Isolation and characterization of polyphosphates from the yeast cell surface

Biochimica et Biophysica Acta, 760 (1983) 143-148

143

Elsevier BBA 21571

ISOLATION AND CHARACTERIZATION OF POLYPHOSPHATES FROM THE YEAST CELL SURFACE J.P.F. TIJSSEN, T.M.A.R. DUBBELMAN and J. VAN STEVENINCK

Department of Medical Biochemistry, Sylvius Laboratories, Wassenaarseweg 72, 2333 AL Leiden (The Netherlands) (Received April 8th, 1983)

Key words: Polyphosphate localization; Osmotic shock," (Yeast cell surface)

When cells of Saccharomyces fragilis are subjected to osmotic shock, they release a limited amount of inorganic polyphosphate into the medium, which represents about 10% of the total cellular content. The osmotic shock procedure causes no substantial membrane damage, as judged from the unimpaired cell viability, limited K + leakage and low percentage of stained cells. It is therefore suggested that this polyphosphate fraction is localized outside the plasma membrane. The released polyphosphate fraction differs from the remaining cellular polyphosphates in two respects: the mean chain length of the shock-sensitive fraction is significantly higher than that of the total cellular polyphosphates and its metabolic turnover rate, subsequent to pulsing with [32p]orthophosphate is much lower compared to the rest of the cellular polyphosphate. Incubation of intact cells with the anion exchange resin Dowex AG I-X4 results in the release of high molecular weight polyphosphates. These results suggest that the osmotic shock-sensitive polyphosphate fraction has specific characteristics in both its cellular localization and metabolism.

Introduction

Several studies suggest that a transport-associated phosphorylation mechanism is involved in the uptake of glucose and glucose derivatives in yeast [1-3]. Circumstantial evidence indicated that a polyphosphate fraction, localized outside the plasma membrane could play an essential role in this phosphotransferase system [4]. Umnov et al. [5] have presented experimental evidence for a similar role of polyphosphates in glucose transport in Neurospora crassa. Several studies suggest the localization of a polyphosphate fraction outside the plasma membrane of some microorganisms including yeast [6-11]. Recently, the existence of a peripherally localized polyphosphate fraction in Saccharomyces fragilis was confirmed by Toluidine blue metachromasia upon binding to yeast cells [12] and by 0304-4165/83/$03.00 © 1983 Elsevier Science Publishers B.V.

4',6-diamJdino-2-phenylindole fluorescence studies [131. Osmotic shock can release extracellular enzymes [ 14] and substrate-binding proteins [ 15] from yeast. In this communication it is shown that this procedure also liberates extracellular polyphosphates. Methods

Saccharomyces fragilis was grown on glucose as carbon source, as described before [16]. The yeast was washed thoroughly 3 times in large volumes of distilled water. Osmotic shock was carried out by preincubating 10% yeast (wet weight/volume) in 3 M NaC1/0.5 mM EDTA/5 mM fl-mercaptoethanol/ 60 mM sodium acetate (pH 6.5) at 30°C for 1 h. The cells were spun down and resuspended in 1

144 mM NaF/0.5 mM MgC12 and incubated at 0°C for 30 min, at a yeast concentration of 20% (w/v). After centrifugation the supernatant (shock fluid) was decanted. Incubation with Dowex AG l-X4 was carried out by gently stirring an unbuffered 20% (w/v) yeast suspension at 0°C with varying amounts of thoroughly washed Dowex AG I-X4. Polyphosphates were extracted from yeast by boiling as described by Weimberg and Orton [7], keeping warming-up times as short as possible, to avoid degradation of the polyphosphates. Polyphosphate was precipitated by adding 0.25 vol. saturated BaC12 and 0.25 vol. 3.5 M sodium acetate/acetic acid (pH 4.5) with subsequent incubation for 15 h at 4°C. The precipitate was suspended in 10 mM EDTA and the polyphosphates were bound to Dowex AG l-X4 [17]. The resin was collected on a glass wool plug and washed thoroughly with 10 mM EDTA. The polyphosphates were eluted with 3 M KC1. This eluate was applied to a 16 x 800 mm column, containing equal volumes of Sephadex G-50 and G-25. Separation of polyphosphates according to chain length took place during elution with a buffer, containing 100 mM KCI/10 mM EDTA/1 mM sodium azide/10 mM Tris-HC1 (pH 7.0). Calibration of the column was achieved by elution with synthetic polyphosphates; the chain length of the eluted fractions was calculated after determination by titration of the ratio between the amount of middle phosphate groups and end groups [ 18]. Orthophosphate in the presence of polyphosphate was determined by adding a 0.4 ml sample (containing up to 1 #mol orthophosphate) to 0.2 ml 10% (w/v) trichloroacetic acid and 0.6 ml 2.5% (w/v) ammonium molybdate (adjusted to pH 2.0 with H2504). The orthophosphate-molybdate complex was extracted with 2.4 ml 2-methylpropan-l-ol; 2 ml extract was added to 1 ml ethanol, 0.9 ml H20 and 1.0 ml 10 mM 1-amino-2-naphthol-4-sulfonic acid. After 10 rain, a stable blue colour had developed, which could be measured spectrophotometrically at 700 nm. Polyphosphate was measured after acid hydrolysis as described by Urech et al. [19]. Cell viability was measured by plating diluted suspensions on 2% agar plates, containing 0.5%

peptone, 0.5% yeast extract and 4% glucose. After 3 days incubation at 30°C, colonies were counted. Damaged cells were stained with uranyl nitrate and Ponceau red, as described previously [20]. Potassium was measured by flame photometry. Semi-permeable yeast cells were prepared, essentially as described by Jaspers et al. [21], by incubating a 10% (w/v) yeast cell suspension in the presence of chitosan at 0°C. Acid phosphatase, alkaline pyrophosphatase and invertase were assayed as described by Schwencke et al. [14], ethanol dehydrogenase as described by Urech et al. [19]. Results

Saccharornyces fragilis, grown for 20 h under standard conditions, contained about 30 /~mol polyphosphate (expressed in Pi equivalents) and 30 /~mol orthophosphate per g yeast (wet weight). The chain-length distribution of the total cellular polyphosphates is shown in Fig. 1. Yeast cells subjected to osmotic shock partially released some extracellular enzymes; 20% of the acid phosphatase activity, 32% of the alkaline pyrophosphatase activity and 18% of the invertase activity were released, all compared to the activity of intact cells. Ethanol dehydrogenase activity could not be detected in the shock fluid. In addition to these enzymes the shock fluid also contained orthophosphate and polyphosphates. This polyphosphate-release amounted to about 3 /xmol/g yeast (10% of the total cellular polyphosphate content). In order to evaluate the origin of the released polyphosphates it was necessary to establish whether membrane damage is induced by the osmotic shock procedure, especially since the yeast cells showed about a 12% leakage of cellular K + when osmotically shocked. A sensitive test for membrane integrity is to stain cells with uranyl nitrate and Ponceau red [20]. It appeared that in fresh, untreated yeast the number of stainable cells was 1-2%, whereas after osmotic shock this increased only to 2-5%. Two other observations also indicate that membrane damage is small: cell viability was unaffected by osmotic shock, as judged from plating experiments, and there was no cytosolic ethanol dehydrogenase activity detectable in the shock fluid.

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A standard procedure to render the yeast cell membrane semi-permeable is to treat the cells with basic marcomolecules such as chitosan and protamine [21,24]. Treatment of the yeast cells with chitosan induced leakage of K +, orthophosphate and polyphosphate, in a time- and chitosan concentration-dependent manner (Fig. 2). At low chitosan concentrations, K ÷ and orthophosphate leakage were much more pronounced than polyphosphate leakage. Treatment of intact yeast cells with relatively low amounts of Dowex AG I-X4 resulted in release of polyphosphate and orthophosphate from the cells, without concomitant K ÷ loss and without membrane damage, as judged from Ponceau red staining of the cells (Table I). At higher Dowexto-cell ratios the cells were severely damaged, as shown by increased staining of cells and leakage of K +, orthophosphate and polyphosphate (Table I). These results strongly suggest that treatment with low amounts of Dowex released a limited amount of polyphosphate from the cells, without inducing any appreciable membrane damage. The polyphosphate fraction, released by osmotic shock or Dowex incubation showed two specific characteristics: first, the mean chain length of this

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Fig. 1. Top: calibration of the Sephadex column utilizing synthetic polyphosphates with a mean chain length of 15 (O) and 35 (@). Bottom: chain length distribution of polyphosphates, isolated from a 20 h batch culture. • •, isolated by osmotic shock; O O , isolated by boiling.

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146 TABLE I EFFECTS OF D O W E X A G I-X4 ON S A C C H A R O M Y C E S FRAGIL1S 2.5 ml 20% unbuffered yeast suspension was incubated at 0°C during 15 min with varying amounts of Dowex A G I-X4, under gentle stirring. Dowex (g added)

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0.0 0.9 4.0 3.7 4,4 10.4 41.2 100

polyphosphate fraction was significantly greater than the mean chain length of the total cellular polyphosphates at all culture times studied, although both the total cellular polyphosphate concentration and the chain length distribution varied with the growth phase of the yeast (Fig. 3). Secondly, when yeast cells were incubated with a trace amount of highly labeled [32P]orthophosphate together with glucose or ethanol, the specific activities of shock-released orthophosphate and polyphosphate were consistently lower than the specific activities of the corresponding total cellular phosphates (Fig. 4). Discussion

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Fig. 4. Specific radioactivity of polyphosphate (top) and orthophosphate (bottom) after aerobic incubation of a 10% ( w / v ) yeast suspension at 25°C with trace amounts of [32p]orthophosphate in the presence of 4% ( w / v ) glucose or 2% ( v / v ) ethanol. The phosphates were isolated by: • 0, boiling after incubation with glucose; O O, osmotic shock after incubation with glucose; • A, boiling after incubation with ethanol; zx zx, osmotic shock after incubation with ethanol.

The localization of polyphosphates outside the plasma membrane of yeast cells has been suggested by many investigators [4,6-13]. A detailed study of this polyphosphate fraction seems indicated, considering its proposed role in transmembrane glucose transport [1,4]. The purpose of this study is to isolate and partially characterize this peripheral polyphosphate fraction. External enzymes and substrate-binding proteins can be selectively released from the yeast cell surface by osmotic shock [14,15,22,23,25]. This was confirmed by the present studies. Based on similar experiments Schwencke et al. [14] showed alkaline pyrophosphatase to be an extracellular

147

enzyme in Saccharomyces chevalieri. Our results indicate a similar extraceUular localization of at least part of this enzym activity in Saccharomyces

fragilis. Schwencke et al. [14] stressed the usefulness of osmotic shock procedures in studies on enzyme localization in yeast. Thus, it seems, a priori, likely that the polyphosphates, released by osmotic shock in our experiments, were localized outside the plasma membrane. Considering the relatively small size of these molecules, however, the possibility that, due to membrane damage, these polyphosphates are of intracellular origin, must be considered. The following arguments suggest that membrane damage is not responsible for the liberation of polyphosphate by osmotic shock. Severe damage must be assumed to render the membrane permeable to highly charged polyelectrolytes, such as polyphosphates. The likelihood of such severe damage is low, considering the low K ÷ leakage, the low percentage of Ponceau red stained cells and the unimpaired cell viability after osmotic shock. Furthermore, if the shock-released polyphosphates had leaked out via a damaged plasma membrane, it would also be expected that the mean chain length of the released fraction would be equal to, or smaller than the mean chain length of total cellular polyphosphates. In fact, the mean chain length of the released polyphosphates exceeds the average (Fig. 3). Leakage of cellular constituents through a damaged plasma membrane was simulated by incubating yeast cells with chitosan. As shown in Fig. 2, chitosan-induced membrane damage leads primarily to leakage of K ÷. Only rather high chitosan concentrations cause an appreciable leakage of polyphosphates, with a concomitant K ÷ loss exceeding 50%. In contrast, osmotic shock causes a much more pronounced polyphosphate release, together with much lower K ÷ leakage. This again suggests that the polyphosphates, released by osmotic shock, did not leak out via the plasma membrane. Most convincing are also the results obtained by incubation of yeast cells with Dowex AG l-X4. Treatment with low amounts of Dowex released high molecular weight polyphosphates from the cells, without any concomitant K ÷ leakage (Table

I). This indicates that these polyphosphates were actually localized outside the plasma membrane. The origin of the relatively large amount of orthophosphate liberated by osmotic shock and treatment with Dowex (Table I) is not yet clear. The fact that the specific activity of this orthophosphate after pulsing with [32p]orthophosphate is lower compared to the total cellular orthophosphate suggests that it may (at least, partially) be generated by degradation of released polyphosphates (Fig. 4). The shock-released polyphosphates differ from the remaining cellular polyphosphates in two respects: the mean chain length is higher, and the metabolic turnover after pulsing with [32p]orthophosphate is lower. These characteristics suggest that biosynthesis of this fraction and the other cellular polyphosphates proceeds via different metabolic pathways. In previous publications on the metabolism of polyphosphates in baker's yeast it was established that pulsing with [32p]orthophosphate first led to the appearance of radioactivity in a high molecular weight polyphosphate fraction, with a subsequent increase in the specific activity of polyphosphates with shorter chain lengths [26,27]. In preliminary experiments with Saccharomyces fragilis we established a similar pattern (results not shown). This suggests that long-chain polyphosphates should be considered as the precursors of shorter-chain polyphosphates, generated by progressive degradation. The low specific activity of the relatively long-chain polyphosphates, released by osmotic shock (Fig. 4) is not in accordance with this general metabolic scheme, suggesting biosynthesis of this fraction via a different metabolic pathway. This is conceivable, since different routes for polyphosphate synthesis in various microorganisms have been described. For instance, a polyphosphate kinase, catalyzing the reaction of ATP + (PP), ~ ADP + (PP),+ and an enzyme, catalyzing the reaction of 1,3-diphosphoglycerate + (PP), ~ 3-phosphoglycerate + (PP)n + l have been identified [6]. If this interpretation is valid, polyphosphates in yeast would not exhibit compartmentation regarding only cellular localization, but also with respect to metabolic behaviour.

148

Acknowledgement We thank Miss H.W. Beekes for skilful technical assistance.

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