FEMS Yeast Research 5 (2005) 823–828
www.fems-microbiology.org
Effects of inactivation of the PPN1 gene on
exopolyphosphatases, inorganic polyphosphates and function
of mitochondria in the yeast Saccharomyces cerevisiae
Nikolay A. Pestov, Tatyana V. Kulakovskaya *, Igor S. Kulaev
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region,
142292, Russian Federation
Received 10 October 2004; received in revised form 18 February 2005; accepted 3 March 2005
First published online 30 March 2005
Abstract
Mutants of Saccharomyces cerevisiae with inactivated endopolyphosphatase gene PPN1 did not grow on lactate and ethanol, and
stopped growth on glucose earlier than the parent strain. Their mitochondria were defective in respiration functions and in metabolism of inorganic polyphosphates. The PPN1 mutants lacked exopolyphosphatase activity and possessed a double level of inorganic polyphosphates in mitochondria. The average chain length of mitochondrial polyphosphates at the stationary growth stage
on glucose was about 15–20 and about 130–180 phosphate residues in the parent strain and PPN1 mutants, respectively. Inactivation of the PPX1 gene encoding exopolyphosphatase had no effect on respiration functions and on polyphosphate level and chain
length in mitochondria.
Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.
Keywords: Mitochondria; Polyphosphate; Exopolyphosphatase; Respiration deficiency; Saccharomyces cerevisiae; PPN gene
1. Introduction
Inorganic polyphosphates (polyP) are linear polymers of many phosphate residues, linked by high-energy
phosphoanhydride bonds. They are widespread in
microorganisms and perform varied functions in cells,
such as phosphate and energy reservation, sequestration
and storage of cations, formation of membrane channels, gene activity control, and regulation of enzyme
activities [1–4].
The study of polyP in mitochondria is of interest in
the light of its possible role in energy metabolism in
yeast cells. Mitochondria of Saccharomyces cerevisiae
*
Corresponding author. Fax: +7 095 923 3602.
E-mail addresses: [email protected], [email protected]
(T.V. Kulakovskaya).
possess their own polyP pool [5,6] and two forms of exopolyphosphatase, soluble and membrane-bound [7].
However, the functions of polyP in mitochondria are
still unclear. Now yeast mutants with deficiency in
polyP-metabolizing enzymes are available: a single mutant with inactivated PPX1 gene encoding major exopolyphosphatase [8], a single mutant with inactivated
PPN1 (PMH5) gene encoding endopolyphosphatase,
and a double PPX1 and PPN1 mutant [9]. It should
be noted that inactivation of the PPX1 gene leads to a
considerable change in exopolyphosphatase activities
and spectrum in mitochondria [10].
Although there is no indication for the presence of
PPN1 in yeast mitochondria [11] and vacuolar localization of this protein has been proposed [12,13], the PPN1
gene has been found to have a substantial effect on the
exopolyphosphatase spectrum in the cytosol [14].
1567-1356/$22.00 Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.
doi:10.1016/j.femsyr.2005.03.002
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N.A. Pestov et al. / FEMS Yeast Research 5 (2005) 823–828
Inactivation of this gene leads to inhibition of the
expression of both exopolyphosphatase PPX1 and
high-molecular-mass exopolyphosphatase of 1000
kDa not encoded by PPX1 in this compartment [14].
The soluble mitochondrial exopolyphosphatase is encoded by PPX1 [10] and therefore the influence of the
PPN1 gene inactivation on exopolyphosphatases of
mitochondria cannot be excluded. Special interest in
the mutants with inactivated PPN1 is due to their reduced viability at the stationary growth stage [9].
The goal of this study was to compare the effects of
inactivation of the PPX1 and PPN1 genes on polyP
metabolism and mitochondrial function in S. cerevisiae.
2.4. Electrophoresis of polyP
2. Materials and methods
2.5. Enzyme assays
2.1. Yeast strains and culture conditions
ATPase activity was assayed in 50 mM Tris–HCl, pH
7.2 and 8.5, with 1 mM ATP and MgSO4 in the presence
of 0.1% Triton X-100. The activity of alkaline phosphatase was measured accordingly [17].
Exopolyphosphatase activities were assayed in soluble and membrane fractions obtained after sonication
of mitochondria [7]. Exopolyphosphatase activities were
determined by the rate of Pi formation at 30 °C for 30–
60 min in 1 ml of reaction mixture containing 50 mM
Tris–HCl, pH 7.2, 2.5 mM MgSO4 and 1 mM polyP188.
An activity unit (U) was defined as a quantity of the enzyme catalyzing the formation of 1 lmol Pi in 1 min.
Succinate dehydrogenase activity was determined by
the rate of potassium ferrocyanide reduction [18].
The strains of the yeast S. cerevisiae (Table 1) were
kindly provided by Dr. N. Rao and Prof. A. Kornberg
(Stanford University, USA) [8,9]. All strains were grown
in a shaker at 30 °C in a medium of the following composition: 1% yeast extract, 2% peptone, and 2% glucose
or 2% lactate as a carbon source.
2.2. Isolation of spheroplasts and mitochondria
The cells were treated by 20 mM dithiotreitol in 100
mM Tris–HCl, pH 9.4 (2 ml per g of wet biomass) for
20 min at 25° and then the spheroplasts were obtained
as described earlier [7]. Mitochondria were isolated from
the spheroplasts according to the literature [7]. The fractions of mitoplasts (mitochondrial matrix surrounded by
inner membrane) connected with outer membrane and
of the intramembrane space were prepared as described
[15].
2.3. Extraction and assay of polyP
Acid-soluble polyP was extracted with 0.5-N HClO4
at 4 °C [6]. After the removal of nucleotide phosphates
by adsorption to Norit A charcoal, the level of polyP
was estimated as a difference in the Pi amount before
and after the hydrolysis of samples in the presence of
1 N HCl for 10 min at 100 °C [6]. Pi was determined with
ascorbic acid and SDS [7].
The acid-soluble polyP fraction was neutralized to
pH 4.5 with NaOH and polyP was precipitated with saturated Ba(NO3)2 by centrifugation at 5000g for 20 min.
The barium salt of polyP was converted to a soluble
form by adding cation-exchange resin Dowex 50 WX 8
in the NHþ
4 form and some distilled water. The obtained
preparation was subjected to electrophoresis in 20%
polyacrylamide gel in the presence of 7 M urea and
the gel was stained with toluidine blue [16]. PolyP with
chain lengths of 15, 25, 45 (Sigma, St. Louis, MO)
and 188 phosphate residues (Monsanto, St. Louis,
MO) were used as standards.
2.6. Other methods
Protein concentration was assayed by the modified
Lowry method [19] using bovine serum albumin as the
standard. Glucose concentration in culture medium
was determined as in [20].
The rate of O2 uptake by mitochondria was estimated
by a Clark-type oxygen electrode using LP-7 Polarograph (Laboratorni Pristroje, Prague, Czechia) with
10-ml reaction chamber at 30 °C. The reaction medium
was as in [21]. 1 mM NADH was used as a substrate.
Respiratory control and P/O ratio were calculated
accordingly [22].
All data in the tables and figures are average values of
three experiments.
Table 1
Saccharomyces cerevisiae strains [8,9]
Strains
Genotype
CRY
CRX
CRN
CNX
MATa,
MATa,
MATa,
MATa,
ade2,
ade2,
ade2,
ade2,
Mutation in polyphosphate metabolism
his3,
his3,
his3,
his3,
leu2, trp1, ura3
trp1, ura3, ppx1D::LEU2
ura3, ppn1D::CgTRP1
ura3, ppn1D::CgTRP1, ppx1D::LEU
Parent strain
Strain with inactivated PPX1 gene
Strain with inactivated PPN1 gene
Strain with inactivated PPX1 and PPN1 genes
N.A. Pestov et al. / FEMS Yeast Research 5 (2005) 823–828
825
3.2. Some properties of mitochondria of the parent
strain and the PPX1 and PPN1 mutants
3. Results
3.1. PPN1 mutants do not grow on lactate and ethanol
The parent CRY strain of S. cerevisiae and the CRX
strain with inactivated PPX1 gene displayed diauxic
growth under glucose consumption (Fig. 1(a)). Strains
CRN and CNX with inactivated PPN1 gene stopped
to grow on glucose earlier than the parent strain (Fig.
1(a)). This growth arrest might be related to their inability to use non-fermentable carbon sources as substrates.
As a matter of fact, the strains CRN and CNX could
not grow on ethanol and lactate even when they were
cultivated for 3–5 d, whereas the growth of CRY and
CRX on lactate (Fig. 1(b)) and ethanol (not shown)
was similar. However, the cells of CRN and CNX grown
to at least 17–24 h on glucose retained their viability
[14].
12
(a)
10
8
6
Cell density, OD600
4
2
0
4
8
8
16
12
20
24
(b)
6
4
2
0
0
4
8
12
16
20
24
For the estimation of some properties of isolated mitochondria, the strains were grown on glucose to the earlystationary growth stage (CRY and CRX for 20 h, and
CRN and CNX for 17 h, Fig. 1(a)). The CRY and CRX
strains were grown also on lactate for 14 h (Fig. 1(b)).
The ATPase activities in isolated mitochondria of the
strains under study are shown in Table 2. At pH 8.5, the
ATPase activities were inhibited by 5 mM of NaN3 for
90% in case of CRY and CRX and for 70–80% in case
of CRN and CNX, respectively. The inhibitor of PATPases, 0.1 mM vanadate [23], at pH 7.2 had no effect
on the ATPase activities in any of the mitochondrial
preparations under study. This indicated their purity
from plasma membranes.
The inhibitor of V-ATPases, 50 mM nitrate [24], had
no effect on ATPase activities at pH 7.2. It should be
noted that nitrate repressed the ATPase of yeast vacuoles by 95% [17]. Besides, the more specific inhibitor of
V-ATPases, bafilomycin A1 [25] (0.3 lM), did not inhibit ATPase activities in any of the mitochondrial fractions obtained. Some authors have indicated alkaline
phosphatase as a vacuolar marker enzyme [15,17]. No
activity of this enzyme was observed in the mitochondrial preparations from strains CNX. In the preparations of other strains under study, the specific activity
of alkaline phosphatase was no more than 50 mU mg
protein1 whereas in yeast vacuoles its activity was
1600 mU mg protein1 [17]. Thus, the preparations were
almost free from contamination by vacuoles.
The criteria for integrity of isolated mitochondria are
presented in Table 2. Respiratory control ratio and P/O
ratio for the mitochondria from CRY and CRX were
close to the known data for S. cerevisiae [21,26]. The
activity of succinate dehydrogenase in the mitochondria
of these strains was 0.55 U (mg of protein)1 and the
enrichment factor of this enzyme as compared with the
cell homogenate was 3. The mitochondria isolated at
the same growth stage from strains CRN and CRX
showed no respiration control (Table 2) and succinate
dehydrogenase activity. The O2 consumption was 26
and 9 nmol O2 min1 mg protein1 for mitochondrial
preparations from CRY and CRX strains and from
PPN1 mutants, respectively.
So, the glucose repression [27] was abolished in case
of strains CRY and CRX (20 h of growth on glucose)
whereas the preparations from CRN and CNX resembled promitochondria [26].
Time of cultivation, h
Fig. 1. The growth of S. cerevisiae strains. (a) Yeast was grown on a
medium with 2% glucose at 30 °C in flasks with 100 ml medium,
shaking at 80 rpm. (b) Yeast was grown on a medium with 2% lactate
at 30 °C in flasks with 100 ml medium, shaking at 220 rpm (–d–, strain
CRY; –s–, strain CRX; –m–, strain CRN; –n–, strain CNX).
3.3. Exopolyphosphatase activities and polyP in
mitochondria prepared from glucose-grown cultures
Exopolyphosphatase activities and polyP levels were
assayed at an early-stationary growth stage on glucose
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N.A. Pestov et al. / FEMS Yeast Research 5 (2005) 823–828
Table 2
Some properties of mitochondria obtained from S. cerevisiae strains grown on glucose and lactate
Carbon source
Some properties of mitochondria
S. cerevisiae strain
CRY
CRX
CRN
CNX
Glucose
Respiratory control ratio
P/O ratio
ATPase activity (U mg protein1)
2.3
1.5
2.14
2.1
1.3
2.15
1a
0
1.6
1a
0
1.4
Lactate
Respiratory control ratio
P/O ratio
ATPase activity (U mg protein1)
1.6
1.3
2.3
2.2
1.1
2.16
–
–
–
–
–
–
–, not measured because the strains did not grow on lactate.
a
The respiratory level was low and did not increase upon addition of ADP.
as in Section 3.2. Inactivation of the PPX1 gene encoding 40-kDa exopolyphosphatase had no effect on membrane-bound but decreased soluble-exopolyphosphatase
activity in mitochondria (Table 3). Earlier, it had been
shown that in the CRX strain mitochondria had no
soluble 40-kDa exopolyphosphatase, which is characteristic of the parent strain CRY [10]. Instead, the highmolecular form of exopolyphosphatase appeared in the
soluble fraction [10].
No membrane-bound exopolyphosphatase activity
was observed in the mitochondria of the strains with
inactivated PPN1 (Table 3). The activity of soluble exopolyphosphatase represented by the 40-kDa enzyme in
the CRN strain was low (Table 3). No exopolyphosphatase activity was detected in the mitochondrial fraction
of double mutant CNX (Table 3).
The acid-soluble polyP levels were similar in the
mitochondria of CRY and CRX, whereas the mitochondria of CRN and CNX had double the amount of polyP
(Table 3). Nevertheless, the Pi levels were similar in all
preparations (Table 3). The chain length of polyP was
15–20 phosphate residues in CRY and CRX and increased to 130–180 phosphate residues in the PPN1
mutants CRN and CNX (Fig. 2).
We compared the polyP levels and chain lengths at
different growth stages on glucose (13 and 20 h of
growth, Fig. 1(a)). The ability of glucose to repress the
development of mitochondria in S. cerevisiae is well
known [27]. The concentration of glucose was 80% of
the initial one by hour 13 and glucose was exhausted
by hour 17 (not shown). At 20 h of growth CRY and
CRX possessed well-developed mitochondria (Table 2),
Fig. 2. The electropherogram of polyP in 20%-polyacrylamide gel in
the presence of 7 M urea. Lanes 1–4: PolyP standards with the chain
lengths of 15 (1), 25 (2), 45 (3) and 188 (4) phosphate residues,
respectively. Lanes 5–12: acid-soluble PolyP of the mitochondria of
S. cerevisiae grown on glucose. Lane 5: the strain CRY 13 h of growth;
lane 6: the strain CRX 13 h of growth; lane 7: the strain CRY 20 h of
growth; lane 8: the strain CRX 20 h of growth; lane 9: the strain CRN
13 h of growth; lane 10: the strain CNX 13 h of growth; lane 11: the
strain CRN 17 h of growth; lane 12: the strain CNX 17 h of growth.
whereas at 13 h of growth these strains had promitochondria (not shown). In the mitochondria of CRY
and CRX, the level of acid-soluble polyP decreased from
1 lmol mg protein1 by hour 13 of growth to
0.3 lmol mg protein1 by hour 20 of growth. The chain
length also decreased (Fig. 2). Quite on the contrary, in
Table 3
Pi, acid-soluble polyP and exopolyphosphatase activities in mitochondria of S. cerevisiae strains (20 h of growth on glucose, Fig. 1(a))
Strain
1
Pi (lmol mg protein )
PolyP (lmol mg protein1)
Activities (mU mg protein1)
Membrane-bound exopolyphosphatase
40-kDa exopolyphosphatase
High-molecular exopolyphosphatase
CRY
CRX
CRN
CNX
0.06 ± 0.00
0.34 ± 0.06
0.06 ± 0.03
0.27 ± 0.06
0.06 ± 0.01
0.73 ± 0.14
0.07 ± 0.01
0.72 ± 0.21
97 ± 8.8
136 ± 20
0
80 ± 2.3
0
35 ± 9.2
0
14 ± 0.07
0
0
0
0
N.A. Pestov et al. / FEMS Yeast Research 5 (2005) 823–828
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Table 4
Exopolyphosphatase activities, Pi and polyP level of mitochondria isolated from the yeast S. cerevisiae grown on lactate (14 h of growth)
Strain
Exopolyphosphatase activity (mU mg protein1)
Soluble form
Membrane-bound form
CRY
CRX
119 ± 1.4
58 ± 18
34 ± 8.1
57 ± 2.8
the mitochondria of CNX and CRN the level of acid-soluble polyP increased from 0.3 lmol mg protein1 by
hour 13 of growth to 0.72 lmol mg protein1 by hour
17 of growth. Its chain length increased (Fig. 2). It should
be noted that in the strains CRN and CNX only promitochondria were observed (Table 2).
3.4. Exopolyphosphatase activities and polyP in
mitochondria prepared from lactate-grown cultures
The soluble mitochondrial exopolyphosphatase activities in CRY and CRX were similar in glucose- and
lactate-grown cultures (Tables 3 and 4). The membrane-bound exopolyphosphatase activity was lower in
lactate-grown cultures. Mitochondria isolated from the
cells grown on lactate had lower polyP levels (Table 4)
and the chain length of polyP was less than 15 phosphate residues (not shown).
4. Discussion
The results suggest that the formation of well-developed mitochondria in the cells of S. cerevisiae after glucose exhaustion is accompanied by decrease in their
polyP level and chain length. On the contrary, in
PPN1 mutants, polyP chain length increased under glucose consumption and the formation of well-developed
mitochondria was not observed. The inability of these
strains to grow on non-fermentable substrates suggests
that the PPN1 gene is essential for mitochondrial functioning in S. cerevisiae. However, the explanation of its
pleiotropic effect needs further investigation.
The inactivation of the PPX1 gene had no effect
on polyP metabolism and functioning of mitochondria
in our experimental conditions. Probably, the
mitochondrial polyPs were inaccessible for soluble
exopolyphosphatase. In our preliminary experiments
on subfractionation of mitochondria, the fraction of
intramembrane space contained 90% of total mitochondrial polyP, while 95% of exopolyphosphatase
activity was observed in the mitoplast fraction. Probably, the main function of the PPX1 enzyme is not participation in the long-chain polyP metabolism but
hydrolysis of other substrates such as tripolyphosphate
and adenosine 5 0 -tetraphosphate [28].
The inactivation of the PPN1 gene resulted in decrease of activity of soluble 40-kDa exopolyphosphatase
Pi (lmol mg protein1)
Acid-soluble polyP (lmol mg protein1)
0.05 ± 0.01
0.04 ± 0.01
0.15 ± 0.03
0.19 ± 0.00
encoded by PPX1 in mitochondria (Table 3), similarly
as in cytosol [14]. Besides, the activity of membranebound mitochondrial exopolyphosphatase encoded by
an unknown gene is abolished in PPN1 mutants. The
absence of PPN1 in mitochondria should be taken into
account [11–13]. Possibly, the expression of both genes
depends on the PPN1 gene or its product in some unknown way. An explanation for the effect of PPN1 disruption on the expression of exopolyphosphatases might
be that this gene is probably responsible for induction of
the expression of the latter.
It is still unclear whether the accumulation and elongation of polyP could directly cause a breakdown of the
functions of mitochondria in PPN1 mutants, or whether
some unknown regulatory mechanisms are involved in
this process. It should be noted that the inactivation of
PPN1 has no effect on the Pi level in mitochondria
(Table 3).
The metabolic integration of phosphorus metabolism
and the PHO regulatory pathway is characteristic of a
yeast cell [29,30]. PolyP and polyP-dependent enzymes
are involved in many regulatory mechanisms and the
pleiotropic effects of mutations in polyP-dependent
enzymes are well-known in bacteria [2]. The transformation of chloroplasts with the polyphosphate kinase gene
of Escherichia coli enhanced polyP accumulation and
changed their energetic parameters [31]. As a whole,
the data on the effects of mutations in polyP-dependent
enzymes have demonstrated the importance of polyP
metabolism for the vital functions of microbial cells.
Acknowledgements
We thank Prof. A. Kornberg and Dr. N. Rao for providing mutant strains and Dr. A. Medentzev for the help
in respiratory experiments. We thank E.V. Makeeva for
help in the preparation of the manuscript. The work was
supported by a grant from the Support Fund of Leading
Science Schools of Russia (Grant 1382.2003.4).
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