Download Vacuolar Function in the Phosphate Homeostasis of the Yeast

Plant Cell Physiol. 37(8): 1090-1093 (1996)
JSPP © 1996
Vacuolar Function in the Phosphate Homeostasis of the Yeast
Saccharomyces cerevisiae
Kanae Shirahama' \ Yoshiaki Yazaki2, Katsuhiro Sakano 2 , Yoh Wada1 and Yoshinori Ohsumi
' Dapartment of Biology, Graduate School of Arts and Sciences, University of Tokyo, Tokyo, 153 Japan
Department of Applied Physiology, National Institute of Agrobiological Resources, Tsukuba, 305 Japan
2
We studied physiological roles of the yeast vacuole
in the phosphate metabolism using 31P-in vivo nuclear
magnetic resonance (NMR) spectroscopy. Under phosphate starvation wild-type yeast cells continued to grow for
two to three generations, implying that wild-type cells contain large phosphate pool to sustain the growth. During the
first four hours under the phosphate starved condition, the
cytosolic phosphate level was maintained almost constant,
while the vacuolar pool of phosphate decreased significantly. "P-NMR spectroscopy on the intact cells and perchloric
acid (PCA) extracts showed that drastic decrease of polyphosphate took place during this phase. In contrast, Aslpl
cells, which were defective in the vacuolar compartment,
thus lacked polyphosphate, ceased their growth immediately when they faced to phosphate starvation. Taken
together, we conclude that vacuolar polyphosphate provides an active pool for phosphate and is mobilized to cytosol during phosphate starvation and sustained cell growth
for a couple rounds of cell cycle.
polyphosphate localizes in the vacuole (Indge 1968, Urech
et al. 1978, Durr et al. 1979, Kornberg 1995). However, the
metabolism and function of polyphosphate have not been
established in yeast.
In this study, we showed a dynamic role of the vacuole
on maintaining the phosphate level in the cytosol. Using
3I
P-NMR, we showed that the levels of cytosolic phosphorous compounds are maintained constant for about four
hours after shift to phosphate starvation medium, whereas
the amount of polyphosphate decreased. This suggests that
the vacuolar polyphosphate is mobilized to the cytosol to
maintain the cytosolic phosphate level. These results showed that the vacuole plays critical roles in the phosphate metabolism.
Materials and Methods
Key words: NMR — Phosphate metabolism — Polyphosphate — Vacuole — Yeast.
Phosphate is one of the most important nutrients for
living organisms. It is a component of nucleotide, phospholipid, and nucleic acid. In addition, chemical energy of
phosphate bonds functions as the major source of free
energy required for many cellular activities. Therefore,
depletion of phosphate is a serious problem to living organisms. For understanding the phosphate metabolism, it is important to consider intracellular compartmentation of phosphorous compounds. Yeast cell accumulates considerable
amounts of phosphate as polyphosphate which is a linear
polymer of orthophosphates with acid anhydride linkage
(Wood and Clark 1988). It accounts for 40% of the total
phosphate content in a certain condition (Kulaev 1979).
The length of polyphosphate varies from three residues to
greater than 1000 residues. In yeast cells, the bulk of the
Abbreviations: MDP, methylene diphosphonate; NMR, nuclear magnetic resonance spectroscopy; PCA, perchloric acid; SD,
synthetic medium; SD( — Pj), synthetic phosphate free medium.
3
Present address: Department of Cell Biology, National Institute
for Basic Biology, Okazaki, 444 Japan.
Strains, media, and culture conditions—Yeast strains used
were X2180-1A (MATa SUC2 mal mel gaI2 CUPJ), YW5-1B
(MA 7a Ieu2 trpl ura3 SUC2 mal mel gal2 CUP1), and YW10-2B
(MA 7a adel slpl::LEU2 SUC2 mal melga/2 CUP1). YEPD medium contained 1% yeast extract (Difco Laboratories, Inc., Detroit,
MI), 2% polypepton (Nippon Seiyaku, Tokyo, Japan), and 2%
glucose. The composition of synthetic medium (SD) was 0.17%
yeast nitrogen base (Difco Laboratories, Inc.), 0.5% ammonium
sulfate, 2% glucose and, if needed, appropriate amino acids. For
synthetic phosphate free medium [SD( — Pj)], potassium phosphate was replaced by potassium chloride. Liquid cultures were
grown in flasks or tubes at 30°C with shaking. For standard starvation-experiments, exponentially growing cells at a density of 2 ~
3x 107 cells ml" 1 were collected by centrifugation, washed twice
with the starvation-medium, and resuspended in the starvation-medium.
3I
P-NMR spectroscopy—"P-NMR spectra were obtained at
202.3 MHz using a Varian VXR-500S spectrometer operating in
the Fourier-transform mode. Each spectrum was acquired with
45° pulses at a repetition rate of 0.35 s and 1024 scans were accumulated. Methylene diphosphonate (MDP) in Tris buffer (pH 8.9)
in a capillary tube was used as an external standard reference.
Temperature was kept at 20°C throughout the measurements.
Cells were transferred to 10-mm NMR tube and suspended at a
density of 2 x 109 cells ml" 1 in SD(—P,) medium with final concentrations of 10% (v/v) D2O and 6 mM EDTA. To maintain aerobic
conditions in the NMR tube, pure oxygen was bubbled at a rate of
4.8 ml min~'.
PCA extract for 3IP-NMR—Cell extracts were prepared by
PCA extraction as described by Navon et al. (1979) with modifications. Cells were collected by centrifugation at 4°C, and washed
once with cold distilled water. Cells were then suspended in cold
distilled water and 60% PCA (prechilled at -20°C) was added at
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Vacuolar phosphate in yeast
1091
NMR, we examined the changes of intracellular phosphorous metabolites both in the cytosol and the vacuole under
the phosphate starvation. Wild-type (YW5-1B) cells were
grown to exponential phase in SD medium, and transferred to SD(—P^ medium, then the amounts of intracellular phosphorous compounds were measured by 3IP-NMR
(Fig. 2A). The amounts of cytosolic phosphorous comResults
pounds did not change during phosphate starvation, inDeprivation of phosphate is a critical situation to liv- dicating that the amount of inorganic phosphate was kept
ing organisms. We examined growth response of yeast to constant (Fig. 2A). The intensity of signal assigned to inorphosphate starvation. Wild-type cells (YW5-1B) grown to ganic phosphate in the cytosol did not change during the
exponential phase in YEPD were transferred to synthetic first four hours. For additional two hours, the intensity of
medium depleted for phosphate [SD( — Pj)]. The growth signal of cytoplasmic inorganic phosphate decreased to
rate during the first four hours in SD(—Pj) medium was about 20% of the initial level. The intensities of signals
similar to that in synthetic medium (SD) (Fig. 1 A). It gradu- assigned to sugar phosphates showed similar decrease to
ally decreased in prolonged incubation in SD( —P,). The that of cytosolic inorganic phosphate.
population of budded cells decreased during phosphate
The "P-NMR spectrum of wild-type (YW5-1B) cells
starvation (Fig. IB). After incubation for 10 hours in grown in SD medium showed a large signal of core phosSD(—Pj) medium, bud index decreased to below 5%, in- phate of polyphosphate (Fig. 2A, peak 11). During phosdicating that cells were arrested in the G, phase of the cell phate starvation, intensity of signal assigned to core phoscycle. The response to phosphate starvation was unique, phate group of polyphosphate decreased (compare a-f),
since yeast cells can not enter the new cell-cycle under the ni- and disappeared after four hours. Examination of PC A
trogen or carbon starvation (Pringle and Hartwell 1982). extracts by 3IP-NMR also confirmed these observations
This obvious contrast in the growth response to the nu- (Fig. 2B). While the terminal phosphate signal (peak 6)
trient starvation conditions may reflect the difference in showed transient increase at one hour and maintained long
the amount of intracellular pool for each nutrient. Yeast period, then disappeared. During incubation of two hours
cells contain a considerable amount of phosphate in in SD( — Pj) medium, the intensity of signal assigned to
various forms (Kulaev 1979). This large pool may allow the core phosphate group of polyphosphate decreased to
cells to undergo two to three cell cycles.
approximately 25%, while the intensity of the terminal
To understand physiological responses to phosphate phosphate signal increased four-fold. This indicates that
starvation, it is necessary to consider the intracellular com- polyphosphate is digested with endopolyphosphatase and
partmentation of phosphorous metabolites because yeast cleaved to shorter chains. We could not observe any change
cells have a large pool for phosphate as polyphosphate (In- in the chemical shift of polyphosphate signal so that the
dge 1968, Diirr et al. 1979, Kornberg 1995). By using 31Pcleavage of polyphosphate should occur within the vacu-
a final concentration of 10%. Cell extracts were prepared by shaking the cell suspension vigorously, freezing and thawing three
times, and pelleting the cell debris by centrifugation. The supernatant was neutralized with 5 M KOH and centrifuged to remove the
deposits. Finally, D2O and EDTA were added at final concentrations of 10% (v/v) and 10 mM, respectively.
(A)
io 8
(B)
3U
wppr
J cells
40
LA
30
20
m
10
" —•—
1
O
2
4
6
Time (h)
8
SD(-P0
1
1
4
6
Time (h)
V.
1
8
10
Fig. 1 Growth and cell-cycle arrest of wild-type cells during phosphate starvation. Cells of a wild-type strain YW5-1B were grown in
YEPD until the exponential phase (2~3 x 107 cells ml"'), harvested, and washed twice with SD(-Pj). The cells were transferred to
SD( —Pi), and their growth (A) and bud index (B) were monitored as a function of time after the shift.
1092
Vacuolar phosphate in yeast
(A)
(B)
-9 -10 -19
- « 3 -SO - 3 3
Fig. 2 The levels of cytoplasmic phosphorus compounds are maintained constant during phosphate starvation. (A) 31P-NMR spectra
of wild-type cells. Exponentially growing cells of wild-type (YW5-1B) were transferred to SD(—P|). At the time indicated, cells were harvested and measured with 31P-NMR. The chemical shift values are expressed in ppm relative to an external standard of MDP. (a) 0 h, (b)
1 h, (c) 2 h, (d) 3 h, (e) 4 h, (f) 5 h. Signals were assigned as follows: 1, MDP; 2, sugar phosphates; 3, cytoplasmic inorganic phosphate;
4, vacuolar inorganic phosphate; 5, NTP-/?,y; 6, terminal phosphate group of polyphosphate; 7, NTP-a; 8, NADP; 9, NTP-/?; 10, the
third phosphate group of polyphosphate; 11, core phosphate group of polyphosphate. (B) Changes in the intensities of phosphorous
compounds measured with 31P-NMR. Wild-type (YW5-1B) cells were grown to the exponential phase in SD, then transferred to
SD(-Pi). At the time indicated, cells were harvested, and 10% PCA extracts were measured by 31P-NMR as described in Materials and
Methods. O, sugar phosphates; •, core phosphate group of polyphosphate; A, inorganic phosphate.
ole.
Aslpl (YW10-2B) cells have no detectable vacuolar
compartment and they also lack vacuolar polyphosphate
(Fig. 3A). We compared the growth response of wild-type
and Aslpl cells to phosphate starvation. Cells of wild-type
(X2180-1 A) and Aslpl (YWI0-2B) were grown to exponential phase in YEPD medium, and then transferred to SD
( — Pi) medium. Wild-type cells continued to grow for more
than six hours, while Aslpl cells immediately stopped their
growth under phosphate-starvation (Fig. 3B). This result
suggests that polyphosphate in the vacuole has certain role
as phosphate reserve and supports cell to proliferate under
phosphate starvation.
Discussion
In this study, we investigated the response of intracellular phosphate levels in yeast under phosphate starvation.
The vacuole of yeast is known to be a storage compartment
for various primary and secondary metabolites. Phosphate
is also stored in the vacuole as a polymeric form, polyphosphate. We used the 31P-NMR spectroscopy to distinguish
the phosphate in the cytosol and the vacuole as well as their
chemical forms. We found that yeast cells continued to
grow for two to three rounds of the cell-cycles during phosphate-starvation. During this phase, the levels of cytoplasmic phosphorous compounds remained constant despite of
the phosphate depriviation, whereas the vacuolar phosphate compounds decreased significantly. Aslpl mutant
cell which lacks the functional vacuolar compartment (Kitamoto et al. 1988, Wada et al. 1990), thus lacks vacuolar
polyphosphate, ceased to grow immediately when faced to
phosphate depletion. The changes in the cytosolic and
vacuolar phosphate levels and the response of the mutant
cells indicate a dynamic flow of phosphorous compounds
during the phosphate starvation.
The exponentially growing cells contain a large
amount of phosphate as polyphosphate in the vacuoles.
Storage of phosphate as polymers may provide an apparent
advantage for reducing osmolarity. It is also likely that the
polyphosphate may participate in the storage of divalent cations and basic amino acids in the vacuole (Diirr et al.
1979). The vacuole is also known to be a storage compartment of calcium (Dunn et al. 1994): it contains several milli
molar order of calcium under certain condition, where the
cytoplasmic calcium level should be regulated within the
submicro molar levels. This large difference in calcium concentration between the two compartments will generate a
large electrochemical potential difference across the vacuolar membrane (Ohsumi and Anraku 1983). Polyphosphate sequesters calcium very effectively, thus the vacuolar
polyphosphate can contribute to reduce the concentration
of free calcium ion in the vacuole. Despite of its physiological importance, the biogenesis of polyphosphate is not well
Vacuolar phosphate in yeast
facts suggest that the cells recognize depletion of extracellular phosphate and induce several responses before significant decrease of phosphate in the cytosol.
(B)
(A)
1093
108
It is important to elucidate the mechanisms of how the
cells sense the extracellular phosphate-level, transduce the
signals and mobilize the vacuolar polyphosphate. In this
paper, we showed polyphosphate was first digested with
endopolyphosphatase in the vacuole. Recently, Wurst et al.
(1995) reported the isolation and the characterization of
cytosolic exopolyphosphatase of yeast. To understand the
polyphosphate metabolism, it is essential to elucidate the
mechanism of phosphate transport across the vacuolar
membrane. Genetic and molecular biological approaches
on enzymes involved in the polyphosphate metabolism,
especially import and export system, will provide clues for
further understanding on the dynamic mechanism.
I I I I • I I
-5
-10 -15 -20 -25 -30 -35
I
ppm
Fig. 3 bslp] cells lack active phosphate pools. (A) 31P-NMR
spectrum of telpl cells. Cells of telpl (YW10-2B) grown to the exponential phase in SD were harvested and measured with 31 PNMR. The chemical shift values are expressed in ppm relative to
an external standard of MDP. Signals were assigned as follows: 1,
MDP; 2, sugar phosphates; 3, cytoplasmic inorganic phosphate;
4, inorganic phosphate in the medium; 5, NTP-/?,y; 6, NTP-a;
7, NADP; 8, NTP-/S. (B) Growth arrest of telpl cells under the
phosphate starved condition. Cells of wild-type (X2180-1A) and
telpl (YW10-2B) were grown in YEPD until the exponential
phase ( 2 ~ 3 x 107 cells ml" 1 ), harvested, and washed twice with
SD(-Pi). The cells were transferred to SD(-Pi), and their growth
was monitored as a function of time after the shift.
examined. Accumulation of polyphosphate requires the
active vacuolar H + -ATPase (Beauvoit et al. 1991), thus
energization of the vacuolar membrane may play an important role.
We showed here that the vacuolar polyphosphate provides a physiologically active pool for phosphate. The
vacuolar polyphosphate dynamically mobilized from the
vacuole to the cytosol during the phosphate starvation and
supported cell growth for a couple rounds of the cell cycle.
This cellular response to phosphate starvation is unique:
we previously showed that the nitrogen and carbon starvation result in the first cell-cycle arrest and under these conditions cells induce autophagy (Takeshige et al. 1992). The
cells already entered into the new round of the cell cycles
stop their growth at the next 'check point' in the G, phase
(Pringle and Hartwell 1982). In contrast, when the cells are
faced to the phosphate starvation, they continued to grow
until their phosphate levels drops significantly. On the
other hand, the cells induce autophagy one hour after shift
to phosphate starvation (manuscript in preparation). These
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(Received July 16, 1996; Accepted August 30, 1996)