Essential arginyl residues in the H+-translocating ATPase of plasma membrane from the yeast Schizosaccharomyces pombe

The H+-translocating adenosine-5'-triphosphatase (ATPase) purified from the yeast Schizosaccharomyces pombe is inactivated upon incubation with the arginine modifier 2,3-butanedione. The inactivation of the enzyme is maximal at pH values above 8.5. The modified enzyme is reactivated when incubated in the absence of borate after removal of 2,3-butanedione. The extent of inactivation is half maximal at 10 mM 2,3-butanedione for an incubation of 30 min at 30 degrees C at pH 7.0. Under the same conditions, the time-dependence of inactivation is biphasic in a semi-logarithmic plot with half-lives of 10.9 min and 65.9 min. Incubation with 2,3-butanedione lowering markedly the maximal rate of ATPase activity does not modify the Km for MgATP. These data suggest that two classes of arginyl residues play essential role in the plasma membrane ATPase activity. Magnesium adenosine 5'-triphosphate (MgATP) and magnesium adenosine 5'-diphosphate (MgADP), the specific substrate and product, protect partially against enzyme inactivation by 2,3-butanedione. Free ATP or MgGTP which are not enzyme substrates do not protect. Free magnesium, another effector of enzyme activity, exhibits partial protection at magnesium concentrations up to 0.5 mM, while increased inactivation is observed at higher Mg2+ concentrations. These protections indicate either the existence of at least one reactive arginyl in the substrate binding site or a general change of enzyme conformation induced by MgATP, MgADP or free magnesium.     

Solubilization and partial purification of ATPase from a rose cell plasma membrane fraction

Some isolates of the fungus Nectria haematococca Berk. and Br. can demethylate pisatin, a phytoalexin from pea (Pisum sativum L.). Pisatin demethylation appears to be necessary for tolerance to pisatin and virulence on pea, and is catalyzed by a microsomal cytochrome P-450. We now report solubilization of this enzyme from N. haematococca microsomes. Pisatin demethylase activity was obtained in the high speed supernatant of detergent treated microsomes, if detergent was removed before assay. The CO-binding spectrum of the soluble enzyme preparation indicated the presence of cytochrome P-450. Cholic acids were the most effective of the detergents tested for solubilizing enzyme activity. Loss of enzyme activity during solubilization was reduced by certain protease inhibitors, but not by substrate, reducing agents, antioxidants, or phospholipids. The most effective solubilization medium tested was 1% sodium cholate, 100 millimolar potassium phosphate, 500 millimolar sucrose, 1 millimolar phenylmethylsulfonyl fluoride, pH 7.5, which yielded approximately 30% of the pisatin demethylase and over 95% of the NADPH-cytochrome c reductase in the soluble fraction. Demethylase activity was lost when the reductase was removed by adsorption on 2′,5′-ADP-agarose. The demethylase activity of reductase-free fractions could be restored by adding a reductase preparation purified approximately 100-fold from microsomes of N. haematococca isolate 74-8-1, which does not demethylate pisatin. We conclude that pisatin demethylase requires NADPH-cytochrome c reductase for activity. The inability of some isolates to demethylate pisatin appears to be due to the absence of a suitable cytochrome P-450, rather than to a lack of functional reductase.     

Solubilization and characterization of a cell wall-bound trehalase from ascospores of the fission yeast Schizosaccharomyces pombe

The genome of the fission yeast Schizosaccharomyces pombe lacks sequence homologs to ath1 genes coding for acid trehalases in other yeasts or filamentous fungi. However, acid trehalase activity is present at the spore stage in the life cycle of the fission yeast. The enzyme responsible for this activity behaves as a surface enzyme covalently linked to the spore cell walls in both wild-type and ntp1 mutant strains devoid of neutral trehalase. Lytic treatment of particulated cell wall fractions allowed the solubilization of the enzyme into an active form. We have characterized this soluble enzyme and found that its kinetic parameters, optimum pH and temperature, thermal denaturation and salt responses are closely similar to other conventional acid trehalases. Hence, this rather unusual enzyme can be recognized as acid trehalase by its biochemical properties although it does not share genetic homology with other known acid trehalases. The potential role of such acid trehalase in the mobilization of trehalose is discussed.     

Mutations induced by x-radiation in the yeast Schizosaccharomyces pombe

Experiments in strains of yeast with different genetic backgrounds were done to evaluate the kinetics of inactivation and mutation induction by X-radiations. A system of forward mutation induction in five loci was used and a specific mutation rate of 0.14·10⁻⁸×locus×rad was evaluated for the wild type.From a comparison of observations with wild type and radiation-sensitive strains, it may be assumed that, in this yeast, mutations are mainly the result of a repair-active process.The range of genotypic and phenotypic influence upon the specific locus mutation rate was evaluated with appropriate biological material and experiments.     

Cyclins of the fission yeast Schizosaccharomyces pombe

Five cyclin-like genes, cig1, cig2/cyc17, mcs2, puc1 and cdc13, have been discovered in S. pombe to date. It is not yet clear what their functions are or even whether they are all involved with control of the cell cycle. Conflicting data for cig1 and cig2/cyc17 have obscured analysis of their function and cig1 remains largely uncharacterized, although clues to the role of cig2/cyc17 have emerged. There is genetic data available for the more distant cyclin homologue mcs2, which has an essential although as yet unspecified role. Puc1 may be involved in regulation of exit from the cell cycle. The first cyclin to be discovered, and the best understood, is cdc13 which with cdc2 promotes mitosis. Studies of the roles of cdc2 and cdc13 in the overall ordering of the cell cycle suggest that cdc13 and probably other cyclins are key regulators, maintaining the order of S phase and mitosis during the cell cycle.     

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.Abbreviations FBPase Fructose-1,6-bisphosphatase - fbp ^- genetic symbol for FBPase deficiency - glr ^- symbol for inability to grow on glycerol A. M. Colson is Research Associate au Fonds National de la Recherche Scientifique     

Acetate assimilation by the fission yeast, Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe utilizes acetate at subinhibitory concentrations in the presence of D-glucose. The nonionized form of acetate is preferentially utilized, oxidized to 14CO2, and assimilated into lipids and proteins. Acetyl CoA synthetase activity greatly increases in the yeast cells grown in media containing acetate. However, glyoxylate cycle enzymes are not detectable in Schizosaccharomyces pombe. [1-14C]Acetate is incorporated into stereols, sterol esters, neutral lipids, and phospholipids. Assimilation of [1-14C]acetate into the peptide structure of proteins was confirmed by a proteolytic digestion experiment.     

A single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe

A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by [gamma-32P] ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors (Ulaszewski, S., Coddington, A., and Goffeau, A. (1986) Curr. Genet. 10, 359-364).     

Topology of the yeast plasma membrane proton-translocating ATPase

Four proteases have been used to assess the topology of the H+-ATPase from Saccharomyces cerevisiae reconstituted into phosphatidylserine vesicles. Limited proteolysis by trypsin and alpha-chymotrypsin inactivates the enzyme and produces stable, membrane-bound fragments. Sequence analyses of these peptides have located the peptide bonds hydrolyzed. The labile bonds are on opposite sides of a central hydrophilic domain containing consensus sequences for the site of phosphorylation and fluorescein isothiocyanate binding of several related ATPases. Limited proteolysis of the ATPase by elastase cuts approximately 50 amino acids from the C terminus, leaving the remaining membrane-bound fragments active. Proteolysis by carboxypeptidase Y in the presence and absence of detergent suggests that the C terminus is on the inside of the vesicle in this reconstitution. A model for the transmembrane arrangement of the polypeptide is proposed. In this model, the C terminus is on the inside of the vesicle, the N terminus is on the outside, the ATP binding region is on the outside, and the polypeptide passes through the membrane a minimum of five times.     

Factors affecting the inhibition of yeast plasma membrane ATPase by vanadate

Inhibition of yeast plasma membrane ATPase by vanadate occurs only if either Mg2+ or MgATP2- is bound to the enzyme. The dissociation constant of the complex of vanadate and inhibitory sites is 0.14-0.20 microM in the presence of optimal concentrations of Mg2+ and of the order of 1 microM if the enzyme is saturated with MgATP2-. The dissociation constants of Mg2+ and MgATP2- for the sites involved are 0.4 and 0.62-0.73 mM, respectively, at pH 7. KCl does not increase the affinity of vanadate to the inhibitory sites as was found with (Na+ + K+)-ATPase. On the other hand, the effect of Mg2+ upon vanadate binding is similar to that upon (Na+ + K+)-ATPase, and the corresponding affinity constants of Mg2+ and vanadate for the two enzymes are of the same order of magnitude.     

High-frequency cotransformation by copolymerization of plasmids in the fission yeast Schizosaccharomyces pombe.

We have developed a high-frequency cotransformation system which is useful in introducing nonreplicating circular DNA plasmids into the fission yeast Schizosaccharomyces pombe. This system depends on two factors: the ability of the ural-complementing helper plasmids pFYM2 and pFYM225 to propagate autonomously in S. pombe, and the intensive recombination activity intrinsic to this yeast. If cotransformed with a helper plasmid, plasmids such as YIp5 or YIp32, Escherichia coli-Saccharomyces cerevisiae shuttle vectors incapable of replication in S. pombe, can enter S. pombe and express the gene carried on them at a frequency comparable to that of autonomously replicating plasmids (10(3) to 10(4) transformants per microgram of DNA). Even if characters of the nonreplicating DNA are not selected directly, 50 to 70% of Ura+ cells transformed with the helper have also incorporated the nonreplicating plasmid. It is shown that these two plasmids have physically recombined at a site of common DNA sequence to form a heteropolymer in the fission yeast. Since any foreign DNA cloned in pBR322 or ColE1 derivatives can be incorporated into S. pombe by using pFYM2 or pFYM225 as a helper, this cotransformation system will serve as a convenient method to examine functional expression of such cloned DNA in S. pombe. This work also demonstrates that the kanamycin resistance gene carried by the bacterial transposon Tn903 can be expressed in S. pombe, as shown by its ability to inactivate the antibiotic G418.     

Cd2+-induced damage to yeast plasma membrane and its alleviation by Zn2+: studies on Schizosaccharomyces pombe cells and reconstituted plasma membrane vesicles

In Schizosaccharomyces pombe, Cd²⁺ shares the same uphill uptake system with Zn²⁺. Both heavy metals inhibited growth, respiration, H⁺/glucose uptake, and glucose-induced proton extrusion, Cd²⁺ being a 10–15-fold stronger inhibitor. In contrast, both had a similar effect on the plasma membrane H⁺-ATPase, enhancing its affinity for ATP and reducing the rate of ATP splitting. Cd²⁺ caused protracted strong fluidization of the plasma membrane of energized cells, whereas deenergized cells, phosphatidylcholine liposomes, and plasma membrane fragments, either purified or incorporated into the liposomes, exhibited only a short initial fluidization. Zn²⁺, which caused only a marginal membrane fluidization, suppressed the fluidizing action of Cd²⁺. The fluidizing effect of both heavy metals on liposomes was reduced by the presence of plasma membrane fragments in the liposome membrane. At 50 μM, Cd²⁺ brought about loss K⁺ (18 K⁺/1 Cd²⁺) from energized, but not from deenergized cells since Cd²⁺ must first accumulate in the cells before causing a detectable effect. A simple membrane disruption by external Cd²⁺ is, therefore, unlikely to be the main mechanism of cadmium-induced potassium loss in intact cells. Zn²⁺ had virtually no effect below 1 mM concentration, and it again weakened the K⁺-releasing effect of Cd²⁺. Cd²⁺ caused a strong loss of K⁺ also from K⁺-containing liposomes, probably because of a direct interaction with liposome phospholipids. Incorporation of plasma membrane fragments into the liposomes reduced the K⁺ loss sixfold. Received: 13 November 1995 / Accepted: 31 January 1996