Regulation of Acid Phosphatase Synthesis in Baker’s Yeast Protoplast by Phosphate Compounds

The regulation of acid phosphatase synthesis by various phosphate compounds was examined in Baker’s yeast protoplasts. Synthesis was repressed by inorganic phosphate and phosphomonoesters. Phosphomonoesters were hydrolysed by a small amount of non-specific acid phosphatase present in the protoplast membrane. The inorganic phosphate that was liberated and incorporated into protoplasts probably repressed acid phosphatase synthesis. Phosphodiesters, such as 3′, 5′-cyclic AMP, 3′, 5′-cyclic CMP and 3′, 5′-cyclic GMP, promoted acid phosphatase synthesis. The effect of 3′, 5′-cyclic AMP was not to overcome hexose repression, because high hexose did not repress acid phosphatase synthesis. 3′, 5′-cyclic AMP did not overcome repression of the enzyme synthesis by inorganic phosphate. From these observations 3′, 5′-cyclic nucleotides probably had some effect on the yeast acid phosphatase-synthesizing system but the exact role of the nucleotides is obscure.     

Ultracytochemical characterization of non-specific acid phosphatase activities in Saccharomyces cerevisiae.

Glutaraldehyde prefixation causes a considerable inactivation of the acid phosphatase of yeast protoplasts in dependence on the duration of aldehyde influence. Lead ions necessary for ultracytochemical demonstration effect a still stronger inhibition of enzymatic activity. Prefixation, however, protects the enzyme from further inhibition by lead. At pH 4.4 in intact cells acid phosphatase activities are mainly localized in the periplasmic space and in vesicles fused with the plasma membrane. The cell wall and cytoplasm usually remain free of reaction products. On the cell surface activities are found in form of globular lead deposits. At pH 5.2 and 6.3 the periplasmic activity appears decreased compared to that at lower pH values and the intracellular activity is increased. The plasma membrane of protoplasts is completely free of precipitates. The intracellular activity sites of protoplasts (cisternae of endoplasmic reticulum and/or Golgi-like system, small vesicles, central vacuole, nuclear envelope) are the same as for intact cells. The occurrence of at least two forms of acid phosphatase in S. cerevisiae id deduced.     

Biosynthesis of acid phosphatase of baker's yeast. Characterization of a protoplast-bound fraction containing precursors of the exo-enzyme.

1. Yeast protoplasts, secreting acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) contain a small amount of firmly bound enzyme, even after lysis (Van Rijn, H.J.M., Boer, P. and Steyn-Parvé, E.P. (1972) Biochim. Biophys. Acta 268, 431-441). The major part (70%) of this protoplast-bound acid phosphatase can be solubilized by nonionic detergents, such as Triton X-100. 2. The kinetics of radioactive amino acid incorporation in the solubilized and in the secreted enzyme has been estimated by pulse-chase labelling of secreting protoplasts, followed by fractionation and counting radioactivity in the enzyme band in polyacrylamide gels after electrophoresis at pH 5.0. A precursor-product relationship between the Triton X-100-extractable fraction of the protoplast-bound acid phosphatase and the secreted enzyme is apparent. 3. The solubilized acid phosphatase is essentially indistinguishable from the secreted enzyme with regard to a number of enzymatic properties and its stability towards pH and temperature. Both enzymes also behave alike on polyacrylamide-gel electrophoresis, producing a single acid phosphatase band with glycoprotein character and comparable mobility. 4. A striking difference is seen in isopycnic equilibrium sedimentation in CsCl: the secreted acid phosphatase is homogeneous, with a buoyant density of p equals 1.47 g/cm3, while the Triton X-100-extractable part of the protoplast-bound acid phosphatase is heterogeneous; besides heavier material a major component with buoyant density of p equals 1.37 g/cm3 is always visible.     

Cytochemical and biochemical determination of acid phosphatase activity in yeasts]

Optimal conditions of the cytochemical assay for acid phosphatase in protoplasts and whole cells of S. cerevisiae have been described. Dimethyl sulfoxide was used to increase the permeability of the yeast cell envelope. In the yeast cells, grown up to the end of the exponential phase, acid phosphatase is shown to be located mainly in the central vacuole and on the cell envelope surface. A considerable activity of acid phosphatase is demonstrable on the surface of the plasma membrane and within adjacent vesicles that represent, presumably, part of the endoplasmic reticulum. Acid phosphatase can be considered as a marker enzyme for yeast cell vacuoles.     

Isolation and characterization of four cell wall purple acid phosphatase genes from tobacco cells

Four full-length cDNAs were isolated from a cDNA library prepared from tobacco cultured cells and designated NtPAP4, NtPAP12, NtPAP19 and NtPAP21, which could correspond to purple acid phosphatase (PAP). Levels of both NtPAP12 and NtPAP21 mRNA in the protoplasts immediately increased after the protoplasts were transferred to a medium for cell wall regeneration, and the accumulation of the mRNA was correlated with cell wall regeneration for 3 h. It is likely that the NtPAP12 and NtPAP21 gene products are wall-bound PAPs at the early stage of regenerating walls in tobacco protoplasts.     

Asparagine-linked carbohydrate does not determine the cellular location of yeast vacuolar nonspecific alkaline phosphatase

The nonspecific alkaline phosphatase of Saccharomyces sp. strain 1710 has been shown by phosphatase cytochemistry to be exclusively located in the vacuole, para-Nitrophenyl phosphate-specific alkaline phosphatase is not detected by this procedure because the activity of this enzyme is sensitive to the fixative agent, glutaraldehyde. To determine whether the oligosaccharide of nonspecific alkaline phosphatase is necessary to transport the enzyme into the vacuole, protoplasts were derepressed in the absence or in the presence of tunicamycin, an antibiotic which interferes with the glycosylation of asparagine residues in proteins. The location of the enzyme in the tunicamycin-treated protoplasts, as determined by electron microscopy and subcellular fractionation, was identical to its location in control protoplasts. In addition, carbohydrate-free alkaline phosphatase was found in vacuoles from tunicamycin-treated protoplasts. Our findings indicate that the asparagine-linked carbohydrate moiety does not determine the cellular location of the enzyme.     

Gibberellic Acid Controls the Secretion of Acid Phosphatase in Aleurone Layers and Isolated Aleurone Protoplasts of Avena fatua

The role of gibberellic acid (GA₃) in controlling the secretion(across the plasma membrane) and release (through the cell wall)of acid phosphatase (E.C. 3.1.3.2 [EC] .) from Avena aleurone layershas been investigated. Evidence from this comparative studywith intact aleurone layers and isolated aleurone protoplastsreveals that the secretion of acid phosphatase is under GA₃control. The mechanism underlying secretion and release of theenzyme from aleurone cells is discussed. Key words: Avena fatua, Acid phosphatase, Aleurone protoplasts, Gibberellic acid, Secretion     

Secretion of an active nonglycosylated form of the repressible acid phosphatase of Saccharomyces cerevisiae in the presence of tunicamycin at low temperatures

The role of mannan chains in the formation and secretion of active acid phosphatase of yeast (Saccharomyces cerevisiae), a repressible cell surface mannoprotein, was studied in yeast protoplast systems by using tunicamycin at various temperatures. At 30 degrees C, tunicamycin-treated protoplasts did not produce active acid phosphatase; however, at 25 or 20 degrees C they formed and secreted active enzyme. This form of acid phosphatase gave 59-, 57-, and 55-kDa bands on SDS-PAGE which neither bound to concanavalin A Sepharose, nor changed in molecular weight upon treatment with endoglycosidase H, indicating that the peptides are nonglycosylated. The nonglycosylated form, like its glycosylated counterpart, is a dimer on the basis of gel permeation chromatography. The Km for para-nitrophenyl-phosphate and Ki for inorganic phosphate of both glycosylated and nonglycosylated acid phosphatases were almost the same. These results suggested that 1) the conformation of the nonglycosylated acid phosphatase secreted at low temperatures is probably identical with that of the glycosylated one, and 2) the conformation of acid phosphatase is very important for its secretion. The rate of intracellular transport of nonglycosylated acid phosphatase is about one-fourth that of the glycosylated enzyme, indicating that glycosylation facilitates the transport of acid phosphatase proteins.     

Glycoprotein nature of yeast alkaline phosphatase. Formation of active enzyme in the presence of tunicamycin.

The nonspecific alkaline phosphatase of yeast (Saccharomyces strain 1710) has been purified by ion exchange, hydrophobic, and affinity chromatography. This vacuolar enzyme has a molecular weight of 130,000 and is composed of subunits (probably of 66,000 molecular weight). It also has a small quantity of covalently associated carbohydrate; hydrolysis yielded mannose and glucosamine. The endo-beta-N-acetylglucosaminidase of Streptomyces plicatus released carbohydrate indicating that the latter was attached to protein through an N-acetylglucosaminylasparginyl bond. Synthesis of active alkaline phosphatase by yeast protoplasts is not depressed by tunicamycin, an inhibitor of dolichol-mediated protein glycosylation. Unlike the enzyme normally produced, the alkaline phosphatase which is formed in the presence of the antibiotic does not interact with concanavalin A and, therefore is deficient in or lacking carbohydrate. We infer that there is no regulatory link in yeast between the glycosylation of a protein and its synthesis. The fact that other Asn-GlcNAc-type glycoprotein enzymes of yeast such as acid phosphatase are not produced in their active forms by tunicamycin-treated protoplasts may mean that, as unglycosylated proteins, they cannot be correctly folded or processed. Protoplasts derepressed for phosphatase production contained substantial amounts of a second alkaline phosphatase which differed from the purified enzyme in substrate specificity, sensitivity to calcium, and reactivity with concanavalin A.     

Gibberellic acid controls specific acid-phosphatase isozymes in aleurone cells and protoplasts of Avena fatua L.

In the presence of gibberellic acid (GA₃) aleurone layers and isolated aleurone protoplasts of Avena fatua accumulate specific isozymes of acid phosphatase (EC 3.1.3.2). Some of these may be involved in mobilizing aleurone-grain phosphate reserves during germination. The hormone also controls secretion of other specific molecular forms of the enzyme that probably assist in endosperm hydrolysis. The accumulation and secretion of putative cell-wall-associated isozymes are stimulated by the action of GA₃ in isolated protoplasts. This effect however, is apparently over-ridden in the intact tissue, possibly by a cell-wall-based feedback mechanism.Abbreviations GA₃ gibberellic acid - pI isoelectric point(s)     

Phosphorylation and Dephosphorylation of Guard-Cell Proteins from Vicia faba L. in Response to Light and Dark

Phosphorylation and dephosphorylation of proteins were investigated in guard-cell protoplasts from Vicia faba L. When guard-cell protoplasts were incubated with 32Pi in the dark for 80 min, several proteins, with molecular masses of 42, 40, 34, 32, 26, and 19 kD, were phosphorylated. Illumination of the dark-adapted protoplasts with red light caused dephosphorylation of the 26-kD protein, but there was no detectable change in levels of phosphorylation in other proteins. In the dephosphorylation of the 26-kD protein, far-red light of 730 nm was most effective, but when the light was turned off, the protein was phosphorylated to the original level within 10 min. Subcellular fractionation of guard-cell protoplasts indicated that the 26-kD protein was located in the chloroplast. The migration pattern of the 26-kD protein was exactly the same as the light-harvesting Chl a/b protein complex of photosystem II (LHCPII) from Vicia mesophyll cells on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dephosphorylated 26-kD protein was phosphorylated by adding sodium hydrosulfite, a strong reducing agent, under the far-red illumination of guard-cell protoplasts. The magnitude of dephosphorylation by red light (660 nm) was increased by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an electron transfer inhibitor of photosystem II (PSII). Light-induced dephosphorylation was inhibited by 1 nM okadaic acid, an inhibitor of serine/threonine protein phosphatase. From these results, it is concluded that the 26-kD protein is LHCPII and that LHCPII is present mostly in the phosphorylated form in the dark and is dephosphorylated by type 2A protein phosphatase under the light absorbed by photosystem I in Vicia guard-cell protoplasts.     

Tunicamycin--an inhibitor of yeast glycoprotein synthesis

Tunicamycin, a glucosamine-containing antibiotic, halted synthesis of the external glycoproteins invertase, acid phosphatase and mannan by yeast protoplasts within 30 min; formation of two intracellular proteins, alpha-glucosidase and alkaline phosphatase, and of glucan continued at the control rate for at least 60–80 min. No accumulation of mannan-free acid phosphatase or invertase was evident in treated cells. Utilization of hexoses and incorporation of ¹⁴C-amino acids into protein were not affected. Incorporation of ³H-glucosamine into trichloroacetic acid-insoluble products was only partially reduced. In yeast tunicamycin acts primarily as an inhibitor of glycoprotein synthesis and not of general glucosamine metabolism.     

Isotopic Determination and Optimum Reaction Conditions of Pantothenic Acid Synthetase

When yeast protoplasts that were producing repressible acid phosphatase (r-APase) were treated with tunicamycin (TM), three specific proteins of 59k, 57k, and 55k daltons were accumulated in the membrane fraction in addition to the usual membrane proteins and these proteins were not detected in the secreted fraction. These proteins were immunoprecipitated with anti r-APase antiserum. Their molecular sizes were almost the same as those endo-H treated r-APase. Therefore these proteins were considered to be nonglycosylated forms of r-APase proteins. These results proved that nonglycosylated forms of r-APase produced by TM-treatment were not secreted by yeast protoplasts.     

Synthesis of beta-glucanase and other extracellular proteins by cells and protoplasts of the yeast Pichia polymorpha: effect of 2-deoxy-D-glucose.

The synthesis of beta-glucanase either by cells or by protoplasts of the yeast Pichia polymorpha has been found to occur in the presence of 2-deoxy-D-glucose in the growth medium. On the other hand, the synthesis of typical extracellular proteins such as invertase and acid phosphatase is strongly affected by the presence of the drug. The degree of inhibition is, however, directly related to the 2-deoxy-D-glucose concentration.     

Involvement of an acid phosphatase on cell wall regeneration of tobacco protoplasts

Tobacco protoplasts begin to regenerate their own cell walls, the major components of which are β-glucans, soon after they are transferred into an adequate medium. During the cell wall regeneration the protoplasts secrete two isoforms of acid phosphatase (APase) in time-dependent manner. We determined that one of the isoforms, the Brefeldin A (BFA) sensitive one, is the cell wall resident APase (WP-II) by immunoblotting of the isoform with anti-WP-II antibody. We hypothesized that the WP-II may participate in the deposition of β-glucan microfibrils on the protoplast surface during cell wall regeneration. In order to examine this hypothesis, the protoplasts were cultivated in the cell wall regeneration medium containing the same amount of the BFA-sensitive APase (230 µg protein) as is secreted by the observed number of protoplasts (1.4 × 10⁵ protoplasts) per plate (30-mm-diameter) during a 3-h cultivation after transfer to the cell wall regeneration medium. The addition of WP-II to the cell wall regeneration medium stimulated the deposition of β-glucan microfibrils on the surface of the protoplasts during cell wall regeneration. To determine the stimulative effect of the 60 kDa polypeptide of WP-II, protoplasts were cultivated in the medium containing the amount of anti-WP-II IgG (230 µg protein) equivalent to the BFA-sensitive APase. These results suggested that the 60 kDa polypeptide of WP-II is the BFA-sensitive APase which is responsible for the enhanced deposition of β-glucan microfibrils on the surface of the protoplasts.     

Electrophoretic Studies on Plant Protoplasts II. Relative Amounts of Various Charged Groups on the Surface of Barley Mesophyll Protoplasts

Electrophoretic mobilities of barley mesophyll cell protoplastsmodified by chemical and enzymatic treatments were measuredin media at various pH values to elucidate the contributionof phosphate, carboxylate and amino groups to the surface chargedensity. Existence of these charged groups was confirmed byresults of treatment of protoplasts with glutaraldehyde (foramino groups), acid phosphatase (for phosphate groups) and l-ethyl-3-(3-dimethylaminopropyl)carbodi-imidetogether with glycine methyl ester (for carboxylate groups).The relative amounts of these groups were estimated from thecurves of surface charge density () vs. surface pH (pH_s) ofthe treated protoplasts, in terms of simplified acid-base dissociationcurves. The estimated ratio of the amounts of phosphate, carboxylateand amino groups was approximate 0.5 :0.5 : 0.7 for the native(unmodified) barley mesophyll cell protoplasts, when the totalnegative charge on the native protoplasts was assumed to be1. (Received January 9, 1989; Accepted April 28, 1989)     

A procedure for isolating vacuoles from leaves of the halophyte Suaeda maritima

Abstract. A method is described here for isolating protoplasts and vacuoles from leaves of the halophyte Suacda maritima. Integrity of the protoplasts and vacuoles was tested by staining and shown to be more than 75%, while use of biochemical markers, staining and light microscopy suggested a high degree of purity of the vacuoles. Phosphatase and NADH cytochrome- c -reductase were associated with vacuoles; phosphatase showed an eight-fold enrichment and NADH cytochrome- c -reductase a 3.5-fold enrichment relative to protoplasts. The vacuoles contained only 15% of the protein in protoplasts.     

Two isoforms of acid phosphatase secreted by tobacco protoplasts: differential effect of brefeldin A on their secretion

Summary The effects of brefeldin A (BFA) on the secretion of acid phosphatase (APase) by tobacco protoplasts were investigated. Secretion of APase was inhibited by BFA in a dose-dependent manner, with a concomitant intracellular accumulation of the enzyme. The secreted APase was composed of two isoforms. BFA (10/ g/ml) inhibited the secretion of one of the isoforms without inhibiting that of the other, and this phenomenon explains the partial inhibition of APase secretion as a whole. The inhibition of APase secretion was accompanied by changes in the morphology of the Golgi apparatus and also by an increment in massdensity of cells.Abbreviations APase acid phosphatase - BFA brefeldin A - CHX cycloheximide - PAGE polyacrylamide gel electrophoresis     

Acid phosphatase and adenosine triphosphatase activities in the cell wall of baker's yeast.

In order to establish whether a specific adenosine triphosphatase is present in yeast cell wall, hydrolysis rates for p-nitrophenylphosphate (acid phosphatase activity) and for ATP (ATPase activity) were compared under various conditions. Rate determinations were made with both, intact cells and with preparations containing secreted enzymes from protoplasts. Acid phosphatase and ATPase activities had the same pH profile and were susceptible in the same way to the repression by orthophosphate and to the inhibition by 2-deoxyglucose. The Lineweaver-Burk plot shows biphasic kinetic behaviour for the hydrolysis of either p-nitrophenylphosphate or ATP. This suggests the existence of two enzymes with different affinities for the substrates, or one enzyme with at least two active sites. The two activities differ in thermostability and only one activity could be completely abolished by heat treatment. The thermostable enzyme activity had K-m values of 0.475 mM for p-nitrophenylphosphate, and 0.040 mM for ATP. ATP behaved as a partially competitive inhibitor of p-nitrophenylphosphate hydrolysis. Substrate competition studies showed that only a non-specific acid phosphatase is responsible for the hydrolysis of ATP.