer.

A simple microcomputer package is described to make the theoretical analysis of protein sequences. Several methods designed to compare two sequences, to model proteolytic reactions and to predict the secondary structure, the hydrophobic/hydrophilic regions and the potential antigenic sites of proteins have been included in an Apple II microcomputer software. The package comprises 21 programs as well as the secondary structure database of Kabsch and Sander (1983).

Evidence from immunological studies of structure-mechanism relationship of F1 and F1F0.

Monoclonal and polyclonal antibodies directed against peptides of F1-ATPase of F1F0-ATPase synthase provide new and efficient tools to study structure-function relationships and mechanisms of such complex membrane enzymes. This review summarizes the main results obtained using this approach. Antibodies have permitted the determination of the nature of subunits involved in the complex, their stoichiometry, their organization, neighboring interactions, and vectorial distribution within or on either face of the membrane.

IF1 inhibition of mitochondrial F1-ATPase is correlated to entrapment of four adenine- or guanine-nucleotides including at least one triphosphate.

This paper demonstrates that the inhibition of F1 ATPase activity by the natural inhibitor protein IF1 is correlated to triphosphate nucleotide entrapment in F1. The complete balance of nucleotides bound after preincubation with Mg-[alpha-32P]GTP or Mg-[alpha-32P]ATP, used to promote IF1 inhibition, has been established on purified F1 containing 0.7 mol of non-exchangeable endogenous nucleotides.

Revertant of the yeast Schizosaccharomyces pombe with modified alpha subunits of mitochondrial ATPase-ATPsynthase: impaired nucleotide interactions with soluble and membrane-bound enzyme.

A partial revertant from a mutant with modified alpha subunits of mitochondrial ATPase-ATPsynthase has been obtained for the first time from the yeast Schizosaccharomyces pombe. The purified F1 contains a lower amount of endogenous nucleotides as compared to the wild-strain enzyme. In contrast to the wild-type, the F1 ATPase activity from the revertant does not exhibit bicarbonate-sensitive negative cooperativity.

Efficient reconstitution of mitochondrial energy-transfer reactions from depleted membranes and F1-ATPase as a function of the amount of bound oligomycin sensitivity-conferring protein (OSCP).

Pig heart mitochondrial membranes depleted of F1 and OSCP by various treatments were analyzed for their content in alpha and beta subunits of F1 and in OSCP using monoclonal antibodies. Membrane treatments and conditions of rebinding of F1 and OSCP were optimized to reconstitute efficient NADH- and ATP-dependent proton fluxes, ATP synthesis and oligomycin-sensitive ATPase activity. F1 and OSCP can be rebound independently to depleted membranes but to avoid unspecific binding of F1 to depleted membranes (ASUA) which is not efficient for ATP synthesis, F1 must be rebound before the addition of OSCP.

Role of phosphate on the ADP-induced hysteretic inhibition of mitochondrial adenosine 5'-triphosphatase. Effects of the natural protein inhibitor.

Preincubation of F1-ATPase with ADP and Mg2+ leads to ADP binding at regulatory site inducing a hysteretic inhibition of ATP hydrolysis, i.e., an inhibition that slowly develops after Mg-ATP addition (Di Pietro, A.

Reversal of glucose-induced inhibition of newborn rat liver mitochondrial maturation by administration of alkylxanthines at birth.

A glucose injection given immediately after birth delays the maturation which normally occurs in rat liver mitochondria and which increases the rate of ATP synthesis coupled to succinate oxidation from a low value at birth to the adult value a few hours after birth [R. Meister, J. Comte, L.