Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Cell growth wikipedia , lookup
Cytoplasmic streaming wikipedia , lookup
Signal transduction wikipedia , lookup
Cellular differentiation wikipedia , lookup
Cell culture wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Tissue engineering wikipedia , lookup
Cell encapsulation wikipedia , lookup
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 1090 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 References Beauvoit, B., Rigoulet, M., Raffard, G., Canioni, P. and Guerin, B. (1991) Differential sensitivity of the cellular compartments of Saccharomyces cerevisiae to protonophoric uncoupler under fermentative and respiratory energy supply. Biochemistry 30: 11212-11220. Dunn, T., Gable. K. and Beeler, T. (1994) Regulation of cellular Ca 2+ by yeast vacuoles. / . Biol. Chem. 269: 7273-7278. Diirr, M., Urech, K., Wiemken, A., Schwencke, J. and Nagy, M. (1979) Sequestration of arginine by polyphosphate in the vacuole. Arch. Microbiol. 121: 169-175. Indge, K.J. (1968) Polyphosphates of the yeast vacuole. J. Gene. Microbiol. 51: 447-455. Kitamoto, K., Yoshizawa, K., Ohsumi, Y. and Anraku, Y. (1988) Mutants of Saccharomyces cerevisiae with defective vacuolar function. J. Bacterial. 170: 2687-2691. Kornberg, A. (1995) Inorganic polyphosphate: toward making a forgotten polymer unforgettable. J. Bacteriol. 177: 491-496. Kulaev, I.S. (1979) The Biochemistry of Inorganic Polyphosphates. John Wiley & Sons Inc., New York. Navon, G., Shulman, R.G., Yamane, T., Eccleshall, T.R. and Lam, K.B. (1979) Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae. Biochemistry 28: 4487-4499. Ohsumi, Y. and Anraku, Y. (1983) Calcium transport driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae. J. Biol. Chem. 258: 5614-5617. Pringle, J.R. and Hartwell, L.H. (1982) The Saccharomyces cerevisiae cell cycle. In The Molecular Biology of the Yeast Saccharomyces cerevisiae. Edited by Strathern, J.N., Jones, E.W. and Broach, J.R. pp. 97-142. Cold Spring Harbor Laboratory, New York. Takeshige, K., Baba, M., Tsuboi, S., Noda, T. and Ohsumi, Y. (1992) Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. J. Cell Biol. 119: 301-311.' Urech, K., Diirr, M., Boiler, T., Wiemken, A. and Schwenche, J. (1978) Localization of polyphosphate in vacuole of Saccharomyces cerevisiae. Arch. Microbiol. 116: 275-278. Wada, Y., Kitamoto, K., Kanbe, T., Tanaka, K. and Anraku, Y. (1990) The SLPI gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function. Mol. Cell. Biol. 10: 2214-2223. Wood, H.G. and Clark, J.E. (1988) Biological aspects of inorganic polyphosphates. Annu. Rev. Biochem. 57: 235-260. Wurst, H., Shiba, T. and Kornberg, A. (1995) The gene for a major exopolyphosphatase of Saccharomyces cerevisiae. J. Bacteriol. 177: 898-906. (Received July 16, 1996; Accepted August 30, 1996)