(Received for publication, April 11, 1995; and in revised form, June 14, 1995)
From the
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
,P
-tetraphosphate
,
-phosphorylase (Ap
A phosphorylase). We also
present direct evidence for the formation of an enzyme-AMP intermediate
in the action of Ap
A phosphorylase.
Inorganic polyphosphates (polyP) ()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
,P
-tetraphosphate
,
-phosphorylase (Ap
A phosphorylase)(26) ,
acting in concert with one or more of the yeast polyphosphatases.
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
exchange activity (see
``Results,'' dashed), and polyphosphatase (dotted). Peak activities were as follows: apparent PPK, 120
nmol/mg/min; ADP-[
P]P
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;
-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.
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 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
[
-
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 [-
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).
To check this possibility, we replaced the
[P]polyP in the assays with an equal amount (in
total P
content) of free
[
P]orthophosphate. In each case,
[
P]ADP was still formed (Table 1; Fig. 3, lane8). In fact,
[
P]P
was a better substrate than
[
P]polyP, resulting in higher levels of
[
P]ADP in every assay, suggesting that P
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
were released, the apparent
specific activity was 9-fold lower than the activity found by assaying
with free [
P]P
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
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
and of an enzyme that can
subsequently catalyze ADP-[
P]P
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
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
) 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.
To confirm this,
we used ApA and AMP, known inhibitors of the
Ap
A phosphorylase exchange activity(30) , to
inhibit the ADP-[
P]P
exchange
activity in our protein preparation (fraction III). Inhibition was 90
± 2% at 2 mM Ap
A, while 4 mM AMP
inhibited to 50 ± 3%. These levels of inhibition are comparable
to those seen with purified Ap
A phosphorylase(30) .
Mg
had opposite effects on the exchange activity,
depending on whether the activity was assayed using
[
P]P
or
[
P]polyP as the substrate. With
[
P]P
, Mg
was
inhibitory, as was reported for purified Ap
A phosphorylase (30) . In contrast, with [
P]polyP, the
activity was 10-fold higher in the presence of 2 mM MgCl
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
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
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 ApA phosphorylases are
present. Protein from these lysates was assayed for exchange activity
using either [
P]polyP or
[
P]P
(Fig. 4). No
P-labeled nucleotide was produced with the lysate from the
cells that lack the Ap
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
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 ApA
phosphorylases. Cell lysates from strains YPALS (APA1 APA2)
and YPALSHU (YPALS apa1
::HIS3 apa2
::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
. 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
A phosphorylase reaction, while the metal ions had
effects both on the ADP-[
P]P
exchange activity and the polyphosphatase activity. In contrast
to the findings of Felter and Stahl(22) . however, we found
that 1 mM
-mercaptoethanol had no inhibitory effect on
the apparent PPK activity. This is consistent with free sulfhydryl
groups being required for Ap
A phosphorylase
activity(26) . It is not clear why
-mercaptoethanol was
found to be inhibitory in the previous study; it is possible that the
-mercaptoethanol used was partially oxidized and that it inhibited
activity by forming mixed disulfides with the crucial free sulfhydryl
groups.
Figure SI: Scheme I.
with ADP-[P]P
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 [
-
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
A phosphorylase action; excess P
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
[
-
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.
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 ApA 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
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
, 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 ApA phosphorylase acting in the direction of
Ap
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
A phosphorylase
is unusual in that it uses ADP as an adenylyl donor. As in the
Ap
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
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.