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.

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.

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.

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).

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.

A novel primary Ca(2+)-transport system from Saccharomyces cerevisiae.

A novel primary Ca(2+)-transport system in membranes from Saccharomyces cerevisiae is described. Ca2+ transport is strictly dependent on the presence of ATP; other nucleotides like GTP, UTP and CTP do not efficiently (< 10% of the rate of ATP) drive uptake. Transport is inhibited by sodium vanadate with an IC50 of 130 microM, but is insensitive to carbonylcyanide p-trifluoromethoxy-phenylhydrazone, valinomycin, gramicidin or calmodulin. Ca2+ accumulates in a free form and can be readily released by the Ca2+ ionophore A-23187 or by osmotic shock. The apparent Km values of transport activity for free Ca2+ was determined to be 0.11 microM and 5 microM for Mg.ATP, respectively. Taken together the results indicate that the Ca2+ transport described here does not belong to the plasma-membrane-type Ca(2+)-ATPase family but rather to the family of endomembrane-type ATPases. Cell-fractionation studies of crude membranes on sucrose gradient centrifugation have shown that the Ca(2+)-transport activity separates from marker enzymes for endoplasmic reticulum, vacuole, or plasma membrane and migrates with GDPase activity, a marker for the yeast Golgi complex.

[H(+)-ATPase from yeast vacuoles is inhibited by heparin]. / H(+)-ATPaza vakuolei drozhzhei ingibiruetsia geparinom.

Heparin (20-40 micrograms/ml) inhibits by 50% both the ATP hydrolase and the delta pH formation in isolated yeast vacuoles. The GTPase activity is not inhibited under these conditions. ATP prevents this inhibition, being added 10 min before heparin. Heparin suppresses the phosphorylation of vacuolar proteins in vitro. It is supposed that the protein kinase, similar to casein kinase I, participates in the regulation of activity of the vacuolar ATPase.

[Characteristics of polyphosphatase activity of vacuoles in Saccharomyces cerevisiae cells]. / Kharkteristika polifosfataznoi aktivnosti vakulei drozhzhevykh kletok Saccharomyces cerevisiae.

Vacuoles of the Saccharomyces cerevisiae yeast possess a polyphosphatase activity which differs from other known vacuolar phosphohydrolase activities by pH-optimum, sensitivity towards inhibitors and distribution between the tonoplast and vacuolar sap. The polyphosphatase activity is inhibited by EDTA, molybdate, ortho-vanadate and fluoride. Nearly 77% of this activity is located in the vacuolar sap, while 25%--in the tonoplast fraction. Both the soluble and membrane-bound polyphosphatase activities are maximal at pH 6.8-7.2. Bivalent metal cations stimulate this activity in the following order: Zn2+ > Mg2+ > Co2+ > Mn2+. Other ions (Fe2+, Cu2+, Ca2+) inhibit this activity at all concentrations tested. The polyphosphatase activity of both the vacuolar sap and the tonoplast increases 2.4-and 2-fold, respectively, with an increase in the degree of substrate polymerization--from n = 3 to n = 208.

Change in transport activities of vacuoles of the yeast Yarrowia lipolytica during its growth on glucose.

Vacuoles were isolated from Yarrowia lipolytica yeast cells taken at various growth phases under carbon or nitrogen limitation. Vacuoles from the cells at the logarithmic growth phase showed a high activity of vacuolar H(+)-ATPase (0.9-1.1 U/mg protein) and efficiently generated chemical proton gradient and membrane potential across the tonoplast. Ca(2+)- and citrate transport were found to be maximal at this growth phase. At growth retardation and then in the stationary phase all the parameters studied decreased irrespective of the method of growth limitation. The citrate-transporting activity of vacuoles completely disappeared at growth retardation, also irrespective of the limitation method and irrespective of whether yeast cells overproduced citrate in the culture medium. The citrate-transporting system of Y. lipolytica vacuolar membrane is concluded not to be involved in citrate efflux and this efflux is probably performed by the plasmalemma transport system.

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.

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.

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.

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.

[Phosphate and glucose accumulation by Pseudomonas cultures in relation to their arsenic resistance]. / Nakoplenie fosfata i gliukozy kul'turami Pseudomonas v zavisimosti ot ikh ustoichivostik mysh'iaku.

The effect of arsenite and arsenate on 14C-glucose and 32-P-phosphate transport was studied in the cells of Pseudomonas aeruginosa 561 sensitive to arsenite and in the cells of Pseudomonas putida 18 oxidizing arsenite and resistant to arsenic. Transport and accumulation of phosphate and glucose were inhibited in the presence of arsenite in the cells of P. aeruginosa 561 whereas arsenate inhibited only phosphate accumulation. Arsenite and arsenate had hardly any effect at the initial transport rate and on the overall accumulation of phosphate and glucose in the cells of P. putida 18. The resistance to arsenite is supposed to be caused by selective impermeability of the cellular membranes to arsenite and arsenate.