©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Alleged Yeast Polyphosphate Kinase Is Actually Diadenosine-5`,5‴-P,P-tetraphosphate ,-Phosphorylase (*)

(Received for publication, April 11, 1995; and in revised form, June 14, 1995)

James W. Booth (§) Guido Guidotti

From the Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Polyphosphates are a major constituent of the yeast Saccharomyces cerevisiae. A purification of the enzyme polyphosphate kinase (E.C. 2.7.4.1) from this organism has been reported (Felter, S., and Stahl, A. J. C.(1973) Biochimie (Paris) 55, 245-251). The assay for activity used in this purification was the production of P-labeled nucleotide, presumed to be ATP, in the presence of [P]polyphosphate and ADP. We have found that this assay does not reflect the activity of a polyphosphate kinase but rather the combination of an exopolyphosphatase, releasing free [P]phosphate from the added [P]polyphosphate, and the ADP-[P]phosphate exchange activity of the enzyme diadenosine 5`,5‴-P^1,P^4-tetraphosphate alpha,beta-phosphorylase (Ap(4)A phosphorylase). We also present direct evidence for the formation of an enzyme-AMP intermediate in the action of Ap(4)A phosphorylase.


INTRODUCTION

Inorganic polyphosphates (polyP) (^1)are linear polymers of phosphate linked by phosphoanhydride bonds. PolyP has been detected in a wide range of prokaryotic and eukaryotic organisms (1) and is particularly abundant in the yeast Saccharomyces cerevisiae, in which it can constitute over 10% of the cellular dry weight(2) . PolyP in yeast is predominantly localized to the vacuole(2) .

There are a number of possible biological functions for polyP in yeast. Foremost, it appears to act as a reservoir or buffer for phosphate. PolyP is hydrolyzed to mobilize free phosphate under conditions of low phosphate availability(3, 4) , and it is produced in very large amounts when phosphate is provided to yeast after a period of phosphate starvation, a phenomenon known as ``polyphosphate overcompensation'' (3, 5) . PolyP also appears to play a role in the regulation of cellular Ca by chelating vacuolar Ca, thereby providing a Ca sink(6) . More generally, polyP is the major anion in the vacuolar lumen and thus is important for charge balance and osmoregulation in the vacuole, a storage compartment for many ions and metabolites in yeast(7) . Other possible physiological functions of polyP in yeast remain to be elucidated.

There have been numerous observations made over the years of the effects of various physiological factors on the polyP pools in yeast. These factors include phosphate levels(3, 4, 8, 9) , aerobic and anaerobic metabolism of various carbon sources(4, 8) , growth phase (10, 11) , nitrogen starvation(11) , Mg concentration (12) , vacuolar acidification(10, 13) , and supply of basic amino acids (3) . The effects on the polyP pools are presumably mediated by effects on the expression or activity of enzymes involved in polyP metabolism. Yet, without a knowledge of these enzymes, no unified picture of polyP function and regulation in yeast has emerged. Three yeast exopolyphosphatases, which catalyze the progressive hydrolysis of polyP to inorganic phosphate, have recently been purified to homogeneity (14, 15, 16) . However, the enzymatic basis of synthesis of polyP in yeast remains unclear.

The only well characterized pathway for polyP synthesis in any organism is via the enzyme polyphosphate kinase (PPK), which catalyzes the following reaction(17) :

PPK activity has been observed in several microorganisms(18, 19) , and the enzymology of polyP synthesis has received growing attention with the purification of PPKs from Escherichia coli and Neisseria meningitidis(19, 20) . The gene encoding E. coli PPK has also recently been sequenced(21) .

The purification of an enzyme identified as a polyphosphate kinase from a soluble extract of yeast cells has been reported(22) . This is the only report to date of a purification of PPK from yeast, and it is widely cited(1, 4, 15, 16, 18, 19, 20, 23) . The enzyme purified was found to be active for the reverse reaction, synthesis of ATP from polyP, but not the forward reaction. This stands in contrast to PPKs that have been purified from other organisms, which are active in both directions (20, 24) . This property of the purified enzyme has led to the suggestion that polyP may play a role as an energy store in yeast by acting as a source of ATP(4, 22, 25) , though in vivo experiments had previously argued against such a role(5) . Moreover, this finding suggests two possibilities for polyP synthesis in yeast. Either polyP synthesis is due to PPK, but some factor required for polyP synthesis was absent in the in vitro experiments, or alternatively, other routes for polyP synthesis exist in yeast.

To better understand polyP metabolism and function in yeast, we attempted to repurify the putative yeast PPK following the protocol of Felter and Stahl (22) and using the same assay for activity, namely the production of P-labeled nucleotide (presumed to be ATP) in the presence of [P]polyP and ADP. We found the activity thus assayed and purified was not due to a PPK but rather to the enzyme diadenosine-5`,5‴-P^1,P^4-tetraphosphate alpha,beta-phosphorylase (Ap(4)A phosphorylase)(26) , acting in concert with one or more of the yeast polyphosphatases.


EXPERIMENTAL PROCEDURES

Materials and Strains

[P]Orthophosphate and [alpha-P]ATP were obtained from DuPont NEN. Polyethyleneimine-cellulose thin layer chromatography sheets were from J.T. Baker. Nitrocellulose filters were from Millipore. Bead-beater was from Biospec (Bartlesville, OK). Protein assay reagent was from Bio-Rad. Protein standards for SDS-PAGE were from Life Technologies, Inc. Unlabeled polyphosphate used for blocking in charcoal-binding experiments was crude phosphate glass (Sigma P8510). All other chemicals were purchased from Sigma. Yeast strain ATCC 32167 was used for all experiments except those with strains YPALS (trp1-Delta1 his3Delta200 ura3-52 ade2-101 lys2-801 can1) and YPALSHU (YPALS apa1Delta::HIS3 apa2Delta::URA3), which were kindly provided by Dr. Pierre Plateau (Ecole Polytechnique, Palaiseau, France).

PPK Assay

The putative PPK activity was assayed by measuring charcoal-absorbable radioactivity after incubation of protein with [P]polyP and ADP(25) . Briefly, the incubation buffer consisted of 50 mM Tris-HCl, pH 7.0, 2 mM MgCl(2), 2 mM ADP, and 4 mM [P]polyP (expressed in P(i) equivalents) (0.5-2 Ci/mol). The total incubation volume was 0.25 ml. The reaction was started with the addition of protein (5-100 µg) and proceeded for 20 min at 37 °C. The reaction was stopped with 0.25 ml of 7% HClO(4), then 0.5 ml of 0.15% bovine serum albumin was added. The tubes were left on ice for >30 min and then spun to precipitate acid-insoluble polyP. Aliquots of the supernatant were added to suspensions of 1% (wt/vol). activated charcoal in 0.1 M LiCl and 1% polyphosphate. The charcoal suspensions were filtered through nitrocellulose filters, and the filters were washed with 3 5 ml of 0.5 M LiCl and then counted in Aquasol scintillation fluid in a Beckman LS-1011 liquid scintillation counter. ADP-[P]P(i) exchange activity (see ``Results'') was measured in the same way as the putative PPK activity but with the [P]polyP replaced by an equal amount (in P(i) equivalents) of sodium [P]phosphate, and the 1% polyphosphate in the charcoal suspension was replaced with 0.1 M NaH(2)PO(4), pH 7.0.

Thin Layer Chromatography of Incubation Mixtures

Aliquots (100 µl) of the supernatants after perchloric acid precipitation were neutralized with K(2)CO(3) and stored at -20 °C overnight. These aliquots were later thawed, and the insoluble KClO(4) was removed by centrifugation. For each mixture, 6 1 µl of the supernatants were spotted on polyethyleneimine-cellulose sheets. The sheets were developed in 0.2, 1.0, and 1.6 M LiCl, for 2 min, 6 min, and 2.5 h, respectively(11) . Under these conditions, ATP, ADP, and phosphate are clearly resolved. TLC sheets were visualized by autoradiography and quantitated with a Bio-imaging analyzer (Fuji).

Preparation of [alpha-P]ADP

[alpha-P]ADP was produced by dephosphorylation of [alpha-P]ATP using adenylate kinase and an excess of AMP over ATP. The incubation buffer consisted of 0.5 mM AMP, pH 7.0, 50 µCi [alpha-P]ATP (30 Ci/mmol), and 2 units of myokinase in a total volume of 50 µl. Incubation was carried out for 50 min at 37 °C, at which time the distribution of radioactivity was 60% ADP, 40% AMP, and <5% ATP, as judged by TLC on polyethyleneimine-cellulose. The mixture was then heated in boiling water for 10 min to inactivate the myokinase. The [alpha-P]ADP thus formed was diluted to the desired specific activity with unlabeled ADP.

Preparation of P-adenylylated Enzyme

For labeling experiments, the same buffer was used as for the PPK assays, except without polyP and containing 10 µM [alpha-P]ADP (2000 Ci/mol). The total incubation volume was 150 µl. The reaction was started with 20 µg of protein from fraction III of the purification. Incubation was for 20 min at 37 °C. The reaction was stopped with 95 µl of 0.045% deoxycholate and 95 µl of 17% trichloroacetic acid, 3.6 mM NaP(i), 3.6 mM ADP. The tubes were held on ice for 30 min and then spun in a microfuge for 10 min. The pellets were washed with 2 0.4 ml of ice-cold dH(2)O, resuspended in Laemmli sample buffer(27) , and analyzed by SDS-PAGE.

Other Methods

Synthesis of long-chain [P]polyP was as described by Kornberg(28) . Protein was assayed by Bradford assays using Bio-Rad reagent and bovine serum albumin as standard. SDS-PAGE was performed according to Laemmli(27) . Gels were stained with silver(29) .


RESULTS

Purification of Alleged PPK

The three step purification of the enzyme identified as yeast PPK was repeated essentially as described by Felter and Stahl (22) ( Table 1and Fig. 1). The apparent specific activities in fractions I-III were similar to those reported(22) , though it should be noted that these are not true specific activities; the apparent specific activities will in general increase with the amount of protein used in the assay for reasons elaborated below. The apparent specific activity in fraction IV was lower than that reported in (22) , probably due to our use of less protein in the assay for activity and to some loss of enzymatic activity.




Figure 1: Elution profile of DEAE-Cellulose column. Activity was eluted from a DEAE-cellulose column as described in the legend to Table 1. The salt gradient from 0.1 to 0.3 M NaCl is shown. Apparent specific activities in each fraction were measured with 10 µg of protein as described under ``Experimental Procedures.'' Relative polyphosphatase activities were calculated by measuring released phosphate by TLC. Activities are expressed as fraction of peak specific activity. Curves represent protein (solid), apparent PPK activity (heavysolid), ADP-[P]P(i) exchange activity (see ``Results,'' dashed), and polyphosphatase (dotted). Peak activities were as follows: apparent PPK, 120 nmol/mg/min; ADP-[P]P(i) exchange, 550 nmol/mg/min; polyphosphatase, 1000 nmol/mg/min.



The protein fractions were analyzed by SDS-PAGE with silver staining (Fig. 2). The end product of this purification, fraction IV, contains a major band at around 40 kDa, but it is clear that fraction IV is not homogeneous as it was concluded to be in the previous work. Comparison of Fig. 2with PAGE analysis of the corresponding fractions in (22) suggests that less sensitive means of visualizing protein did not reveal numerous minor bands.


Figure 2: Analysis of protein fractions from purification by SDS-PAGE. Samples as described in Table 1were fractionated by 10% SDS-PAGE. Lane 1, markers are as follows: myosin, 205 kDa; phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 29 kDa; beta-lactoglobulin, 18 kDa. Lane 2, 5 µg of fraction I; lane 3, 5 µg of fraction II; lane 4, 5 µg of fraction III; lane 5, 2 µg of fraction IV.



Analysis of Reaction Products

In addition to measuring the production of P-labeled nucleotide by adsorption on charcoal, we analyzed aliquots of the reaction mixtures by thin layer chromatography on polyethyleneimine-cellulose sheets and autoradiography (Fig. 3). Two important observations can be made from the autoradiographs. First, while the chemically synthesized [P]polyP used in the assays contains very little (<1%) contaminating free phosphate (lane1), incubation with the protein fractions results in release of [P]P(i), indicating the presence of one or more polyphosphatases (lanes2-7).


Figure 3: Analysis of reaction products by thin layer chromatography. TLC was carried out on incubation mixtures as described under ``Experimental Procedures.'' The elution positions of ATP, ADP, and P(i) are indicated. The material that remains at the origin consists of acid-soluble polyphosphates. Unlabeled ADP was either present or absent in the incubation buffer as indicated. The incubations in lanes1-7 and 9 were with [P]polyphosphate; lane8 was with [P]phosphate. The incubations contained protein as follows: lanes 1 and 9, no protein; lane 2, 100 µg of fraction I; lane 3, 100 µg of fraction II; lanes 4 and 5, 20 µg of fraction III; lanes 6, 7, and 8, 5 µg of fraction IV. PanelsA and B are from different TLC runs.



Second, analysis of the reaction products from incubation with fractions III and IV reveals that [P]ADP is the major labeled nucleotide product, not [-P]ATP as would be expected for the action of a polyphosphate kinase (lanes5 and 7). Formation of [P]ADP is dependent on the presence of unlabeled ADP in the reaction mixture (lane4versuslane5 and lane6versuslane7); neither AMP nor ATP can substitute for ADP (not shown). Incubation with protein from fractions I or II does produce a mixture of both labeled ADP and ATP (lanes2 and 3); however, it appears that this [P]ATP is produced secondarily from synthesized [beta-P]ADP by the action of adenylate kinase present in the preparation:

The adenylate kinase activity is present in large amounts in the starting yeast extract but is largely purified away during the DEAE-cellulose column step, eluting at the beginning of the salt gradient (not shown) so that [P]ADP is the major nucleotide product obtained even with the earliest eluting fractions that contain the apparent PPK activity. The fact that the principal product with the most purified protein fractions is [P]ADP shows that ADP, not ATP, is the actual product of the reaction being assayed. Thus, the reaction can be characterized as an exchange of radioactivity into ADP rather than as a phosphorylation of ADP.

The identification of the observed product as ADP was confirmed in three ways. Incubation of the quenched, neutralized reaction mixture with charcoal resulted in disappearance of the ADP spot. Incubation with potato apyrase also resulted in a disappearance of the spot, with a concomitant increase in the radioactivity in the phosphate spot, corresponding to phosphate released by hydrolysis of the [beta-P]ADP. Finally, incubation with pyruvate kinase and phosphoenolpyruvate resulted in a shift of the spot from the ADP position to the ATP position (data not shown).

Phosphate Is the Substrate for the Exchange Reaction

As noted above, we observed that a significant fraction of the phosphate residues in the [P]polyP added were released as free P(i) in the course of the incubations. This observation, along with the characterization of the reaction as an exchange process rather than an energy-dependent phosphorylation, suggested to us that the reaction might not involve polyphosphate at all, but rather could be the following:

To check this possibility, we replaced the [P]polyP in the assays with an equal amount (in total P(i) content) of free [P]orthophosphate. In each case, [P]ADP was still formed (Table 1; Fig. 3, lane8). In fact, [P]P(i) was a better substrate than [P]polyP, resulting in higher levels of [P]ADP in every assay, suggesting that P(i) is the true substrate for the exchange reaction. Indeed, when [P]polyP was incubated with 5 µg of protein from fraction IV, conditions under which only 20 nmol of free [P]P(i) were released, the apparent specific activity was 9-fold lower than the activity found by assaying with free [P]P(i) under the same conditions (Table 1). Furthermore, the peak of activity from the DEAE-cellulose column, when assayed with [P]polyP, appeared at the end of the NaCl gradient, where the highest levels of polyphosphatase activity were also eluted (Fig. 1). When the fractions from this column were reassayed using [P]P(i) instead of [P]polyP, the activity profile shifted to earlier fractions. Therefore, the activity measured with [P]polyP appears to depend on the presence both of a polyphosphatase that can release free [P]P(i) and of an enzyme that can subsequently catalyze ADP-[P]P(i) exchange.

The activity profile of the apparent PPK eluted from the DEAE-cellulose column reported by Felter and Stahl (22) peaks somewhat earlier than does that of the [P]polyP-dependent activity that we assay; it more closely resembles the profile of the ADP-[P]P(i) exchange activity that we measure. This difference and the higher recovery of [P]polyP-dependent activity from the final polyphosphate-cellulose column in the previous study suggest that there was better survival of polyphosphatase activity and higher levels of polyphosphatase activity in earlier DEAE-cellulose fractions during their purification. Felter and Stahl (22) may also simply have been using different amounts of protein in their assays; since the amount of substrate ([P]P(i)) released and available for exchange will increase with the amount of protein used, the apparent specific activity measured will also increase with increasing protein. Differences in the amounts of protein used for assaying the fractions could thus change the elution profile of the apparent PPK activity. Finally, some differences in elution profiles between the two studies are likely due to differences between the size of columns used and amounts of protein loaded.

The Exchange Activity Is Due to Diadenosine Tetraphosphate Phosphorylase

The yeast enzyme Ap(4)A phosphorylase catalyzes the phosphorolysis of Ap(4)A to ATP and ADP(26) . It also catalyzes the following exchange reaction between nucleoside diphosphates (NDP) and phosphate: NDP + [P]P(i) [beta-P]NDP + P(i), where N is A, T, C, or G(30) . Two isoforms of the enzyme, Ap(4)A phosphorylase I and II, have been identified and are encoded by the APA1 and APA2 genes(31) . Ap(4)A phosphorylase I appears to be responsible for >90% of the ADP-[P]P(i) exchange activity measured in crude yeast extracts(32) . Moreover, the Ap(4)A phosphorylases have apparent molecular masses on SDS-PAGE of 40 kDa, the same size as the major band in fraction IV. Finally, the initial steps in the reported purification of Ap(4)A phosphorylase I (in which Ap(4)A phosphorylase II was not detected) are virtually identical to those used in this study(26) . Thus, the Ap(4)A phosphorylases are likely candidates for the source of the ADP-[P]P(i) exchange activity purified by this protocol.

To confirm this, we used Ap(4)A and AMP, known inhibitors of the Ap(4)A phosphorylase exchange activity(30) , to inhibit the ADP-[P]P(i) exchange activity in our protein preparation (fraction III). Inhibition was 90 ± 2% at 2 mM Ap(4)A, while 4 mM AMP inhibited to 50 ± 3%. These levels of inhibition are comparable to those seen with purified Ap(4)A phosphorylase(30) . Mg had opposite effects on the exchange activity, depending on whether the activity was assayed using [P]P(i) or [P]polyP as the substrate. With [P]P(i), Mg was inhibitory, as was reported for purified Ap(4)A phosphorylase (30) . In contrast, with [P]polyP, the activity was 10-fold higher in the presence of 2 mM MgCl(2) than in its absence, consistent with the requirement for Mg for apparent PPK activity reported in(22) . This stimulatory effect of Mg in the PPK assay is explained by our observation that the polyphosphatase activity of the protein preparation increases more than 10-fold in the presence of Mg, consistent with the Mg dependence of the known yeast exopolyphosphatases(14, 15, 16) . These effects of Mg again support the conclusion that [P]P(i) must be released from [P]polyP to be incorporated into ADP. Raising [Mg] to 0.1 M led to complete inhibition of the [P]polyP-dependent activity, consistent with the observations of Felter and Stahl (22) and with the inhibitory effect of Mg on Ap(4)A phosphorylase.

Finally, we acquired a yeast strain in which both the APA1 and APA2 genes were disrupted(31) . Lysates were prepared from cells both of this strain and of its parental strain in which both Ap(4)A phosphorylases are present. Protein from these lysates was assayed for exchange activity using either [P]polyP or [P]P(i) (Fig. 4). No P-labeled nucleotide was produced with the lysate from the cells that lack the Ap(4)A phosphorylases. The absence of labeled nucleotide was confirmed by TLC analysis of the products (not shown). Thus, while one might expect that this assay would be able to detect an actual PPK activity in the absence of the interfering Ap(4)A phosphorylase activity, we were unable to detect such an activity in these crude lysates. We were also unable to detect any substantial PPK activity using this assay in lysates prepared from YPALSHU cells that had been starved for phosphate (which has been suggested to derepress PPK expression(5) ). Omitting the 10,000 g centrifugation step during preparation of the lysates also failed to result in detection of PPK activity by this assay in starved or non-starved cells.


Figure 4: Lack of exchange activity in yeast strain lacking Ap(4)A phosphorylases. Cell lysates from strains YPALS (APA1 APA2) and YPALSHU (YPALS apa1Delta::HIS3 apa2Delta::URA3) were prepared as described under ``Experimental Procedures.'' 100 µg of protein from the lysates was used for each assay. Activity was measured in the presence of either 4 mM [P]polyphosphate (2.2 Ci/mol) or 4 mM [P]phosphate (1.5 Ci/mol), indicated as Poly or P(i). Charcoal-absorbed radioactivity (mean ± S.D., n = 4) is shown in the absence (background) and presence of ADP.



As a further demonstration that the activity which we are assaying is the same activity described by Felter and Stahl(22) , we measured the effects of addition of salts and metal ions on the apparent PPK activity in fraction III. As noted above, Mg has the same effects on our apparent PPK activity as on the activity previously reported. We also found, as reported by Felter and Stahl(22) , that high concentrations (0.1-0.3 M) of NaCl or KCl inhibited the apparent PPK activity, with KCl giving higher levels of inhibition than NaCl. Addition of 1 mM Zn, Fe, or Mn (added as sulfate salts) inhibited the activity by 100, 85, and 68%, respectively, as compared to 98, 97, and 69% in the previous work. Analysis of reaction products revealed that the effects of NaCl and KCl were due to inhibition of the Ap(4)A phosphorylase reaction, while the metal ions had effects both on the ADP-[P]P(i) exchange activity and the polyphosphatase activity. In contrast to the findings of Felter and Stahl(22) . however, we found that 1 mM beta-mercaptoethanol had no inhibitory effect on the apparent PPK activity. This is consistent with free sulfhydryl groups being required for Ap(4)A phosphorylase activity(26) . It is not clear why beta-mercaptoethanol was found to be inhibitory in the previous study; it is possible that the beta-mercaptoethanol used was partially oxidized and that it inhibited activity by forming mixed disulfides with the crucial free sulfhydryl groups.

Demonstration of an Enzyme-AMP Intermediate

It has been suggested that Ap(4)A phosphorylase probably acts via an enzyme-AMP intermediate (30) as shown in Fig. SI:


Figure SI: Scheme I.



with ADP-[P]P(i) exchange occurring due to the reversibility of enzyme-AMP formation. We attempted to detect formation of a covalent enzyme-AMP species by incubating protein from fraction III with [alpha-P]ADP and analyzing the products by SDS-PAGE and autoradiography (Fig. 5). A single band with an apparent molecular mass of 40 kDa was labeled. Labeling was abolished by addition of 10 mM phosphate or arsenate to the incubation buffer. Addition of ATP resulted in an almost complete abolition of labeling, while 10 mM sulfate had little if any effect. These effects are consistent with the model of Ap(4)A phosphorylase action; excess P(i) or ATP reduces the appearance of labeled species by shifting the equilibrium away from the enzyme-AMP form, while arsenate irreversibly displaces the AMP from the enzyme by forming an unstable adenosine phosphoarsenate species(30) . Sulfate is known to be unable to similarly displace AMP(30) .


Figure 5: Formation of P-adenylylated enzyme. Incubation of 20 µg of protein from fraction III with [alpha-P]ADP was performed as described under ``Experimental Procedures.'' Labeled protein was fractionated by 10% SDS-PAGE. The positions of the 97-, 66-, 43-, and 29-kDa markers are shown. Additions to the incubation mixtures were as follows. Lane 1, no addition; lane 2, 10 mM sodium phosphate; lane 3, 10 mM sodium arsenate; lane 4, 10 mM sodium sulfate; lane 5, 10 mM ATP.




DISCUSSION

A number of points from the report of the apparent purification of yeast PPK may be addressed in light of the identification of the activity as Ap(4)A phosphorylase. Most important, the previous investigators were unable to observe substantial PPK activity in the opposite direction, i.e. synthesis of polyP from ATP. They reported that no more than 4% of radioactivity added as [-P]ATP could be incorporated into a fraction that was attributed to polyphosphate. This fraction was characterized solely as radioactivity that was not adsorbed by charcoal and that was precipitated by BaCl(2) at pH 2. Since no further analysis was conducted to demonstrate that this radioactivity actually was in polyP, we believe that it most likely represents a nonspecific background. The difficulty in observing polyP synthesis is explained by the fact that the activity being assayed in the protein preparation is not in fact polyphosphate kinase. The inference, based on this apparent irreversibility, that PPK in yeast acts primarily in the direction of ATP synthesis(22, 25) , is thus unsupported. Previous in vivo experiments were unable to detect direct synthesis of ATP from polyP in yeast cells, suggesting that polyP is not used as an energy store by yeast(5) .

Furthermore, it was reported that the supposed PPK activity was inhibited in a competitive manner by phosphate(22) . This is explained by the demonstration that phosphate is actually the substrate of the exchange reaction being observed; addition of unlabeled phosphate will dilute the radioactivity of the released [P]P(i), giving the appearance of competitive inhibition. In fact, phosphate stimulates the activity of most of the polyphosphate kinases that have been characterized(19, 24, 33, 34) . This may reflect a mechanistic requirement for phosphate to act as a primer or an allosteric effect(19) . Given the role of polyP as a phosphate store, a stimulation of PPK by phosphate would provide a direct means of inducing polyP synthesis in response to high phosphate levels(33) . The large burst of polyP synthesis in yeast vacuoles during polyphosphate overcompensation is in fact preceded by a rapid increase in vacuolar phosphate concentration(9) .

In the case of Ap(4)A phosphorylase acting in the direction of Ap(4)A synthesis, the enzyme performs a nucleotidyl transfer to the 5`-phosphate of a nucleotide acceptor, namely ATP. In this respect, its action is similar to that of the guanylyltransferases of eukaryotic mRNA capping enzymes, though Ap(4)A phosphorylase is unusual in that it uses ADP as an adenylyl donor. As in the Ap(4)A phosphorylase reaction in which the enzyme is reversibly adenylylated, the RNA capping reaction proceeds via guanylylation of the guanylyltransferase(35) . In the enzyme-GMP intermediate, the GMP is attached to the -amino group of a lysine, as is also the case for RNA and DNA ligases. An attachment motif common to both classes of enzymes has been identified, consisting of KXBG(35) . It is possible that the Ap(4)A phosphorylases act via a similar mechanism. Examination of the sequence of the APA2 gene reveals the presence of this motif, and the region containing the motif shows some similarity to the region surrounding the attachment motif in the RNA-capping guanylyltransferase CEG1 of S. cerevisiae:

A tyrosine or phenylalanine appears four residues upstream of the conserved lysine in the guanylyltransferases and is also present in APA2.

While yeast has been an organism in which many studies of polyphosphate metabolism have been conducted, no polyphosphate kinase from yeast has yet been purified. A small amount of PPK activity was reported to be detected in yeast vacuoles(23) , but it remains to be determined whether a PPK is responsible for the synthesis of the large vacuolar polyP pool or whether alternative or multiple synthetic routes may exist(36) . Identification of the enzymes involved in polyP synthesis in yeast will shed light on the metabolism and physiological functions of polyP and may suggest mechanisms of polyP synthesis in other eukaryotes.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL08893. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Howard Hughes Medical Institute Predoctoral Fellowship. To whom correspondence should be addressed: 7 Divinity Ave., Cambridge, MA 02138. Tel.: 617-495-2301; Fax: 617-495-8308; booth{at}husc.harvard.edu.

(^1)
The abbreviations used are: polyP, inorganic polyphosphates; PPK, polyphosphate kinase; Ap(4)A, diadenosine-5`,5‴-tetraphosphate; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We are grateful to Dr. Pierre Plateau for the yeast strains YPALS and YPALSHU and to Dr. Anthony Morielli and Larry Coury for critical reading of the manuscript.


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