Legume vicilins (7S storage globulins) inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells.

Vicilin (7S storage proteins) isolated from different legume seeds were shown to inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. The degree of growth inhibition varied with the origin of vicilins. It was more than 90% for vicilins from cowpea (Vigna unguiculata, cultivar pitiuba) and equal to 65% for vicilins from Vigna radiata, in the case of Saccharomyces cerevisae. Vicilins from cowpea seeds inhibited the glucose stimulated acidification of the medium by S. cerevisae up to 60%. We have also observed that vicilins bind to yeast cells. We suggest that vicilins bind to chitin-containing structures of yeast cells and that such association could result in inhibition of H+ pumping, cell growth and spore formation. A final consequence of the yeast growth inhibition by vicilins is (probably) the formation of spores.

What family of ATPases does the vacuolar H+-ATPase belong to?

Polyacrylamide gel electrophoresis (PAGE) of partially purified ATPase from vacuoles of Saccharomyces carlsbergensis under non-dissociating conditions revealed 3 bands with ATPase activity. Further PAGE in dissociating conditions showed the similarity in composition of these 3 ATPase preparations. They were assumed to contain the same vacuolar ATPase exhibiting, however, different electrophoretic mobility due to the formation of enzyme complexes with different proteins and phospholipids. The ATPase preparation with the highest electrophoretic mobility contained 6 subunits of 75, 62, 16, 14, 12 and 9 kDa. Inhibitors of vacuolar ATPase [14C]DCCD and [14C]NEM bound to a 9 kDa polypeptide, while [14C]DES associated with a polypeptide of 75 kDa. A partially purified preparation of the vacuolar ATPase was not phosphorylated by [gamma-32P]ATP under conditions when plasma membrane ATPase formed a phosphorylated intermediate. Our results show that vacuolar H+-ATPase consists of several polypeptides, does not form the phosphorylated intermediate, and evidently represents a new type of H+-ATPase of yeast.

Some properties of membrane-bound, solubilized and reconstituted into liposomes H+-ATPase of vacuoles of Saccharomyces carlsbergensis.

Vacuoles of yeast grown in peptone medium possessed high ATPase activity (up to 1 mumol X mg protein-1 X min-1). Membrane-bound and solubilized ATPase activities were insensitive to vanadate and azide, but were inhibited by NO-3 . K+ and cyclic AMP stimulated both membrane-bound and solubilized ATPase activities. Dio-9 activated the membrane form of vacuolar ATPase 1.5-2-fold and did not affect the solubilized enzyme. Solubilized and partially purified vacuolar ATPase was reconstituted with soy-bean phospholipids by a freeze-thaw procedure. ATPase activities in native vacuoles and proteoliposomes were stimulated effectively by Dio-9, the protonophore FCCP and ionophores valinomycin and nigericin. ATP-dependent H+ transport into proteoliposomes was also shown by quenching of ACMA fluorescence. Vacuolar and partially purified ATPase preparations possessed also GTPase activity. Unlike ATPase, however, GTPase was not incorporated as a proton pump into liposomes.

Ca(2+) and H+ homeostasis in fission yeast: a role of Ca(2+)/H+ exchange and distinct V-H+-ATPases of the secretory pathway organelles.

We determined the H+ and Ca(2+) uptake by fission yeast membranes separated on sucrose gradient and found that (i) Ca(2+) sequestering is due to Ca(2+)/H+ antiporter(s) localized to secretory pathway organelles while Ca(2+)-ATPase activity is not detectable in their membranes; (ii) immunochemically distinct V-H+-ATPases acidify the lumen of the secretory pathway organelles. The data indicate that the endoplasmic reticulum, Golgi and vacuole form a network of Ca(2+) and H+ stores in the single cell, providing favorable conditions for such key processes as protein folding/sorting, membrane fusion, ion homeostasis and Ca(2+) signaling in a differential and local manner.

H+/ion antiport as the principal mechanism of transport systems in the vacuolar membrane of the yeast Saccharomyces carlsbergensis.

The secondary transport systems of the yeast vacuolar membrane have been investigated by the method of radioactive isotopes [( 14C]arginine); activation of H+-ATPase by cations (Cat+), when the enzyme is under H+ control and measurement of changes in the proton gradient (delta pH) and membrane potential (Em) due to the supposed substrates of the transporters. The main mechanism of cation transport across the yeast tonoplast is probably H+/Cat+ antiport. The apparent Km of antiporters for Ca2+, Mg2+, Mn2+, Zn2+ and Pi are 0.06, 0.3, 0.8, 0.055-0.17 and 1.5 mM, respectively.

Several compartments of Saccharomyces cerevisiae are equipped with Ca(2+)-ATPase(s)

Sucrose density fractionation of yeast membranes revealed two major and two minor peaks of 45Ca2+ transport activity which all co-migrate with marker enzymes of the endoplasmic reticulum, Golgi and membranes associated with these compartments as well as with ATPase activity measured when all other known ATPase are inhibited. Co-migration of 45Ca2+ transport and ATPase activities was also found after removal of plasma membranes by concanavalin A treatment. SDS-PAGE at pH 6.3 shows the Ca(2+)-dependent formation of acyl phosphate polypeptides of about 110 and 200 kDa. It is concluded that several compartments or sub-compartments of yeast are equipped with Ca(2+)-ATPase(s). It is proposed that these compartments are derived from the protein secretory apparatus of yeast.

Ca(2+)-ATPases of Saccharomyces cerevisiae: diversity and possible role in protein sorting.

The PMR1 gene of Saccharomyces cerevisiae is thought to encode a putative Ca(2+)-ATPase [1]. Membranes isolated from wild-type cells and from pmr1 null mutant of S. cerevisiae were fractionated on sucrose density gradients. In the pmr1 mutant we found a decrease in activity of the P-type ATPase and of ATP-dependent, protonophore-insensitive Ca2+ transport in light membranes, that comigrate with the Golgi marker GDPase. We conclude that the product of the PMR1 gene (Pmr1p) is indeed a Ca(2+)-ATPase of the Golgi and Golgi-like membranes. Surprisingly, the pmr1 null mutation abolished Ca(2+)-ATPase activity in Golgi and/or Golgi-like membranes only to 50% under conditions where they are separated from vacuolar membranes. This indicates that an additional Ca(2+)-ATPase is localized in Golgi and/or Golgi-like membranes. Moreover, a third Ca(2+)-ATPase is found in the ER and ER-like membranes. The data are consistent with the assumption that these Ca(2+)-ATPases are encoded by gene(s) different from PMR1. Disruption of PMR1 Ca(2+)-ATPase causes significant redistribution of enzyme activities and of total protein in compartments of the secretory pathway. A decrease in activity is observed for three integral membrane proteins: NADPH cytochrome c reductase, dolichyl phosphate mannose synthase, and Ca(2+)-ATPase, and also for total protein in Golgi, Golgi-like compartments and in vacuoles, whereas a corresponding increase of these activities is observed in endoplasmic reticulum and endoplasmic reticulum-like membranes. We assume that Ca(2+)-ATPases and sufficient Ca2+ gradients across the organellar membranes are important for the correct sorting of proteins to the various compartments of the secretory apparatus.

Ca(2+)-transporting ATPase(s) of the reticulum type in intracellular membranes of Saccharomyces cerevisiae: biochemical identification.

Several lines of evidence are presented to show that the Ca(2+)-ATPase activity of total yeast membranes is due to the reticulum (R) type of Ca(2+)-ATPase: (1) Neither calmodulin nor low concentrations of calmodulin antagonists change Ca2+ uptake; (2) removal of plasma membranes (PM) following Con A treatment of spheroplasts (SP) does not significantly alter Ca2+ uptake by the remaining membranes, but increases its specific activity 3.5-fold; (3) after incubation of membranes with [gamma-32P]ATP, SDS-PAGE shows the formation of acyl phosphate intermediates with molecular masses of around 100, 180-190 and 205 kDa; formation of these acyl phosphates requires Ca2+ and is blocked by cyclopiazonic acid, La3+ ions and in the absence of Ca2+. The data on fractionation of yeast membranes are consistent with the suggestion that both the ER and the Golgi are equipped with Ca(2+)-ATPase(s).

Transport of manganese into Saccharomyces cerevisiae.

The uptake of Mn2+ by Saccharomyces cerevisiae at the expense of endogenous sources of energy depends on the stage of culture development and is maximum in the middle of the exponential phase. The ability of cells to take up Mn+ is related to the content of intracellular potassium at all stages of growth, to the content of ATP during the exponential phase and it is not related to the content of inorganic polyphosphates. The uptake is inhibited by oligomycin (25 microgram/ml) by 50-85% and under anaerobic conditions by 10-50%, depending on the stage of growth, indicating the role of aerobic phosphorylation in the process. The uptake of Mn+ is apparently associated with a hydrolysis of low-molecular weight polyphosphates and ATP, as well as with the exit of K+ from cells.

Free and bound magnesium in fungi and yeasts.

The content of total, bound and osmotically free magnesium was estimated in various fungi and in the yeast Saccharomyces cerevisiae. Total magnesium increases at lower growth rates of Endomyces magnusii and Penicillium chrysogenum 140A as well as during the logarithmic stage of growth of Penicillium chrysogenum Q-176. The binding of magnesium requires orthophosphate, decreasing during lack of external phosphate when the intracellular concentration of free magnesium rises. The fungi were found to contain a novel form of bound magnesium, a polymeric magnesium orthophosphate (PO Mg), which appears to take part in the control of free magnesium level in Penicillium chrysogenum Q-176. The level of free magnesium is proportional to the growth rate of Endomyces magnusii and Penicillium chrysogenum Q-176 and 140A. Total, as well as free, magnesium changes less than three-fold as external Mg concentration is changed 13,000-fold. The magnesium is taken up against concentration gradients of 1 : 25 to 1 : 1300, the metal being distributed non-uniformly in the cells of Saccharomyces cerevisiae.

Increase of the anion and proton permeability of Saccharomyces carlsbergensis plasmalemma by n-alcohols as a possible cause of its de-energization.

The effect of n-alcohols on ATP-dependent generation of delta pH and Em across the plasma membrane vesicles of the yeast Saccharomyces carlsbergensis was investigated. The alcohols were shown to collapse delta pH and Em in the order C2 less than C3 less than C4 less than C5 less than or equal to C6 greater than or equal to C7 greater than C8 greater than C11, the dissipation of Em being more pronounced. Inhibition of the plasmalemma H(+)-ATPase was insignificant; at low alcohol concentrations its activity even increased. The basic reason for the toxic effect of the alcohols on the yeast cells was suggested to be due to the increase in the anion and proton permeability of the plasma membrane. Mg2+ partially prevented the increase in the plasmalemma ion permeability by the alcohols investigated.

Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope.

Saccharomyces cerevisiae cell envelope polyphosphatase was isolated in highly active and stable form by extraction from cells with zwittergent TM-314 followed by chromatography of the extract on phosphocellulose and QAE-Sephadex in the presence of 5 mM-MgCl2, 0.5 mM-EDTA and 0.1% Triton X-100. The enzyme possessed a specific activity of 220 U/mg and after 30 days retained 87% of its activity at -20 degrees C. Polyphosphatase molecular mass was determined to be about 40 kDa by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme hydrolysed polyphosphates with various chain lengths (n = 3-208), had low activity for GTP and did not split pyrophosphate, ATP and p-nitrophenylphosphate. On polyphosphates with chain lengths n = 3, 9 and 208, Km values were 1.7 x 10(-4), 1.5 x 10(-5) and 8.8 x 10(-7) M respectively. Polyphosphatase was most active and stable at pH 6.0-8.0. The enzyme showed maximal activity at 50 degrees C. The time of half inactivation of polyphosphatase at 40, 45 and 50 degrees C was 45, 10 and 3 min, respectively. In the absence of divalent cations and also with Ca2+ or Cu2+, the enzyme showed practically no activity. The ability of divalent cations to activate polyphosphatase was reduced in the following order: Co2+ > Mg2+ > Mn2+ > Fe2+ > Zn2+. Polyphosphatase was completely inhibited by 1 mM-ammonium molybdate and 50 microM-Zn2+ or Cu2+ (in the presence of Mg2+).

[Energizing plasmalemma of yeasts is necessary for activation of its H+-ATPase by glucose]. / Energizatsiia plazmalemmy drozhzhei neobkhodima dlia aktivatsii ee H+-ATPazy gliukozoi.

ATPase of yeast plasmalemma is known to be activated during incubation of cells or protoplasts with glucose. It has been shown that the level of ATPase activation is sharply decreased after pretreatment of cells or protoplasts with mercaptoethanol, dinitrophenol, gramicidin D, nigericin, or monensin. It is suggested that deenergization of yeast plasmalemma by monensin, nigericin, and mercaptoethanol as uncoupler plays a crucial role in the prevention of in vivo activation of plasma membrane ATPase by glucose. It is concluded that energization of yeast plasmalemma is necessary for activation of ATPase by glucose.

Mercaptoethanol and dithiothreitol decrease the difference of electrochemical proton potentials across the yeast plasma and vacuolar membranes and activate their H(+)-ATPases.

Mercaptoethanol and dithiothreitol (DTT) inhibited the acidification of external medium by Saccharomyces carlsbergensis cells and protoplasts during glucose oxidation. The inhibition was also observed when cells were incubated with mercaptoethanol or when mercaptoethanol and DTT were used to prepare protoplasts. Experiments with S. carlsbergensis plasma membrane vesicles and vacuoles showed these thiol reagents to inhibit ATP-dependent generation of delta pH and Em across plasma membrane vesicles and vacuoles but to activate their H(+)-ATPases. Mercaptoethanol and DTT are suggested to de-energize plasmalemma as well as tonoplast by increasing their H(+)-permeability and to disturb the cell ion homeostasis.

Vacuoles: main compartments of potassium, magnesium, and phosphate ions in Saccharomyces carlsbergenis cells.

The uneven distribution of Mg2+, K+, and phosphate in Saccharomyces carlsbergensis was demonstrated by the differential extraction of ions. Their concentrations were 5, 60, and 1 mM in the cytoplasm and 73, 470, and 110 mM in vacuoles, respectively. The intracellular gradients of these ions were 1:15, 1:8, and 1:110, respectively, across the tonoplast. The determination of free Mg2+ (1.35 mM in the cytosol and 20 mM in vacuoles) showed that the ion accumulation in vacuoles could not be explained by the higher degree of ion complexing in these organelles.