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Polyphosphate synthesis in yeast
Biochimica et Biophysica Acta, 1010 (1989) 191-198 Elsevier
191
BBA12399
Polyphosphate synthesis in yeast J. Sehuddemat, R. de Boo, C.C.M. van Leeuwen, P.J.A. van den Broek and J. van Steveninek Sylvius Laboratories, Department of Medical Biochemistry, Leiden (The Netherlands) (Received 18 August 1988)
Key words: Polyphosphate synthesis: Phosphate starvation; (S. cerevisiae); (K. marxianus) Polyphosphate synthesis was studied in phosphate-starved cells of Saccharomyces cerecMae and gluyveromyces marxianus. Incubation of these yeasts for a short time with phosphate and either glucose or ethanol resulted in the formation of pulyphosphate with a short chaGn length. With increasing incubation times, polypbosphates with longer chain lengths were formed. Pulypbosphates were synthes|Ted faster during incubation with glucose than with ethanol. Antimycin did not affect the glucose-induced polypbosphate synthesis in either yeast. Using ethanol as an energy source, antimycin A treatment blocked both polypbosphate synthesis and accumulation of ortbopbosphate in the yeast $. cereo~/ae. However, in K. marManus, pulyphosphate synthesis and orthopbosphate accumulation proceeded n,~rmaily in antimyein-treated ce|is, suggesting that endogenous reserves were used as energy source. This was confil~ned in experiments, conducted in the absence of an exogenous energy source.
introduction Polyphosphates, which are linear polymers of orthophosphate in anhydrous linkage, are present in large amounts in yeasts, growing on a complete medium with a high orthophosphate content. Conversely, when phosphate-rich cells are grown in a medium lacking phosphate (phosphate starvation), polyphosphate disappears. Phosphorus starvation also causes derepression of an acid phosphatase located at the cell surface [1,2]. Simultaneously, a high-affinity phosphate transport system appears [3] (for a model of phosphate metabolism and phosphatase regulation in S. cerevisiae see Ref. 2). When phosphate-starved cells are inoculated in a medium containing orthophosphate and an energy source, the cells assimilate phosphate and condense it to polyphosphate [4-7]. The presence of K + and Mg 2+ stimulates this process [6-8]. Under these conditions, the polyphosphate content can exceed 2370 of the cellular dry weight [7]. This effect is known as hypercompensation. Polyphosphates in yeast can be divided in two general classes: the acid-soluble polyphosphates, with a mean chain length of four orthophosphate units, and acid-insoluble polyphosphates, reaching chain lengths Correspondence: P.J.A. van den Brock, Sylvius Laboratories, Department of Medical Biochemistry, P.O. Box 9503, 2300 RA Leiden, The Netherlands.
of more than 260 orthophosphate units [7,9]. From in vivo experiments, Langen and Liss reported that during polyphosphate biosynthesis, exogenous [32P]orthophosphate ([32p]Pi) was first incorporated mainly in he most polymerized polyphosphate (h = 260). The shorter polyphosphates would be formed as secondary products, by degradation of the high molecular weight polyphosphate. Hydrolysis of the most polymerized form would yield the lower molecular weight polyphosphate with a mean chain length of 55 orthophosphate units. This fraction would be hydrolyzed to a polyphosphate with a mean chain length of 20 units. Finally, the lowest molecular weight polyphosphate would be formed (h = 4), which is hydrolyzed to orthophosphate [10,11]. These results, viz. formation of short chains from long chains of polyphosphate, were confirmed by Lusby and McLaughlin [12]. However, in more recent years it has become clear that specific fractions of polyphosphate represent various pools of polyphosphate, differing in their localization in the cell [1,13,14]. Besides the synthesis of the polyphosphate fractions as described above [10,11], biogenesis related to the metabolism of specific cellular compartments is also found [1,13,14]. The group with mean chain length of 20 orthophosphate residues, which is, at least partly, located in the nucleus, is suggested to be sy,thesized from pyrophosphate, formed during RNA synthesis [1,13,14], High molecular weight polyphosphates are supposed to be located at the cell pc-
0167-4889/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
192 riphery. Their biosynthesis is apparently related to cell wall synthesis [1,13,15]. In the fungus Neurospora crassa, the low molecular weight polyphosphate (~ = 4), which is located in the cytoplasm, seems to be synthesized by means of the glycolytic enzyme 1 3-diphosphoglycerate: polyphosphate phosphotransferase. This enzyme transfers a phosphoryl group from 1,3-:~iphosphoglycerate to polyphosphate instead of to ADP [1,13,14]. In this paper, the biosynthesis of polyphosphate was studied in phosphate-starved cells of S. cerevisiae and K. marxianus, supplied with different phe~sphate concentrations and energy sources. In all cases, polyphosphate appeared to be synthesized from chains with short lengths to chains with long lengths. The rate of polyphosphate synthesis was dependent on the energy source. Materials and Methods
The yeasts S. cerevisiae strain Delft I (CBS 1172) and K. marxianus (CBS 397) were grown for 20 h on a synthetic medium with 270 glucose as a carbon source, as described before [16]. For phosphate starvation, 0.5 g wet weight of yeast was incubated for 15-16 h in 250 ml of this synthetic medium, lacking phosphate, with 170 glucose as carbon source. The suspension was incubated at 20°C on a GFL rotary shaker. Subsequently, the cells were collected by centrifugation and washed three times with distilled water. To study polyphosphate synthesis, a 1070 (wet w / v ) suspension of phosphate-starved cells was made in incomplete synthetic medium, lacking phosphate and vitamins. In some experiments, the respiratory capacity was blocked by adding 20/tg antimycin A per g wet wt. of yeast to the incubate. The suspensions were pre-incubated for 7 min under aerobic conditions before 170 (w/v) glucose or 270 (v/v) ethanol was added as energy source. After another 6 rain of pre-incubation, polyphosphate synthesis was started by addition of Kt~.2PO4 and 10/tCi [32PIpi per ml. Polyphosphates were extracted and purified as described by Clark et al. [171 with small modifications. The acid-soluble polyphosphates were extractec~ by suspending 0.1 g (wet wt) of yeast in 0.3 ml 270 uichloroacetic acid. The suspension was centrifugated fer 5 rain at 1200 × g and the pellet was resuspended in 1 ~1 0°770 trichloroacetic acid/6770 acet,ne. The suspension was centrifuged for 5 min at 1200 × g and the pellet was washed with 1 ml 6770 acetone. The three supernatants were combined and extracted with 2.3 ml phenol/ chloroform (90 g pl'enol solubilized in 10 ml water plus 100 ml chloroform and subsequently saturated with 0.1 M ammonium acetate pH 6.5). This fraction is called the trichloroacetic acid/acetone fraction. The long-chain soluble polyphosphates and the polyphosphates from
the volutine granule were extracted by suspending the remaining pellet in 0.5 ml 2 mM EDTA, followed by titration with LiOH to pH = 7. Subsequently, 0.2 ml phenol/chloroform was added and the suspension was shaken vigorously. The supernatant obtained after 10 min centrifugation at 1200 × g will be indicated as the EDTA/LiOH fraction. Polyphosphates were separated on 1570 polyacrylamide gels as described by Wood and co-workers [18-20]. The ratio acrylamide to bis-acrylamide was always 20: 1. As the chain lengths of polyphosphate can be estimated by counting bands [18], a sample containing high molecular •iveight polyphosphate was subjected to partial hydrolysis in 25 mM HCI for 15 min at 100°C and used as a marker. The different lanes contain the same amount of 32p. Therefore, each lane shows the relative ratio between the different chain lengths of polyphosphates. For autoradiography, the gels were packed in plastic foil and exposed to X-ray film at - 7 0 o C. Parallel with the radioactive experiment, the changes in the polyphosphate, orthophosphate and ATP concentrations were measured :in a similar experiment, but without addition of [32pIPt. For ATP determination, samples from the incubate were mixed with 4 volumes of ethanol. For determination of the polyphosphate and orthophosphate concentrations, the cells were washed with ice-cold water and resuspended in water. After boiling the suspension for 2 rain, the polyphosphate and orthophosphate concentrations were determined in the supernatant. The amount of polyphosphate was determined spectrophotometrically, after acid hydrolysis to orthophosphate at 100 °C as described by Lohmann and Langen [21], by mixing 2 ml of sample with 1 ml 2.570 ammonium molybdate in 2 M HCI and 200 ttl 2 mM 1-amino-2-naphthol-4-sulfonic acid. After 30 rain, the absorbance was measured at 700 nm. The amount of orthophosphate was measured in the same way as polyphosphate, but without hydrolysis at 100°C. The ATP concentrations in extracts were measured with the luciferin/luciferase method [22]. The presence of glucose in the medium was determined, using the Boehringer Combur test (detection level 2.2 raM). [ 32P]Orthophosphate and [32P]ATP were purchased from Amersham International. Ribonuclease A (RNAse) was obtained from Boehringer. Results
To study polyphosphate synthesis in yeast, a mild extraction procedure and a good method for separating the polyphosphates with different chain length are indispensible. Recently, a relatively mild polyphosphate isolation method, coupled to a separation of the poly-.
193 TABLE l The amounts of polyphosphate and orthophosphate extracted from S. cereoisiae and K. marxianus using different extraction methods
Three different extraction procedures are compared. Extraction 1: the extraction procedure according to Clark et el. [17]. Extraction 2." bolting the cells in water as described by Weimberg and Orton [24]. Extraction 3: the extraction in five steps, described by Liss and Langen [23]. Orthophosphate (Pi) is expressed in /tmol/g wet wt of yeast. Polyphosphate (Pn) is expressed in /tmol orthophosphate units/g wet wt. of yeast. Extraction method
S. cerevisiae
K. marxianus
Pi
Pn
Pi
Pn
15.8
0
22
0
1.8
19.2
0.5
14.7
2. (Boiling)
21.4
20.5
28.6
14.6
3. (Liss and Langen)
24
11.2
29.8
13.8
1. Trichloroacetic acid/ acetone fraction 1. E D T A / L i O H fraction
phosphates by gel electrophoresis has been described by Wood and co-workers [17-20] for Propionibacterium shermanii. To determine whether this extraction procedure can also be used for the isolation of polyphosphates from K. marxianus and S. cerevisiae, a comparison was made between this method, the extraction method of Liss and Langen [23] and the extraction with boiling water, described by Weimberg and Orton [24]. The data from Table I indicate that there is no significant difference between the amounts of polyphosphate and orthophosphate extracted with boiling water or by the method of Clark et al. [17]. The isolation procedure of Liss and Langen extracts the same amount of phosphate from the cells as the other two methods, but there seems to be a shift in the amount of polyphosphate to orthophosphate in S. cerevisiae. This may be caused by hydrolysis of polyphosphate. Table I also shows that, in contrast to the results found for Propionibacterium shermanii, in yeast the tricMoroacetic acid/acetone fraction contained no polyphosphates. Incubating phosphate-starved yeast cells with an energy source orthophosphate and [a2p]p i followed by extraction and gel electrophoresis also confirmed that the trichloroacetic acid/acetone fraction contained a negligible amount of labeled polyphosphates. Incubating the polyphosphate containing EDTA/LiOH fractions with RNAse did not alter the gel pattern, indicating that no contaminant labeled RNA was present on the gel. These data show that this mild extraction and separation procedure is readily applicable to yeast. In further experiments, it appeared that the fractions, obtained with the extraction method of Langen and Liss [23], could not be analyzed by gel electrophoresis, due to the high ionic strength of the fractions. When phosphate-starved cells of S. cerevisiae or K.
marxianus were transferred to a medium containing 10 mM phosphate, [32P]Pi and 170 glucose, the cells almost immediately started to synthesize polyphosphate. Fig. 1 shows that synthesis proceeded flom chains with a short length to chains with a long length. After approx. 17 min, when all the glucose was consumed, the composition of the polyphosphate pool rearranged, shifting to a shorter mean chain length. When the respiratory capacity of the phosphate-starved cells was blocked by treating the cells with antimycin A, the pattern of polyphosphate synthesis was exactly the same as in the absence of antimycin A (not shown). In a parallel experiment, the changes in polyphosphate, orthophospimte and ATP concentrations were measured. Fig. 2 shows that there was no net consumption of polyphosphate when the glucose was exhausted from the medium. Moreover, antimycin did not significantly influence the polyphosphate and orthophosphate levels. It should be noticed, however, that in contrast to K. marxianus, in S. cerevisiae the ATP content was sensitive to the presence of antimycin.
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Fig. 1. Polyphosphate synthesis in phosphate-starved cells of S. cerevisiae (A) and K. marxianus (B) incubated with 1% glucose, 10 mM phosphate and [32PIPi, for various lengths of time. The compounds in the extracts were separated on 15% polyacrylamide gels containing 1 M urea. The lanes of each gel contain the same amount of label. Lane 1, t = 5 min; lane 2, t = 10 mira lane 3, t = 17 min: lane 4, t = 30 min; lane 5, t = 50 min; lane 6, t = 80 min; lane 7, partly hydrolyzed sample as marker.
194
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Fig. 2. Changes in ATP, polyphosphate and orthophosphate levels in phosphate-starved cells of S. cerevisiae (A) and K. marxianus (B), after addition of 10 mM phosphate and 1% glucose. Upper frames: the ATP levels; o, ATP; ®, ATP in antimycin A-treated cells. Lower frames: polyphosphate and orthophosphate levels; D, orthophosphate; m, orthophosphate in antimycin A-treated cells; o, polyphosphate; @, polyphosphate in antimycin A-treated cells. The amount of polyphosphate is expressed in/~mol orthophosphate units per g wet wt. of yeast.
The rearrangement of chain lengths of the polyphosphate started at the moment that the glucose was exhausted from the medium (17 min). However, incubating $. cerevisiae cells with 10 mM phosphate and 10% (instead of 1%) glucose did not alter this pattern. When the experiment was performed with 50 mM phosphate and 1~ glucose, the rearrangement started after about 30 rain. At that time, the cells contained 77 /~mol polyphosphate per g wet wt. of yet~st (indicated in/tmol orthophosphate units) and 28/~mol orthophosphate per g wet wt of yeast. Only increasing both the glucose and the phosphate concentration to 10% and :~0 raM, respectively, resulted in a continuous synthesis of polyphosphates with higher molecular weights during 80 rain of incubation. Polyphosphate synthesis with ethanol as energy source is shown in Fig. 3. It differs from the synthesis with glucose only in the synthesis rate. Also in this case, polyphosphates were built up from short chains to long chains. In S. cerevisiae, antimycin A completely blocked ethanol-driven polyphosphate synthesis (see also Fig. :), whereas in K. marxianus, polyphosphate was still built up with the same chair, length pattern as in untreated cells. In a parallel experiment, the concentrations of poly-
phosphate, orthophosphate and ATP were measured (Fig. 4). In S. cerevisiae, antimycin A treatment not only abolished polyphosphate synthesis, but it also lowered the intracellular orthophosphate and ATP levels. In K. marxianus, antimycin A treatment reduced the amount of polyphosphate in the cell, but orthophosphate reached the same concentration as in untreated cells, although more slowly. In this yeast antimycin appeared to influence the ATP level only after a longer incubation period. For polyphosphate synthesis, an energy source must be present [4]. Experiments with the yeast Saccharomyces mellis indicated that the endogenous reserves of this yeast are adequate to supply the energy for polyphosphate synthesis [24]. To investigate whether this is also the case in our yeasts, the changes in orthophosphate, polyphosphate and ATP were studied in phosphate-starved cells after addition of 10 mM phosphate without an external energy source. Fig. 5 demonstrates that S. cerevisiae cells undergo very slow polyphosphate synthesis under these circumstances. This polyphosphate synthesis is almost completely blocked by antimycin A. At the same time, this treatment reduces the increase in the internal orthophosphate and ATP concentration. In the yeast K. marxianus, on the
195 other hand, the endogenous substrate seems t o be adequate for polyphosphate synthesis, even in the presence of antimycin A.
phate seems to be formed via chains of short length to chains of long length. This seems to contradict earUer results of Langen et al. [7,10,11], which indicated that a long-chain polyphosphate fraction was formed first, that was subsequently degraded to fractions with a shorter mean chain length. It should be noted, however, that this apparent contradiction only holds for their results with phosphate-starved cells, whereas in part of their experiment, Langen et al. used yeast cells that were not starved of phosphate [10,11]. To explain the results presented in this paper, an alternative processive mechanism can not be fully excluded. According to this mechanism, polyphosphates are first synthesized as (very) long chains. This process should, in the present experiments, be followed by a rapid conversion of the synthesized high molecular weight polyphosphates to
Discussion
Phosphate-starved cells of K. marxianus and S. cerevisiae contain virtually no polyphosphates. Incubating these cells with phosphate, [32PIPi and an energy source, followed by extraction and separation of the polyphosphates by gel electrophoresis, gives an indication of the mechanism of polyphosphate biosynthesis. As the different lanes in Figs. 1 and 3 contain the same amount of 32p, each lane shows the relative ratio between the different chain lengths of polyphosphates, but not the absolute concentration. In all cases, polyphos-
A 1
2
3
4
5
[3 6
7
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4
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?
1
2
3
4= 5
6
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B o O
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Fig. 3. Polyphosphate synthesis in phosphate-starved cells of S. cerevisiae (A), K. marxianus (B, C) incut:,ated with 2% ethanol, 10 m M phosphate a n d [ 32F]p i for different lengths of time. Incubate C contained antimycin. The extracts were separated on a 15% polyacrylamide gel containing I M urea. T h e lanes of each gel contain the same amount of label. Lane 1, t = 5 min; lane 2, t = 10 rain; lane 3, t = 17 vain; lane 4, t = 30 rain, lane 5, t = 50 min; lane 6, t = 80 min; lane 7, partly hydrolyzed sample used as marker.
incubation time Iminutes]
incubation time ImWwtesl
Fig, 4. Changes in ATP, polyphosphate and orthophosphate levels in phosphate-starved 5’. cereuisiue (A) and K. marxiunw (B), after addition of 16 r frames: the polyphosphate ATP in antimycin A-treated cells. mM phosphate and 2% ethanol. Upper frames: he ATP levels; 0, ATP; polyphosphate in antimycin orthophosphate in anti in A-treated cells; o, polyphospha and orthophosphate levels; 0, orthophosphate; A-treated &Is. The amount of polyphosphate is expressed in pmol orthophosphate units per g wet wt. of yeast.
I
A
10
30
50
incubatran time (minutes)
70
10
30 60 incubation time htinuted
70
Fig. 5. Changes in ATP, polyphosphate and orthophosphate levels in phosphate-starved S. cerevisiue (A) and K. murxianus (B), after addition of 10 mM phosphate atone. Upper frames: the P curves; o, ATP; ATP in antimycin A-treated cells. Lower : the polyphosphate and orthophosphate levels; 0, orthophosphate; orthophosphate in timycin A-treated cells; o, polyphosphate; yphosphate in antimycin A-treated cells. The amount of polyphosphate is expressed in pmol orthophosphate units per g wet wt. of yeast.
197 shorter chains. However, the gradual increase of chain length observed in the time course makes the possibility of such a mechanism unlikely. The mean chain length of the polyphosphate pool, after incubating phosphate-starved cells with I0 m M phosphate and 17O glucose, decreased after all the external glucose and most of the orthophosphate had been consumed (Fig. 1). However, Fig. 2 demonstrates that the amount of pnlyp_hosphate is not diminished. Therefore, this rea~Tangement in the polyphosphate pool indicates conversion of polyphosphate with a long chain length to shorter polyphosphates. This rearrangement is probably related to the low external phosphate concentration. This is supported by results,obtained with the yeast S. cerevisiae, showing that increasing the glucose concentration does not prevent this rearrangement, whereas increasing the external phosphate concentration, at a 17O glucose concentration, delays this rearrangement. Under the latter conditions, the total amount of orthophosphate and polyphosphate increases, even though the glucose is exhausted from the medium after 17 rain. It seems likely that after glucose exhaustion intermediate products, formed during glucose consumption, such as organic acids or ethanol, are used as an energy source, since S. cerevisiae does not seem to contain endogenous substrate (Fig. 5A). However, since thisrearrangement does not appear within 80 rain when phosphate-starved cells are incubated with 107o glucose and 50 m M phosphate, whereas it does with cellsincubated with 17O glucose and 50 m M phosphate, it follows that shortage of energy substrate also leads to polyphosphate splitting.It should bc noted that this process of polyphosphate splitting is probably catalyzed by a polyphosphate depolymerase. This kind of enzyme has been shown to be present in yeast [1,13,141. Both with S. cerevisiae and with K. marxianus, experiments with glucose as energy source show that antimycin A, blocking respiration, has no influence on polyphosphate synthesis. This seems to indicate that fermentation rather than respiration provides the energy for polyphosphate synthesis. This is in accordance with previous investigations of Wiame and Langen and Liss [4,7]. With ethanol as energy source, the two yeast strains behave differently (Figs. 3 and 4). The synthesis of polyphosphate in the presence of ethanol is slower in the yeast S. cerevisiae than it. K. marxianus. Further, antimycin A, which blocks ethanol respiration, completely prevents polyphosphate synthesis and greatly reduces orthophosphate assimilation in S. cerevisiae. Since the ATP concentration of the antimycin A-treated S. cerevisiae cells is very low, while transport of orthophosphate needs energy [25,26], abolition of ethanol respiration will thus reduce orthophosphate transport in this yeast. In K. marxianus cells, on the other hand, antimycin A also inhibits polyphosphate synthesis, but
not completely in this case. This suggests that fermentation of endogenous reserves can be used to drive polyphosphate synthesis in this yeast. This is confirmed by the results shown in Fig. 5B: without external energy source there is still polyphosphate synthesis, with about the same velocity as when ethanol and antimycin A are present simultaneously. Contrarily, in S. cerevisiae endogenous reserves can not, apparently, be used for polyphosphate synthesis (Fig. 5A). Summarizing these results, together with the model of Bostian et al. [2], lead to the following hypothesis: polyphosphate synthesis starts only when orthophosphate is accumulated. That polyphosphate ldnase, which forms polyphosphate at the expense of ATP, may play a role cannot be excluded, because this enzyme is present in elevated concentrations in phosphate-starved cells (see Ref. 2). However, if this enzyme were solely responsible for polyphosphate synthesis, a correlation would be observed between the ATP concentration and the rate of polyphosphate synthesis. Since such a strict relation was not observed (compare Figs. 2 and 4), it seems likely that, besides polyphosphate kinase, more enzymes are involved in the synthesis of polyphosphates. Polyphosphate synthesis was not inhibited by antimycin A indicating that, apparently, most energy needed for polyphosphate synthesis, is derived from the glycolytic pathway. Further experiments are required to show whether this is caused by the activity of, for example, the enzyme 1,3-diphosphoglycerate: polyphosphate phosphotransferase, which has been found in Neurospora [1,13,14]. References 1 Kulaev, I.S. (1979) The Biochemistry of Inorganic Polyphosphates pp. 122-192, John Wiley & Sons, New York. 2 Bostian, K.A., Lemire, J.M. and Halvorson, H.O. (1983) Mol. Cell. Biol. 3, 839-853. 3 Tamai, Y., Toh-E, A. and Oshima, Y. (1985) J. Bacteriol. 164, 964-968. 4 Wiame, J.M. (1947) Biochim. Biophys. Acta 1,234-255. 5 Wiame, J.M. (1949) J. Biol. Chem. 178, 919-929. 6 Schmidt, G., Hecht, L. and Tannhauser, S.J. (1949) J. Biol. Chem. 178, 733-742. 7 Liss, E. and Langen, P. (1962) Arch. Mikrobiol. 41, 383-392. 8 Weimberg, R. (1975)J. Bacteriol. 121, 1122-1130. 9 Liss, E. and Langen, P. (1959) Naturwissenschaften 46, 15~. 10 Langen, P. and Liss, E. (1958) Biochem. Z. 330, 455-466. 11 Langen, P., Liss, E. and Lohmann, K. (1962) in Acides Ribonucleiques et Polyphosphates: Structure, Synthese et Fonctions, pp. 603-612, Colloq. Int. CNRS, Strasbourg, 1961, CNRS Paris. 12 Lusby, E.W. and McLaughlin, C.S. (1980) M'ol. Gen. Oenet. 178, 69-76. 13 Kulaev, I.S. and Vagabov, V.M. 0983) in Advances in Microbial Physiology, (Rose, A.H. and Morris, J.,eds.),Vol. 24, pp. 83-171, Academic Press, N e w York. 14 Kulaev, I.S. (1985) in Enviroxunental Regulation of Microbial Metabofism (Kulaev, I.S.,Dawes, E.A. and Tempest, D.W., eds.), pp. 1-25, Academic Press, London.
198 15 Trilisenko, L.V., Irinskaya, O.N., Vagabov, V.M. and Kulaev, I.S. (1985) Biokhimiya 50, 1120-1126. 16 Jaspers, H.T.A. and Van Steveninck, J. (1976) Biochirn. Biophys. Acta 443, 2,*3-253. 17 Clark, J.E., Beegen, H. and Wood, H.G. (1986) J. Bacteriol. 168, 1212-1219. 18 Clark, J.E. and Wood, H.G. (1987) Anal Biochem. 161, 280-291. 19 Robinson, N.A., Goss, N.H. and Wood, H.G. (1984) Biochem. Int. 8, 757-769. 20 Peppin, T.C.A., Wood, H.G. and Robinson, N.A. (1986) Biochem. Int. 12, 111-123.
2! Lohmann, K. and Langen, P. (1956) Biochem. Z. 328, 1-11. 22 Addanki, S., Sotos, J.F. and Rearick, P.D. (1966) Anal. Biochem. 14, 261-264. 23 Liss, E. and Langen, P. (1960) Biochem. Z. 333, 193-201. 24 Weimberg, R. and Orton, W.L. (1965) J. Bacteriol. 89, 740-747. 25 Borst Pauwels, G.W.F.H. and Jager, S. (1969) Biochim. Biophys. Acta 172, 399-406. 26 Roomans, G.M., Blasco, F. and Borst Pauwels, G.W.F.H. (1977) Biochim. •iophys. Acta 467, 65-71.
191
BBA12399
Polyphosphate synthesis in yeast J. Sehuddemat, R. de Boo, C.C.M. van Leeuwen, P.J.A. van den Broek and J. van Steveninek Sylvius Laboratories, Department of Medical Biochemistry, Leiden (The Netherlands) (Received 18 August 1988)
Key words: Polyphosphate synthesis: Phosphate starvation; (S. cerevisiae); (K. marxianus) Polyphosphate synthesis was studied in phosphate-starved cells of Saccharomyces cerecMae and gluyveromyces marxianus. Incubation of these yeasts for a short time with phosphate and either glucose or ethanol resulted in the formation of pulyphosphate with a short chaGn length. With increasing incubation times, polypbosphates with longer chain lengths were formed. Pulypbosphates were synthes|Ted faster during incubation with glucose than with ethanol. Antimycin did not affect the glucose-induced polypbosphate synthesis in either yeast. Using ethanol as an energy source, antimycin A treatment blocked both polypbosphate synthesis and accumulation of ortbopbosphate in the yeast $. cereo~/ae. However, in K. marManus, pulyphosphate synthesis and orthopbosphate accumulation proceeded n,~rmaily in antimyein-treated ce|is, suggesting that endogenous reserves were used as energy source. This was confil~ned in experiments, conducted in the absence of an exogenous energy source.
introduction Polyphosphates, which are linear polymers of orthophosphate in anhydrous linkage, are present in large amounts in yeasts, growing on a complete medium with a high orthophosphate content. Conversely, when phosphate-rich cells are grown in a medium lacking phosphate (phosphate starvation), polyphosphate disappears. Phosphorus starvation also causes derepression of an acid phosphatase located at the cell surface [1,2]. Simultaneously, a high-affinity phosphate transport system appears [3] (for a model of phosphate metabolism and phosphatase regulation in S. cerevisiae see Ref. 2). When phosphate-starved cells are inoculated in a medium containing orthophosphate and an energy source, the cells assimilate phosphate and condense it to polyphosphate [4-7]. The presence of K + and Mg 2+ stimulates this process [6-8]. Under these conditions, the polyphosphate content can exceed 2370 of the cellular dry weight [7]. This effect is known as hypercompensation. Polyphosphates in yeast can be divided in two general classes: the acid-soluble polyphosphates, with a mean chain length of four orthophosphate units, and acid-insoluble polyphosphates, reaching chain lengths Correspondence: P.J.A. van den Brock, Sylvius Laboratories, Department of Medical Biochemistry, P.O. Box 9503, 2300 RA Leiden, The Netherlands.
of more than 260 orthophosphate units [7,9]. From in vivo experiments, Langen and Liss reported that during polyphosphate biosynthesis, exogenous [32P]orthophosphate ([32p]Pi) was first incorporated mainly in he most polymerized polyphosphate (h = 260). The shorter polyphosphates would be formed as secondary products, by degradation of the high molecular weight polyphosphate. Hydrolysis of the most polymerized form would yield the lower molecular weight polyphosphate with a mean chain length of 55 orthophosphate units. This fraction would be hydrolyzed to a polyphosphate with a mean chain length of 20 units. Finally, the lowest molecular weight polyphosphate would be formed (h = 4), which is hydrolyzed to orthophosphate [10,11]. These results, viz. formation of short chains from long chains of polyphosphate, were confirmed by Lusby and McLaughlin [12]. However, in more recent years it has become clear that specific fractions of polyphosphate represent various pools of polyphosphate, differing in their localization in the cell [1,13,14]. Besides the synthesis of the polyphosphate fractions as described above [10,11], biogenesis related to the metabolism of specific cellular compartments is also found [1,13,14]. The group with mean chain length of 20 orthophosphate residues, which is, at least partly, located in the nucleus, is suggested to be sy,thesized from pyrophosphate, formed during RNA synthesis [1,13,14], High molecular weight polyphosphates are supposed to be located at the cell pc-
0167-4889/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
192 riphery. Their biosynthesis is apparently related to cell wall synthesis [1,13,15]. In the fungus Neurospora crassa, the low molecular weight polyphosphate (~ = 4), which is located in the cytoplasm, seems to be synthesized by means of the glycolytic enzyme 1 3-diphosphoglycerate: polyphosphate phosphotransferase. This enzyme transfers a phosphoryl group from 1,3-:~iphosphoglycerate to polyphosphate instead of to ADP [1,13,14]. In this paper, the biosynthesis of polyphosphate was studied in phosphate-starved cells of S. cerevisiae and K. marxianus, supplied with different phe~sphate concentrations and energy sources. In all cases, polyphosphate appeared to be synthesized from chains with short lengths to chains with long lengths. The rate of polyphosphate synthesis was dependent on the energy source. Materials and Methods
The yeasts S. cerevisiae strain Delft I (CBS 1172) and K. marxianus (CBS 397) were grown for 20 h on a synthetic medium with 270 glucose as a carbon source, as described before [16]. For phosphate starvation, 0.5 g wet weight of yeast was incubated for 15-16 h in 250 ml of this synthetic medium, lacking phosphate, with 170 glucose as carbon source. The suspension was incubated at 20°C on a GFL rotary shaker. Subsequently, the cells were collected by centrifugation and washed three times with distilled water. To study polyphosphate synthesis, a 1070 (wet w / v ) suspension of phosphate-starved cells was made in incomplete synthetic medium, lacking phosphate and vitamins. In some experiments, the respiratory capacity was blocked by adding 20/tg antimycin A per g wet wt. of yeast to the incubate. The suspensions were pre-incubated for 7 min under aerobic conditions before 170 (w/v) glucose or 270 (v/v) ethanol was added as energy source. After another 6 rain of pre-incubation, polyphosphate synthesis was started by addition of Kt~.2PO4 and 10/tCi [32PIpi per ml. Polyphosphates were extracted and purified as described by Clark et al. [171 with small modifications. The acid-soluble polyphosphates were extractec~ by suspending 0.1 g (wet wt) of yeast in 0.3 ml 270 uichloroacetic acid. The suspension was centrifugated fer 5 rain at 1200 × g and the pellet was resuspended in 1 ~1 0°770 trichloroacetic acid/6770 acet,ne. The suspension was centrifuged for 5 min at 1200 × g and the pellet was washed with 1 ml 6770 acetone. The three supernatants were combined and extracted with 2.3 ml phenol/ chloroform (90 g pl'enol solubilized in 10 ml water plus 100 ml chloroform and subsequently saturated with 0.1 M ammonium acetate pH 6.5). This fraction is called the trichloroacetic acid/acetone fraction. The long-chain soluble polyphosphates and the polyphosphates from
the volutine granule were extracted by suspending the remaining pellet in 0.5 ml 2 mM EDTA, followed by titration with LiOH to pH = 7. Subsequently, 0.2 ml phenol/chloroform was added and the suspension was shaken vigorously. The supernatant obtained after 10 min centrifugation at 1200 × g will be indicated as the EDTA/LiOH fraction. Polyphosphates were separated on 1570 polyacrylamide gels as described by Wood and co-workers [18-20]. The ratio acrylamide to bis-acrylamide was always 20: 1. As the chain lengths of polyphosphate can be estimated by counting bands [18], a sample containing high molecular •iveight polyphosphate was subjected to partial hydrolysis in 25 mM HCI for 15 min at 100°C and used as a marker. The different lanes contain the same amount of 32p. Therefore, each lane shows the relative ratio between the different chain lengths of polyphosphates. For autoradiography, the gels were packed in plastic foil and exposed to X-ray film at - 7 0 o C. Parallel with the radioactive experiment, the changes in the polyphosphate, orthophosphate and ATP concentrations were measured :in a similar experiment, but without addition of [32pIPt. For ATP determination, samples from the incubate were mixed with 4 volumes of ethanol. For determination of the polyphosphate and orthophosphate concentrations, the cells were washed with ice-cold water and resuspended in water. After boiling the suspension for 2 rain, the polyphosphate and orthophosphate concentrations were determined in the supernatant. The amount of polyphosphate was determined spectrophotometrically, after acid hydrolysis to orthophosphate at 100 °C as described by Lohmann and Langen [21], by mixing 2 ml of sample with 1 ml 2.570 ammonium molybdate in 2 M HCI and 200 ttl 2 mM 1-amino-2-naphthol-4-sulfonic acid. After 30 rain, the absorbance was measured at 700 nm. The amount of orthophosphate was measured in the same way as polyphosphate, but without hydrolysis at 100°C. The ATP concentrations in extracts were measured with the luciferin/luciferase method [22]. The presence of glucose in the medium was determined, using the Boehringer Combur test (detection level 2.2 raM). [ 32P]Orthophosphate and [32P]ATP were purchased from Amersham International. Ribonuclease A (RNAse) was obtained from Boehringer. Results
To study polyphosphate synthesis in yeast, a mild extraction procedure and a good method for separating the polyphosphates with different chain length are indispensible. Recently, a relatively mild polyphosphate isolation method, coupled to a separation of the poly-.
193 TABLE l The amounts of polyphosphate and orthophosphate extracted from S. cereoisiae and K. marxianus using different extraction methods
Three different extraction procedures are compared. Extraction 1: the extraction procedure according to Clark et el. [17]. Extraction 2." bolting the cells in water as described by Weimberg and Orton [24]. Extraction 3: the extraction in five steps, described by Liss and Langen [23]. Orthophosphate (Pi) is expressed in /tmol/g wet wt of yeast. Polyphosphate (Pn) is expressed in /tmol orthophosphate units/g wet wt. of yeast. Extraction method
S. cerevisiae
K. marxianus
Pi
Pn
Pi
Pn
15.8
0
22
0
1.8
19.2
0.5
14.7
2. (Boiling)
21.4
20.5
28.6
14.6
3. (Liss and Langen)
24
11.2
29.8
13.8
1. Trichloroacetic acid/ acetone fraction 1. E D T A / L i O H fraction
phosphates by gel electrophoresis has been described by Wood and co-workers [17-20] for Propionibacterium shermanii. To determine whether this extraction procedure can also be used for the isolation of polyphosphates from K. marxianus and S. cerevisiae, a comparison was made between this method, the extraction method of Liss and Langen [23] and the extraction with boiling water, described by Weimberg and Orton [24]. The data from Table I indicate that there is no significant difference between the amounts of polyphosphate and orthophosphate extracted with boiling water or by the method of Clark et al. [17]. The isolation procedure of Liss and Langen extracts the same amount of phosphate from the cells as the other two methods, but there seems to be a shift in the amount of polyphosphate to orthophosphate in S. cerevisiae. This may be caused by hydrolysis of polyphosphate. Table I also shows that, in contrast to the results found for Propionibacterium shermanii, in yeast the tricMoroacetic acid/acetone fraction contained no polyphosphates. Incubating phosphate-starved yeast cells with an energy source orthophosphate and [a2p]p i followed by extraction and gel electrophoresis also confirmed that the trichloroacetic acid/acetone fraction contained a negligible amount of labeled polyphosphates. Incubating the polyphosphate containing EDTA/LiOH fractions with RNAse did not alter the gel pattern, indicating that no contaminant labeled RNA was present on the gel. These data show that this mild extraction and separation procedure is readily applicable to yeast. In further experiments, it appeared that the fractions, obtained with the extraction method of Langen and Liss [23], could not be analyzed by gel electrophoresis, due to the high ionic strength of the fractions. When phosphate-starved cells of S. cerevisiae or K.
marxianus were transferred to a medium containing 10 mM phosphate, [32P]Pi and 170 glucose, the cells almost immediately started to synthesize polyphosphate. Fig. 1 shows that synthesis proceeded flom chains with a short length to chains with a long length. After approx. 17 min, when all the glucose was consumed, the composition of the polyphosphate pool rearranged, shifting to a shorter mean chain length. When the respiratory capacity of the phosphate-starved cells was blocked by treating the cells with antimycin A, the pattern of polyphosphate synthesis was exactly the same as in the absence of antimycin A (not shown). In a parallel experiment, the changes in polyphosphate, orthophospimte and ATP concentrations were measured. Fig. 2 shows that there was no net consumption of polyphosphate when the glucose was exhausted from the medium. Moreover, antimycin did not significantly influence the polyphosphate and orthophosphate levels. It should be noticed, however, that in contrast to K. marxianus, in S. cerevisiae the ATP content was sensitive to the presence of antimycin.
A .[
A'FP
2
3
,'l
5
B 6
7
12.~3,~156
--¢p
Q
~
® 0 0 0
O
o
O O
6 Q
Q Q
Fig. 1. Polyphosphate synthesis in phosphate-starved cells of S. cerevisiae (A) and K. marxianus (B) incubated with 1% glucose, 10 mM phosphate and [32PIPi, for various lengths of time. The compounds in the extracts were separated on 15% polyacrylamide gels containing 1 M urea. The lanes of each gel contain the same amount of label. Lane 1, t = 5 min; lane 2, t = 10 mira lane 3, t = 17 min: lane 4, t = 30 min; lane 5, t = 50 min; lane 6, t = 80 min; lane 7, partly hydrolyzed sample as marker.
194
-t~ B
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0 . 8
0.6
mO. § "6 E ~o.,~
_~..o
o. k.. < 0.2
< 0.2
.
~
~
,
;o
'
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'"
s'o
'
10
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SO
~-
.
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30
r
t~
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o
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Jr1
. ....
-e......
TJ •
0
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iiIIII ,~,
30
30 .=,,. I
fl
O
,IC
Q.
i
10
10 e~
3'0 incubation
' s'o ' time (minutes)
7'o
'
' 3'0 incubation
' s'o ' time (minutes}
Fig. 2. Changes in ATP, polyphosphate and orthophosphate levels in phosphate-starved cells of S. cerevisiae (A) and K. marxianus (B), after addition of 10 mM phosphate and 1% glucose. Upper frames: the ATP levels; o, ATP; ®, ATP in antimycin A-treated cells. Lower frames: polyphosphate and orthophosphate levels; D, orthophosphate; m, orthophosphate in antimycin A-treated cells; o, polyphosphate; @, polyphosphate in antimycin A-treated cells. The amount of polyphosphate is expressed in/~mol orthophosphate units per g wet wt. of yeast.
The rearrangement of chain lengths of the polyphosphate started at the moment that the glucose was exhausted from the medium (17 min). However, incubating $. cerevisiae cells with 10 mM phosphate and 10% (instead of 1%) glucose did not alter this pattern. When the experiment was performed with 50 mM phosphate and 1~ glucose, the rearrangement started after about 30 rain. At that time, the cells contained 77 /~mol polyphosphate per g wet wt. of yet~st (indicated in/tmol orthophosphate units) and 28/~mol orthophosphate per g wet wt of yeast. Only increasing both the glucose and the phosphate concentration to 10% and :~0 raM, respectively, resulted in a continuous synthesis of polyphosphates with higher molecular weights during 80 rain of incubation. Polyphosphate synthesis with ethanol as energy source is shown in Fig. 3. It differs from the synthesis with glucose only in the synthesis rate. Also in this case, polyphosphates were built up from short chains to long chains. In S. cerevisiae, antimycin A completely blocked ethanol-driven polyphosphate synthesis (see also Fig. :), whereas in K. marxianus, polyphosphate was still built up with the same chair, length pattern as in untreated cells. In a parallel experiment, the concentrations of poly-
phosphate, orthophosphate and ATP were measured (Fig. 4). In S. cerevisiae, antimycin A treatment not only abolished polyphosphate synthesis, but it also lowered the intracellular orthophosphate and ATP levels. In K. marxianus, antimycin A treatment reduced the amount of polyphosphate in the cell, but orthophosphate reached the same concentration as in untreated cells, although more slowly. In this yeast antimycin appeared to influence the ATP level only after a longer incubation period. For polyphosphate synthesis, an energy source must be present [4]. Experiments with the yeast Saccharomyces mellis indicated that the endogenous reserves of this yeast are adequate to supply the energy for polyphosphate synthesis [24]. To investigate whether this is also the case in our yeasts, the changes in orthophosphate, polyphosphate and ATP were studied in phosphate-starved cells after addition of 10 mM phosphate without an external energy source. Fig. 5 demonstrates that S. cerevisiae cells undergo very slow polyphosphate synthesis under these circumstances. This polyphosphate synthesis is almost completely blocked by antimycin A. At the same time, this treatment reduces the increase in the internal orthophosphate and ATP concentration. In the yeast K. marxianus, on the
195 other hand, the endogenous substrate seems t o be adequate for polyphosphate synthesis, even in the presence of antimycin A.
phate seems to be formed via chains of short length to chains of long length. This seems to contradict earUer results of Langen et al. [7,10,11], which indicated that a long-chain polyphosphate fraction was formed first, that was subsequently degraded to fractions with a shorter mean chain length. It should be noted, however, that this apparent contradiction only holds for their results with phosphate-starved cells, whereas in part of their experiment, Langen et al. used yeast cells that were not starved of phosphate [10,11]. To explain the results presented in this paper, an alternative processive mechanism can not be fully excluded. According to this mechanism, polyphosphates are first synthesized as (very) long chains. This process should, in the present experiments, be followed by a rapid conversion of the synthesized high molecular weight polyphosphates to
Discussion
Phosphate-starved cells of K. marxianus and S. cerevisiae contain virtually no polyphosphates. Incubating these cells with phosphate, [32PIPi and an energy source, followed by extraction and separation of the polyphosphates by gel electrophoresis, gives an indication of the mechanism of polyphosphate biosynthesis. As the different lanes in Figs. 1 and 3 contain the same amount of 32p, each lane shows the relative ratio between the different chain lengths of polyphosphates, but not the absolute concentration. In all cases, polyphos-
A 1
2
3
4
5
[3 6
7
3_ 2
3
4
5
C 6
?
1
2
3
4= 5
6
7
B o O
O
o°o®
O
¢b
0
....
O
0
~:~ ~
Fig. 3. Polyphosphate synthesis in phosphate-starved cells of S. cerevisiae (A), K. marxianus (B, C) incut:,ated with 2% ethanol, 10 m M phosphate a n d [ 32F]p i for different lengths of time. Incubate C contained antimycin. The extracts were separated on a 15% polyacrylamide gel containing I M urea. T h e lanes of each gel contain the same amount of label. Lane 1, t = 5 min; lane 2, t = 10 rain; lane 3, t = 17 vain; lane 4, t = 30 rain, lane 5, t = 50 min; lane 6, t = 80 min; lane 7, partly hydrolyzed sample used as marker.
incubation time Iminutes]
incubation time ImWwtesl
Fig, 4. Changes in ATP, polyphosphate and orthophosphate levels in phosphate-starved 5’. cereuisiue (A) and K. marxiunw (B), after addition of 16 r frames: the polyphosphate ATP in antimycin A-treated cells. mM phosphate and 2% ethanol. Upper frames: he ATP levels; 0, ATP; polyphosphate in antimycin orthophosphate in anti in A-treated cells; o, polyphospha and orthophosphate levels; 0, orthophosphate; A-treated &Is. The amount of polyphosphate is expressed in pmol orthophosphate units per g wet wt. of yeast.
I
A
10
30
50
incubatran time (minutes)
70
10
30 60 incubation time htinuted
70
Fig. 5. Changes in ATP, polyphosphate and orthophosphate levels in phosphate-starved S. cerevisiue (A) and K. murxianus (B), after addition of 10 mM phosphate atone. Upper frames: the P curves; o, ATP; ATP in antimycin A-treated cells. Lower : the polyphosphate and orthophosphate levels; 0, orthophosphate; orthophosphate in timycin A-treated cells; o, polyphosphate; yphosphate in antimycin A-treated cells. The amount of polyphosphate is expressed in pmol orthophosphate units per g wet wt. of yeast.
197 shorter chains. However, the gradual increase of chain length observed in the time course makes the possibility of such a mechanism unlikely. The mean chain length of the polyphosphate pool, after incubating phosphate-starved cells with I0 m M phosphate and 17O glucose, decreased after all the external glucose and most of the orthophosphate had been consumed (Fig. 1). However, Fig. 2 demonstrates that the amount of pnlyp_hosphate is not diminished. Therefore, this rea~Tangement in the polyphosphate pool indicates conversion of polyphosphate with a long chain length to shorter polyphosphates. This rearrangement is probably related to the low external phosphate concentration. This is supported by results,obtained with the yeast S. cerevisiae, showing that increasing the glucose concentration does not prevent this rearrangement, whereas increasing the external phosphate concentration, at a 17O glucose concentration, delays this rearrangement. Under the latter conditions, the total amount of orthophosphate and polyphosphate increases, even though the glucose is exhausted from the medium after 17 rain. It seems likely that after glucose exhaustion intermediate products, formed during glucose consumption, such as organic acids or ethanol, are used as an energy source, since S. cerevisiae does not seem to contain endogenous substrate (Fig. 5A). However, since thisrearrangement does not appear within 80 rain when phosphate-starved cells are incubated with 107o glucose and 50 m M phosphate, whereas it does with cellsincubated with 17O glucose and 50 m M phosphate, it follows that shortage of energy substrate also leads to polyphosphate splitting.It should bc noted that this process of polyphosphate splitting is probably catalyzed by a polyphosphate depolymerase. This kind of enzyme has been shown to be present in yeast [1,13,141. Both with S. cerevisiae and with K. marxianus, experiments with glucose as energy source show that antimycin A, blocking respiration, has no influence on polyphosphate synthesis. This seems to indicate that fermentation rather than respiration provides the energy for polyphosphate synthesis. This is in accordance with previous investigations of Wiame and Langen and Liss [4,7]. With ethanol as energy source, the two yeast strains behave differently (Figs. 3 and 4). The synthesis of polyphosphate in the presence of ethanol is slower in the yeast S. cerevisiae than it. K. marxianus. Further, antimycin A, which blocks ethanol respiration, completely prevents polyphosphate synthesis and greatly reduces orthophosphate assimilation in S. cerevisiae. Since the ATP concentration of the antimycin A-treated S. cerevisiae cells is very low, while transport of orthophosphate needs energy [25,26], abolition of ethanol respiration will thus reduce orthophosphate transport in this yeast. In K. marxianus cells, on the other hand, antimycin A also inhibits polyphosphate synthesis, but
not completely in this case. This suggests that fermentation of endogenous reserves can be used to drive polyphosphate synthesis in this yeast. This is confirmed by the results shown in Fig. 5B: without external energy source there is still polyphosphate synthesis, with about the same velocity as when ethanol and antimycin A are present simultaneously. Contrarily, in S. cerevisiae endogenous reserves can not, apparently, be used for polyphosphate synthesis (Fig. 5A). Summarizing these results, together with the model of Bostian et al. [2], lead to the following hypothesis: polyphosphate synthesis starts only when orthophosphate is accumulated. That polyphosphate ldnase, which forms polyphosphate at the expense of ATP, may play a role cannot be excluded, because this enzyme is present in elevated concentrations in phosphate-starved cells (see Ref. 2). However, if this enzyme were solely responsible for polyphosphate synthesis, a correlation would be observed between the ATP concentration and the rate of polyphosphate synthesis. Since such a strict relation was not observed (compare Figs. 2 and 4), it seems likely that, besides polyphosphate kinase, more enzymes are involved in the synthesis of polyphosphates. Polyphosphate synthesis was not inhibited by antimycin A indicating that, apparently, most energy needed for polyphosphate synthesis, is derived from the glycolytic pathway. Further experiments are required to show whether this is caused by the activity of, for example, the enzyme 1,3-diphosphoglycerate: polyphosphate phosphotransferase, which has been found in Neurospora [1,13,14]. References 1 Kulaev, I.S. (1979) The Biochemistry of Inorganic Polyphosphates pp. 122-192, John Wiley & Sons, New York. 2 Bostian, K.A., Lemire, J.M. and Halvorson, H.O. (1983) Mol. Cell. Biol. 3, 839-853. 3 Tamai, Y., Toh-E, A. and Oshima, Y. (1985) J. Bacteriol. 164, 964-968. 4 Wiame, J.M. (1947) Biochim. Biophys. Acta 1,234-255. 5 Wiame, J.M. (1949) J. Biol. Chem. 178, 919-929. 6 Schmidt, G., Hecht, L. and Tannhauser, S.J. (1949) J. Biol. Chem. 178, 733-742. 7 Liss, E. and Langen, P. (1962) Arch. Mikrobiol. 41, 383-392. 8 Weimberg, R. (1975)J. Bacteriol. 121, 1122-1130. 9 Liss, E. and Langen, P. (1959) Naturwissenschaften 46, 15~. 10 Langen, P. and Liss, E. (1958) Biochem. Z. 330, 455-466. 11 Langen, P., Liss, E. and Lohmann, K. (1962) in Acides Ribonucleiques et Polyphosphates: Structure, Synthese et Fonctions, pp. 603-612, Colloq. Int. CNRS, Strasbourg, 1961, CNRS Paris. 12 Lusby, E.W. and McLaughlin, C.S. (1980) M'ol. Gen. Oenet. 178, 69-76. 13 Kulaev, I.S. and Vagabov, V.M. 0983) in Advances in Microbial Physiology, (Rose, A.H. and Morris, J.,eds.),Vol. 24, pp. 83-171, Academic Press, N e w York. 14 Kulaev, I.S. (1985) in Enviroxunental Regulation of Microbial Metabofism (Kulaev, I.S.,Dawes, E.A. and Tempest, D.W., eds.), pp. 1-25, Academic Press, London.
198 15 Trilisenko, L.V., Irinskaya, O.N., Vagabov, V.M. and Kulaev, I.S. (1985) Biokhimiya 50, 1120-1126. 16 Jaspers, H.T.A. and Van Steveninck, J. (1976) Biochirn. Biophys. Acta 443, 2,*3-253. 17 Clark, J.E., Beegen, H. and Wood, H.G. (1986) J. Bacteriol. 168, 1212-1219. 18 Clark, J.E. and Wood, H.G. (1987) Anal Biochem. 161, 280-291. 19 Robinson, N.A., Goss, N.H. and Wood, H.G. (1984) Biochem. Int. 8, 757-769. 20 Peppin, T.C.A., Wood, H.G. and Robinson, N.A. (1986) Biochem. Int. 12, 111-123.
2! Lohmann, K. and Langen, P. (1956) Biochem. Z. 328, 1-11. 22 Addanki, S., Sotos, J.F. and Rearick, P.D. (1966) Anal. Biochem. 14, 261-264. 23 Liss, E. and Langen, P. (1960) Biochem. Z. 333, 193-201. 24 Weimberg, R. and Orton, W.L. (1965) J. Bacteriol. 89, 740-747. 25 Borst Pauwels, G.W.F.H. and Jager, S. (1969) Biochim. Biophys. Acta 172, 399-406. 26 Roomans, G.M., Blasco, F. and Borst Pauwels, G.W.F.H. (1977) Biochim. •iophys. Acta 467, 65-71.