A 31P-NMR study of phosphate transport and compartmentation in Candida utilis

Biochimica et Biophysica Acta, 1055 (1990) 1-9 Elsevier

1

BBAMCR 12785

A 31p-NMR study of phosphate transport and compartmentation in Candida utilis Roger M. Bourne Department of Microbiology, University of Queensland, St. Lucia (Australia) (Received 11 January 1990) (Revised manuscript received 23 May 1990)

Key words: Phosphate transport; Polyphosphate synthesis; Phosphate starvation; NMR, 31p.; (C. utilis)

The recovery of Candida utilis from phosphate starvation was studied using 3tP-NMR. The phosphate analogue methylphosphonate was found to be a useful indicator of cytosol pH. Added orthophosphate was rapidly accumulated by the cells and stored mainly In a stable pool of polyphosphate of mean chain-length at least 200 units. Observed pH changes in the medium and cytosol during uptake of orthophosphate and methylphosphonate are consistent with the transport of these compounds across the plasma membrane by a proton/phosphate symport. However, transport of phosphate across the vacuole membrane occurs by a mechanism for which methylphosphonate is not a substrate. In the cytosoi pH changes are strongly correlated with changes in orthophosphate concentration, however, this is not the case in the vacuole.

Introduction Through a fortuitous combination of physical and biological circumstances the in vivo study of phosphate metabolism in yeasts is exceptionally well served by 31p-NMR. In a suitably acidic environment it is possible to resolve and quantitate separate orthophosphate resonances in the medium, cytosol and vacuole compartments. Furthermore, changes in the chemical shifts of these and other resonances indicate the pH of these compartments and provide useful information about membrane transport processes of the plasma membrane and tonoplast and about the ionic environment of the phosphate metabolites. 3~p-NMR has been used to study phosphate metabolism and subcellular compartmentation in several yeast species under a variety of metabolic conditions [1-5]. Ion transport in yeasts has been studied using 39K-, 23Na- and 31P-NMR [5-7]. The studies reported here concern the recovery of Candida utilis from phosphate starvation. This is a particularly good system for study of phosphate transport processes and polyphosphate synthesis because

Abbreviations: NMR, nuclear magnetic resonance; FID, free induction decay. Correspondence: R.M. Bourne, Department of Microbiology, University of Queensland, St. Lucia 4067, Australia.

phosphate-starved cells are capable of rapid phosphate uptake and synthesis of large quantities of polyphosphate. By implementing a new technique for aeration and mixing of the sample cell suspension [8,9] and including an in situ probe for extracellular pH, it has been possible to obtain experimental data which sheds new light on the mechanism and energization of phosphate accumulation. The in situ pH probe provides a continuous and accurate record of the rapid changes In extracellular pH which could not be provided by the chemical shift of an extracellular phosphate compound even if a suitable compound were available. Materials and Methods

Preparation of phosphate-starved yeast Starter cultures of C. utilis UQMCC-23Y were grown at 30°C in batch culture in a mineral salts medium containing, per litre of reverse-osmosis water: 300 mg MgSO4.7H20, 23 mg CaCI 2, 1.0 g KH2PO4, 3.5 g (NH4)2SOa, 10 g D-glucose, 600 mg NazEDTA, 5 mg ZnSO4.7H10, 5 mg MnSO4-HzO, 1.2 mg CuSO4. 5HEO, 1.5 mg M o O 3 and 9 mg FeSO4-7HzO. pH was maintained in the range 4.8-5.2 by automatic addition of 2 M KOH. Washed cells from this medium were subsequently phosphate starved by repeated batch culture and resuspension In a phosphate-deficient medium which was the same as above except KH2PO4 was replaced with 1.0 g KC1.

0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

After phosphate starvation the culture was centrifuged and washed twice by resuspension and recentrifugation in cold reverse-osmosis water. The final pellet (50-100 g) was weighed and resuspended in a measured amount (about half the pellet mass) of NMR buffer (10 mM succinic acid, 10 mM KCI, 2.5 mM CaC12, pH adjusted to 5.0 with Tris base) and the suspension was stored at 0-4°C until used.

Experimental apparatus The cell suspension was aerated and mixed in a 20 mm NMR sample tube using the Loop Mixer apparatus described previously [8,9]. This device eliminates bubbles from the sensitive region of the probe and thus reduces line broadening due to field inhomogeneity. Sample flow also gives rise to an improvement in sensitivity [8]. The apparatus was modified slightly to include an in situ pH probe for continuous measurement of extracellular pH (Fig. 1). The parts were made from standard glassworkers' fittings. The 6 × 280 mm epoxy body pH probe (Activon BJ432, Activon Scientific Products Company, Thornleigh, N.S.W.) was connected

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to a TPS meter (TPS Electronics, Brisbane, Qld.) and the signal recorded with a Shimadzu CR3-A chromatography recorder/integrator. The tip of the pH probe was 100 mm above the top of the N MR probe receiver coils. This arrangement introduced no significant noise into either the NMR or the pH signal. 30 ml of cell suspension plus approx. 50/~1 antifoam (1% glycerol monooleate) was maintained at 30°C and aerated with 95% 02/5% CO 2 at approx. 70 ml rain -1. At this aeration rate the maximum oxygen transfer rate is approx. 600/~M min -1 [9]. Experiments were performed at the Brisbane N M R Centre, Griffith University, Nathan, Brisbane. The instrument used was a Bruker CXP 300 with a 20 mm broad band probe. The spectrometer was operated in the Fourier-transform mode at 121.47 MHz. Proton decoupling was not employed. Transients were averaged using either 2 or 4K of memory over a spectral width of 15 kHz. 60 ° pulses were applied at 1-s intervals and free induction decays (FIDs) accumulated in 2-rain blocks. Chemical shifts were referenced relative to an external standard of 85% orthophosphoric acid measured at the conclusion of the experiment. Intensities were measured by computer integration of the spectra. When peaks were partially overlapped, the integration limits were set at the lowest point of the valley between the peaks. Overlaps were mostly small (Fig. 2b) and were estimated to contribute approx. + 5% to the uncertainty in intensity measurements.

Estimation of saturation At the end of a typical phosphate uptake experiment a fully relaxed FID was obtained. This was preceded and followed by FIDs at normal pulse rate to check for metabolic changes in resonance intensities during the accumulation of the unsaturated FID. All intensities were first normalized with respect to the intensity of the middle-P of polyphosphate resonance which is known to be unsaturated in all spectra (a previous inversion recovery experiment had established T1 = 0.3 s for the middle phosphates of intracellular polyphosphate). Saturation factors were calculated by comparison of the fully relaxed spectrum with the spectra obtained both before and after, and the average of these two values was used for calculations.

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Fig. 1. Diagram of the sample chamber showingthe position of the in situ pH probe and the arrangementof the gas and addition tubes.

Intracellular volume was calculated to be 30% of the suspension volume at the beginning of the experiment according to the assumption that 50% of the wet cell pellet volume is intracellular water [10]. Vacuole volume was taken as 25% of intracellular volume [11,12]. These estimates were found to be reasonable when compared with microscopic measurements. As additions were made to the suspension, the cells were diluted and the fraction of the total volume constituted by the medium in-

creased. At the same time the fraction of the total volume within the sensitive region of the probe decreased. Intensities and concentrations have been adjusted accordingly.

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Extracellular pH (pH~x) was measured directly with the in situ pH probe. Cytosol pH (pHeyt) was calculated from the chemical shift of the methylphosphonate resonance (see Discussion), and vacuole pH (PHv~) from the chemical shift of vacuolar orthophosphate [13]. Titration of the chemical shifts of methylphosphonate and orthophosphate was performed in a solution containing 150 mM KCI, 10 mM (NH4)2HPO 4, 5 mM NaCl and 10 mM methylphosphonic acid.

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Fig. 3. Intensity information derived from the phosphate-uptake experiment partially illustrated in Fig. 2. The vertical scale represents approximate micromoles of phosphate in the total sample. Methylphosphonate (MeP) and orthophosphate (Pi) were added as indicated in the figure. (A) Changes in the pools of orthophosphate in the medium (P~x), A; cytosol (P~yt), o ; and vacuole (Pv,c), II. (B) Changes in the pools of phosphomonoestcrs (PME), ra; polyphosphate (polyP), t ; P=x, z~; and the sum of PME, polyP, Pex, Pcyt and Pvac (SUM), ~ .

with the titration of the chemical shift of MeP, the above observations are seen to be consistent with the transport of MeP from the medium (pH 5) to the cytosol (pH 7). For the remainder of the experiment the intensity of the MeI5 peak was essentially constant and the changes in its chemical shift indicate changes in cytosol pH (see Discussion). At t = 15 rain 800/~mol of KH2PO4 (Pi) was added to the suspension and showed in the spectrum as a peak at 0.9 ppm, the expected position for extracellular orthophosphate (Pe~) at pH 5. Another peak at 2.3 ppm is in the expected position for cytosol orthophosphate (P~yt) at pH 6.8. A phosphomonoester (PME) peak appears at approx. 4 ppm and maintains approximately constant intensity for the rest of the experiment. The estimated concentration of phosphomonoesters is approx. 20 mM. During the next 14 min the extracellular P¢~ peak decreases in intensity and two further peaks appear: one, at 1.5 ppm, between Pcyt and P,~ is in the expected

between pH of the orthophosphate solution and the buffered suspension caused no significant extracellular pH change upon addition of orthophosphate. Methylphosphonic acid was obtained from Aldrich and was added to the cell suspension as a 1 M solution adjusted to pH 5.0 with Tris base. All other reagents were of analytical grade. Results

Fig. 2a shows full 3]p-NMR spectra from the first 34 min of the first phosphate uptake experiment. Fig. 2b shows an expanded view of the orthophosphate region from t = 14 to 42 min. At t = 2 min 200 /Jmol methylphosphonate (MeP) was added to the suspension and two peaks appeared in the next spectrum at 25.2 and 24.4 ppm. In the following two spectra the peak at 24.4 ppm increased in intensity while the peak at 25.2 ppm decreased to zero. When the positions of these two peaks are compared 200

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position for vacuolar orthophosphate (Pva¢) at pH = approx. 6.2. The other peak, at - 2 2 . 4 ppm, is in the position expected for the middle phosphates of longchain polyphosphate and continues to increase in intensity until the extracellular Pi is exhausted. As the polyphosphate signal increases in intensity its resonance moves gradually downfield to a final position of - 2 1 . 9 ppm. Throughout the experiment nucleotide phosphate levels (peaks at approx. - 5 , - 1 0 and - 1 9 ppm) remain relatively low compared with the levels seen in cells which are not phosphate starved (results not shown, but see Nicolay et al. [1,2]). These peaks are barely distinguishable from the baseline noise and it is impossible to obtain a useful measure of either their position or their intensity. Fig. 3a shows the changes in intensity of the Pcyt, Pvac and P¢~ resonances during the experiment partially il-

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Fig. 5. Measured pH changes in the medium (PHex) and calculated pH changes in the cytosol (PHcyt) and vacuole (pHvac), and parallel changes in orthophosphate concentration in the corresponding compartments during phosphate accumulation in an experiment similar to that illustrated in Figs 2-4. P-starved cells were from the same culture as those used for the previous experiment. Methylphosphonate (MeP) and orthophosphate (Pi) were added as indicated in the figure. (A) medium (B) cytosol; (C) vacuole.

the intensities indicates that the saturation factors used are correct and that there is no net loss of phosphate into NMR-invisible pools. Furthermore, the increments in the sum of the intensities are approximately equal to the increments in Pcx when Pi is added to the system. The initial discrepancy can be attributed to the small amount of PME and Pcyt present in the phosphatestarved ceils. At no time during the experiment is the resonance for the terminal phosphates of polyphosphate observed (approx. - 6 ppm). The mean chain length of the polyphosphate can be calculated from the ratio of intensities of the terminal and middle phosphate resonances [14]. In this experiment the intensity of the terminal P resonance is less than or equal to the noise level and it is calculated that the mean chain length is at least 200 units. Fig. 4 shows the measured pH changes in the medium (pHex) and calculated pH changes in the cytosol (pHeyt) and vacuole (pHw~) during the experiment illustrated in Figs. 2 and 3. For comparison these pH changes are plotted with the concurrent changes in orthophosphate concentration in the same compartment. Orthophosphate concentrations were calculated according to the estimated compartment volumes (Materials and Methods). Fig. 5 illustrates similar results for a second experiment with cells from the same phosphate-starved culture and is included to illustrate the reproducibility of the patterns observed in Fig. 4. Discussion

Energy metabolism If the cell suspension is aerated for several hours in succinate buffer, the succinate is eventually exhausted and the pH of the medium rises to about 7. The exhaustion of the succinate is indicated by the loss of buffer capacity of the medium. In the absence of a fermentable substrate the energy metabolism of the cells will be limited by the rather slow process of oxygen transfer from the gas phase to the liquid. This rate of transfer has been estimated to be approx. 600 #M min-1 for the apparatus and aeration rate used in the present study [9]. If one assumes that 4-6 moles of ATP are generated for each mole of oxygen reduced by respiratory electron transport [15], then the maximum rate of ATP synthesis would be 70-110/xmol • rain -1 in the 30 ml of cell suspension. The rate of polyphosphate synthesis after addition of the first and second aliquots of Pi was approx. 67 #mol. rain -1 in the first experiment (Fig. 3b) and approx. 51 # m o l . m i n -1 in the second experiment (not shown). Since a significant amount of ATP would be required by the cell for maintenance processes other than the synthesis of polyphosphate, it is possible that the observed polyphos-

phate synthesis rate is limited by the supply of oxygen to the system. ATP levels remain very low throughout both experiments. In cells grown in complete medium cytosol ATP levels are typically about 3-4 mM [2,16], but they are apparently less than 1 mM in the present study. Yeast transferred from complete to phosphate-free medium can typically increase its biomass 10-times before growth ceases due to phosphate limitation [17], at which time 95% of cellular phosphate is found in the nucleic acid pool. These phosphate-deficient cells have probably hydrolysed a large part of their nucleotide-phosphate pool and cannot replenish this pool until returned to a complete medium.

Compartmentation of orthophosphate Upon addition of orthophosphate to the medium there is a rapid uptake of this phosphate into the cytosol and vacuole. Extracellular phosphate is taken up at an approx, constant rate until exhausted, however, in the first experiment the rate of uptake slowed after addition of the third aliquot of phosphate. In both experiments there was an initial peak in Pcyt, following addition of the first aliquot of orthophosphate, which was not attained following succeeding additions of phosphate. Apparently the mechanism which regulates cytosol orthophosphate levels requires 'priming' with phosphate before it is fully functional. The observed concentration of phosphomonoesters (approx. 20 mM) and range of cytosol orthophosphate (5-20 mM alter the initial peak) compares well with earlier estimates in C. utilis [2]. The concentration variations in vacuole orthophosphate are much greater in magnitude and range than those in the cytosol. The maximum concentration of Pvac of 130 mM is similar to the 110 mM measured in Saccharomyces cerevisiae [12], but much higher than the 5-15 mM measured by Nicolay et al. in nonphosphatestarved C. utilis [2]. The vacuole/cytosol concentration gradient of orthophosphate has a maximum of 12 and is mostly in the range 4-8, a figure which also compares well with the range 0.4-25 reported previously [1]. It appears that, as suggested by Nicolay et al., the vacuole may participate in the regulation of cytosol orthophosphate levels by functioning as a reservoir for excess orthophosphate before its conversion to polyphosphate. However, Pvac is never completely depleted by polyphosphate synthesis and the concentration of Pv~ is always greater than Pcyt" There appears to be no correlation between the vacuole/cytosol orthophosphate concentration gradient and the pH gradient across the tonoplast. Similar observations were made by Nicolay et al. [1,2] and led to the conclusion that the mechanism of tonoplast phosphate transport was different from the H ÷/ phosphate symport operating at the plasma membrane. Further

support for this conclusion is lent by the absence of detectable methylphosphonate accumulation in the vacuole. If a significant amount of methylphosphonate is accumulated in the vacuole it should be detected as a peak separate from cytosol MeP and located at approx. 25 ppm when pH vac = 6.2.

Polyphosphate synthesis The chain length of the newly synthesized polyphosphate appears to be unusually long compared with the range of 20-40 [2] and 10-15 [18] residues reported for C. utilis grown on complete medium. The estimated chain length of at least 200 is consistent with the observations of Liss and Langen [19], who found that the first pool of polyphosphate synthesized alter Pstarvation had a mean chain length of 260, but is at variance with the findings of Schuddemat et al. [20] who found that short chains were synthesized first when S. cereoisiae and Kluyveromyces marxianus were recovering from P-starvation. The relatively long chain length may result from the absence in P-starved cells of any polyphosphate primers for polyphosphate kinase [21]. It was reported by Bostain et al. [22] that alkaline phosphatase, polyphosphate kinase, polyphosphatase and exocellular acid phosphatase are derepressed in phosphate-starved S. cerevisiae. It was suggested by these authors that the phosphatases are induced in order to obtain orthophosphate from extracellular and intracellular phosphate esters. Phosphate-starved C. utilis rapidly hydrolyses extracellular pyrophosphate and tripolyphosphate (not shown), however, there is no evidence for the activity of intracelhilar polyphosphatases in the present study. There was no net hydrolysis of polyphosphate at any time during the experiment nor any apparent reduction in the mean chain length of the polyphosphate. Possibly the activity of the intracellular phosphatases is lost when P-starvation becomes so severe that all available intracellular phosphate esters have been hydrolysed. There is no obvious explanation for the reduced rate of polyphosphate synthesis in the first experiment following addition of the third aliquot of P~. Phosphate uptake rate was also reduced although not as severely. The excess uptake of orthophosphate did not affect the cytosol phosphate concentration but resulted in a steady accumulation of orthophosphate in the vacuole. Obviously polyphosphate synthesis is not limited by a shortage of orthophosphate. Possibly there was some inhibition of energy metabolism as Peyt did not return to its normal post-uptake level (8-10 mM) and pncy t was slow to recover despite very slow phosphate uptake (see discussion below). The gradual downfield shift of the polyphosphate resonance from -22.4 to -21.9 ppm can be attributed to the decreasing intracellular Mg/polyphosphate ratio [23].

Transport processes and p H changes Since many membrane transport processes involve the simultaneous transport of protons with the substrate of interest, pH changes occur on either side of the membrane during transport. By measuring pH changes during transport it is possible to obtain some insight into the transport mechanisms operating. Methylphosphonic acid has previously been used as an indicator of intracellular pH in erythrocytes [24,25] and bacteria [26] and does not appear to affect normal metabolism. In NMR experiments with C. utilis it has been found that the chemical shift changes of intracellular MeP and Peyt are closely correlated and give calculated pH values which differ by only 0.1 pH units on average (0.11 +0.03 in the second experiment). However, the chemical shift of MeP exhibits more regular and less noisy changes than the shift of Pcyt, and titrates more with pH. Once absorbed by the cell MeP is not consumed in any metabolic reactions and thus has a constant concentration, pH calculated from the chemical shift of MeP is less affected by changes in magnesium concentration than pH calculated from the chemical shift of Pi- The regularity of the extracellular pH changes also suggests that cytosol pH is not changing as erratically as the chemical shift of Pcyt might indicate. Once absorbed the intracellular MeP peak is always clearly visible, whereas Pcyt sometimes has very low intensity and uncertain position. In the light of these considerations it is assumed that the chemical shift of MeP is a more reliable indicator of cytosol pH than is the shift of Pcyt and for this reason the shift of MeP was used for the calculation of PHcy t in the present study. Transport of Pi across the plasma membrane at extracellular pH 5 is mediated by a proton/phosphate symport [27]. In P-starved cells a high affinity phosphate transport system is induced which is also a H + / phosphate symport. There is also a sodium/phosphate symport in the plasma membrane which has an alkaline pH optimum but this system is essentially inactive at pH 5. The observed pH increase in the medium and decrease in the cytosol during the first few minutes of phosphate uptake (Figs. 4 and 5) are consistent with phosphate transport via a proton symport. The same pH changes also occur during the transport of MeP, indicating that it also enters the cell via the proton symport. However, the transport system has a preference for Pi- If both MeP and Pi are added to the sample at the same time no significant uptake of MeP occurs until all of the extracellular Pi is depleted (not shown). This occurs even when there is a 5:1 excess of MeP over PiConsiderable cytoplasmic acidification occurs during the uptake of Pi. pHcyt decreased approx. 0.3 units after addition of the first aliquot of Pi. This observation compares well with a measured decrease of pHcy t from

7.17 to 6.85 during phosphate uptake by S. cerevisiae [27]. The changes in extracelhilar p H shown bear a remarkable similarity to those recorded by Cockburn et al. [28] during the uptake of phosphate by P-starved Saccharomyces sp. When MeP is added to the suspension there follows a steady increase in PHex as MeP is transported into the cytosol. As extracellular MeP is depleted the rate of p H increase slows and eventually, when extracelhilar MeP is exhausted, pHex begins to decrease slowly. This same pattern of p H change was recorded by Cockburn et al. when orthophosphate was taken up by cells whose energy metabolism had been inhibited by deoxyglucose and antimycin. In the absence of deoxyglucose and antimycin the p H increase following orthophosphate addition ceased abruptly after about 1 min and a rapid p H decrease ensued. This same pattern is evident in Figs. 4a and 5a following addition of the first aliquot of Pi- The period of rapid decrease of pH~x is not accompanied by an increase in pHcy t as would be expected if the p H change were due to the activity of the plasma m e m b r a n e H + / A T P a s e . it is more likely the result of an increased rate of respiration or glycolysis [29] and consequent bicarbonate production following resupply of phosphate to the P-starved cells. Uptake of phosphate via the p r o t o n / p h o s p h a t e symport results in a decrease in both the p H gradient and the m e m b r a n e potential across the plasma membrane because of the excess of protons which are cotransported with the phosphate [28]. T h e cell can act to restore the m e m b r a n e potential and p H gradient by activation of the plasma m e m b r a n e H + / A T P a s e . This process can be clearly seen in Figs. 4 and 5 after uptake of MeP. The process appears to be relatively slow in comparison with the rate of phosphate uptake so that pncy t does not fully recover until some minutes alter phosphate uptake has ceased. The decrease in PHcy t following addition of the second and subsequent aliquots of Pi is less than after the first and can be e x p l a i n e d by a primed energy metabolism being able to readily supply the plasma m e m b r a n e H + / A T P a s e . Before the exhaustion of P~x the ATPase is able to compete with H + / P i influx, causing pH~x to decrease and pHcy t to increase. Except after addition of the first aliquot of Pi the increase in pHex following Pi addition appears to be essentially independent of the amount of Pi added and is the same after MeP addition. This may indicate a delay or a certain amount of p H gradient collapse before the plasma m e m b r a n e ATPase is activated. The absence of any visible pool of methylphosphonate in the vacuole (even after 18 h aeration of the suspension) suggests that phosphate transport into the vacuole is mediated by a different and more selective system than phosphate transport across the plasma

membrane. As Nicolay et al. [2] noted, a lower p H in the vacuole than the cytosol would tend to favour the accumulation of orthophosphate in the cytosol if a p r o t o n / p h o s p h a t e symport were active. Consequently, the electrochemical proton gradient developed by the tonoplast ATPase [16] cannot be responsible for the development and maintenance of the orthophosphate concentration gradient via a H + / P i symport. However, an electrical phosphate uniport equilibrating the phosphate concentration gradient with the tonoplast membrane potential is a possibility.

Acknowledgements This work was funded in part by grants from the Mayne Bequest Fund and Uniquest Limited, University of Queensland. I am grateful to John H a n n a for operation of the N M R spectrometer.

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