FEMS Microbiology Letters 92 (1992) 15 I- 15(~ t':~ 1992 Federation of European Microbiological S~r:icties (1378. !1J07/92/$05.1~,| Published by Elsevier

151

FEMSLE 0,1841

Synthesis and degradation of polyphosphate in the fission yeast Schizosaccharomyces pombe: mutations in phosphatase genes

do not affect polyphosphate metabolism

Joachim Miiller, Beate Westenberg, Thomas Boiler and Andres Wiemken

Botani.whes In.~titut ,ler Uni~cr.~itiit Ba.wl. Ba.wl. Swit:crhmd

Received 4 February 1t)92 Accepted 4 February. 1~92

Key words: Polyphosphate; Phosphate starvation; Phosphatase; Schizosaccharonzyces pombe

1. SUMMARY

The fission yeast Schizo~accharomyces pombe was found to accumulate large amounts of polyphosphate, particularly when grown on argi- nine as the nitrogen source. Upon transfer to a medium without phosphate, polyphosphate was degraded and served as at~ endogenous phos- phate reserve. When phosphate was added again after a prolonged period of phosphate starvation, fission yeast cells synthesized more polyphos- phate than they had contained before starvation, a phenomenon known as over-compensation. Strains carrying mutated structural genes for three different phosphatases, phol, pho2 or pho3, de- graded polyphosphate at the same rate as the wild-type strain during phosphate starvation and showed the same type of over-compensation when phosphate was added again.

Correspondence to: A. Wiemken. Botanisches lnstilut der Uni- versiL~it Basel, Hebelsxrasse i, CH-4056 Basel, Switzerland.

2. INTRODUCTION

Polyphosphate (polyP), a linear polymer of or- thophosphate (Pi) linked by ener~-rich phospho- anhydride bonds, occurs in many fungi and other microorganisms, often the main phosphate (P)- containing metabolite apart from RNA [1-3]. In yeasts [4-7], other fungi [8,91 and algae [10l, polyp accumulates primarily in the vacuole and plays a role there in P storage [1,6,7,9,I0] as well as in the retention of N-rich cationic amino acids, such as arginine [7,9,11,12] and lysine [13]. Under con- ditions of P starvation, the vacuolar polyp is rapidly degraded, supplying a large part of the phosphate needed for growth [6,7,9].

Little is known about the enzymes involved in polyphosphate synthesis and degradation in yeast and other fungi [I-3], Some of the key enzymes in polyP metabolism have been identified in prokaryotes [3]. However, although some of these activities have also been described in extracts from fungi [2], including polyp kinase [14], the significance of these activities for fungal polyP

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metabolism in vivo remains unknown. Since fun- gal vacuoles contain several phosphatascs [15,161, none of which have been convincingly assigned a biological function, wc became interested in the possibility that these enzymes might contribute to polyphosphatc metabolism. One of the vacuolar phosphatascs of the yeast CamtMa utilis, a phos- phate-repressible, unspecific alkaline phospha- tasc, has been shown to degrade polyp in vitro, and this type of phosphatase could be important in polyP degradation in vivo [17]. Alkaline phos- phatases of a similar type also occur in Schizosac- charomyces pombe, named phosphatase (PHO) 3 [16], and in Sacdlaromyces cererisiae, named PHO8 [18], where the enzyme is vacuolar [15].

To test the possibility of an involvement of the vacuolar alkaline phosphatase in polyP metabo- lism as well as the possible contribution of other phosphatascs, wc took advant.age of the well-de- fined phosphatase mutants available for Schizo- saccharomyces pombe [16,19]. We report that wild-type cells of Schizosaccharomyces pombe, as other yeasts, accumulate polyP when supplied with phosphate, particularly in media containing arginine as the only nitrogen source. They rapidly degrade polyP upon P starvation and accumulate particularly high levels of polyP when supplied with phosphate after a period of P starvation. However, mutations in the structural gene of the unspecific alkaline phosphatase (pho3), as well as mutations in the genes for specific pNPP phos- phatase (pho2) and for the acidic phosphatase (phol), do not affect mobilization of polyphos- phate during P starvation or over-compensation in Schizosaccharomyces pombe, indicating that none of these phosphatases is important for polyphosphate metabolism.

3. MATERIAL AND METHODS

3. I. Organisms and cultiration Schizosaccharomyces pombe, strain 972h-, and

three phosphatase mutants [19] (Table 1), all gen- erously given by Dr. E. Schweingruber (Bern), were grown on the synthetic medium EMM-2 with glucose (2%) as described by Nurse [20], using either ammonium chloride (20 raM) or argi-

nine (10 mM) as sole nitrogen source. This medium contains 16 mM of P (as Na2HPO4). Cultures ( 150 ml) were grown in Erlenmeyer flasks (600 ml total volume) on a rotary shaker (130 rpm) at 27°C. The phenotypc of the mutants was verified by measuring phosphatases [19].

3.2. Phosphate starration and re-addition Cultures in the exponential growth phase (ap-

prox. 5 × 10" cells/ml) were harvested by filtra- tion (Whatman GF/C), washed twice with pre- warmed medium lacking P and then resuspended in an equal volume of medium lacking P. After 6 h of P starvation, the remaining culture was mixed with an equal volume of pre-warmed medium containing 32 mM P to restore the normal P concentration.

3.3. Analytical methods For quantitation of inorganic phosphate and

polyphosphate, cells were harvested by filtration (Whatman GF/C), fixed by boiling for 10 min and extracted as previously described [7,11]. Aliquots of the extracts were subjected to acid hydrolysis (1 N H2SO 4, 7 rain) and neutralized. The Pi content in hydrolysed and non-hydrolysed samples was determined as already described [11]. The P~ content of the samples before acid hydrol- ysis was taken to correspond to Pi, and the differ- ence between the P~ in hydrolysed and non-hy- drolysed samples to polyP [21].

For polyacrylamide gel electrophoresis (PAGE), polyphosphate was extracted and frac- tionated by the method of Schuddemat et al, [22]. Ceils harvested, as described above, were resus- pended in ice-cold trichloric acid (TCA; 2% w/v). After centrifugation (10(~l × g, l0 rain), the pel- lets were washed with ice-cold TCA (0,7% w/v in acetone 67% v/v) and with acetone (67% v/v). The combined supernatant fluids (TCA-acetone fraction) contained all P~ and a small amount ef polyP (less than 10% of the total) and were r~ot further analysed. The pellets containing the high molecular mass polyP (> 10 Pi units) were rcsus- pended in 2 mM ethylene EDTA and subse- quently neutralized with ! M LiOH. This suspen- sion was mixed with an equal volume of phenol- chloroform (1:1) and centrifuged. The upper

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phase contained the high molecular mass polyP (EDTA/LiOH fraction), This fraction was sepa- rated by PAGE and stained with Toluidine blue as described by Clark and Wood [23].

Protein was measured [24] using bovine serum albumin as standard.

4. RESULTS AND DISCUSSION

S. pombe, like many other microorganisms, was found to contain polyP, When grown on ammo- nium as a nitrogen source, wild-type cells con- tained about 200 nmol P per mg protein in the form of polyP throughout the growth cycle (Fig. 1). As in other fungi [4,9], the polyp content was much higher when the cells were grown on argi- nine as nitrogen source, reaching about 800 ntnol per mg protein during the exponential growth phase (Fig. i). In contrast to S. cerevisiae, in which the polyP pool decreased strongly upon entry into the stationary phase [25], cells of S. pombe continued to accumulate polyP in the sta- tiona~ phase (Fig. 1). In eight-day-old arginine cultures, the polyp reached a level of 35011-4000 neq P per mg protein (data not shown). The phosphatase mutants had comparable levels of polyp during the growth cycle (data not shown).

To study the dynamics of polyp metabolism, exponentially growing cells of the wild-type and of the phosphatase mutants were transferred to a medium lacking P and re-supplied with P after 6 h of starvation. During the period of P starvation, cells of the wild-type as well as of the mutants

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"rime (n) Fig. I. Growth (A) and polyphosphate content (B) of wild-type ceils of S. pombe on media with arginine (o) or ammonium

( & ) as N-source.

Table l

Designation and relevant phenotypes of the strains used in this work, All were kindly provided by Dr. E. Schweingruber (Bern).

Strain Phenotype

972h - Wildtype 972h- pho 1.132 Lacking the extraeellular, P-repressible,

acid phosphatase 972h - #ho 2-1 Lacking the pNPP-speeific, alkaline

phosphatase 972h - pho 3.1 Lacking the unspecific, alkaline

phosphatase

continued to grow with only slightly increased generation times (Table !). Thus, endogenous P reserves could be used almost as efficiently as exogenous Pi, even in the phosphatase mutants. In wild-type ceils, the polyp level started to de- crease rapidly upon transfer into the starvation medium and was almost compk':ely consumed after 6 h (Fig. 2A). The cellular Pi pool was initially maintained at about 200 neq P per mg protein and dropped to about 100 neq P per mg protein after 6 h (Fig. 2A), All three phosphatase

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Table 2

Average generation times of the investigated strains on nor- mal EMM-2 medium with arginine as sole N-source and on the same medium lacking phosphate (P).

Strain Generation time (h)

On normal medium On medium lacking P

972h 3.1 972h- pho 1-132 3.8 972h- pho 2-1 4.0 972h- pho3-1 3.2

3.8 4,9 4.5 3,7

mutants showed similarly rapid consumption of the polyp during P starvation (Fig. 2B-D).

When wild-type cells were supplied with Pi again after 6 h of P starvation, the normal level of Pi was almost immediately restituted, and polyp accumulated very rapidly (Fig. 2A). Within 1-2 h, the intracellular amount of polyP exceeded the amount before P starvation by a factor 2 to 2.5 (Fig. 2A). This phenomenon has been described as 'polyP over-compensation' in many unicellular organisms storing polyp [26]. Al l three phos-

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Time after transfer to P Fig. 2. Polyphosphate (e) and orthophosphate ( t, ) content of S, pombe after transfer to a medium lacking P and after subsequent re-supply of phosphate, Time given in hours. A. Wild-type, strain 972h-. B. Mutant pho3-1. C. Mutant pho2-1, D. Mutant

phol- 132, Phosphate was added back to the media at the time indicated by the arrow.

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phatase mutants showed similar kinetics of polyP over-compensation (Fk~. 2B-D).

The size range of acid-insoluble polyp molecules in S. pom0e was analysed by PAGE, followed by staining with Toluidine blue accord- ing to the technique of Clark and Wood [23] (Fig. 3). Acid-insoluble polyP accounted for more than 90% of the total polyp in all extracts analysed. In extracts from exponentially growing cells of both wild-type cells and the phosphatase mutants, Toluidlne-blue staining was observed in a sLze range corresponding to polyp cf 20 to more than 100 phosphate units, as compared with polyp size markers (Fig. 3, lane a). Extracts from both wild- type cells and phosphatase mutants subjected to

P starvation for 6 h contained no detectable polyP (lane b). When P was added again to these P- starved cells, polyp rapidly re-appeared in the extracts from all strains (lanes e-h), As discussed elsewhere [2], this finding indicates that the en- zymes involved in polyp synthesis remain present and may perhaps even be induced during P star- vation. Interestingly, even at the earliest time points (5-10 min, lanes c,d) staining was most prominent in the region containing large polyp molecules with more than 100 phosphate units. This indicates that very large molecules are formed immediately during biosynthesis of polyp de novo in S. pombe, as previously suggested for other microorganisms [26].

wildtype pho 3-1 pho 2-1 pho 1-123

q b c d e l g h o b c d e t ~ h ob c d e f g h a b c d e f g h

100

50

3O

Fig, 3, Analysis of polyphosphate in wild-type cells and phosphatase mutants of S. pombe by electrophoresis and staining with Toluidine blue. Cells were transferred to media lacking P and subsequently re-supplied with P as described in Fig. 2, Extracts corresponding to !,5 × 107 cells were loaded per hme. The lanes represent cells subjected to the following conditions: a, growing on P-containing medium: b, deprived of P for 6 h: c-h, deprived of P for 6 h and then re-supplied with P for 5, 10, 15, 20, 30 or 60 rain.

Scale at the right side: average chain length is given in phosphale units.

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The kinetics of polyP degradation during P starvation and of polyp synthesis during a subse- quent incubation with P were also studied in the budding yeast, S. ceret'isiae. Wild-type cells of S. cerevisiae showed similar dynamics of polyp as £ pombe, and S. ceret'isiac mutants defective in either both acid phosphatases (pho3 and pho5) or in the repressible alkatine phosphatase (p ha 8) [18], kindly provided by Dr. A. Hinnen and by Dr. G. Pohlig (CIBA Geigy, Basel) and Dr. T. Stevens (Eugene, Oregon) respectively, were in- distinguishable from wild-type cells with respect to polyp degradatior, during starvation and polyP over-compensation when P was supplied again (data not shown).

From these results, we conclude that in S. pombe and S. cerevisiae, none of the phos- phatases examined by analysis of their mutants is important for polyp synthesis or degradation, in particular, in contrast to the hypothesis put for- ward by Fernandez et al. [16] for C utilis, the unspecific, P-repressible alkaline phosphatase is unnecessary for polyp breakdown. It remains to be seen which enzymatic activities are responsible for polyp metabolism in yeast.

ACKNOWLEDGEMENTS

We thank Dr. E. Schweingruber (Bern) for the S. pombe strains, Dr. A. Hinnen and Dr. G. Pohllg (CIBA Geigy, Basel) and Dr. T. Stevens (Eugene, Oregon) for the S. cerevisiae strains. We are grateful to Dr. V. Wiemken for her help in preparation of the graphics. This work was sup- ported by the Swiss National Science Foundation.

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