Kinetic Characterization the Two Phosphate Uptake Systems ...jb.asm.org/content/jb/132/2/511.full.pdfCuSO4, 0.32,M; (NH4)6Mo7O04, 0.032 tkM; d-biotin, 0.02 AM. BM was prepared at 2.5

JOURNAL OF BACTERIOLOGY, Nov. 1977, p. 511-519Copyright © 1977 American Society for Microbiology

Vol. 132, No. 2Printed in U.S.A.

Kinetic Characterization of the Two Phosphate UptakeSystems in the Fungus Neurospora crassa

DONALD J. W. BURNS* AND ROSS E. BEEVERPlant Diseases Division, Department of Scientific and Industrial Research, Auckland, New Zealand

Received for publication 17 August 1977

The kinetics of phosphate uptake by exponentially growing Neurosporacrassa were studied to determine the nature of the differences in uptakeactivity associated with growth at different external phosphate concentrations.Conidia, grown in liquid medium containing either 10 mM or 50 ,AM phosphate,were harvested, and their phosphate uptake ability was measured. Initialexperiments, where uptake was examined over a narrow concentration rangenear that of the growth medium, indicated the presence of a low-affinity (highKmn) system in germlings from 10 mM phosphate and a high-affinity (low K,,,)system in germlings from 50 ,uM phosphate. Uptake by each system wasenergy dependent and sensitive to inhibitors of membrane function. No effluxof phosphate or phosphorus-containing compounds could be detected. Whenexamined over a wide concentration range, uptake was consistent with thesimultaneous operation of low- and high-affinity systems in both types ofgermlings. The Vmax estimates for the two systems were higher in germlingsfrom 50 AtM phosphate than for the corresponding systems in germlings from 10mM phosphate. The K,, of the high-affinity system was the same in both typesof germlings, whereas the K,,, of the low-affinity system in germlings from 10mM phosphate was about three times that of the system in germlings from 50,uM phosphate.

Relatively little is known of the mechanismswhereby microorganisms, particularly fungi,achieve and regulate uptake of phosphate (13,24). There are conflicting reports regarding thenumber and the kinetic properties of the up-take systems of the yeast Saccharomyces cere-visiae (2, 3, 8, 15). The differences may be due,in part, to differences in the culture conditionsbefore assay, since, in studies with other fungi,phosphate uptake ability has been found to beincreased after growth in low-phosphate media(4, 27, 28). The most extensive study of phos-phate uptake by a filamentous fungus has beencarried out recently with Neurospora crassa(18-20). This fungus was reported to possesstwo uptake systems, one of low affinity (highK,,,) and one of high affinity (low K,, ). Phospho-rus starvation resulted in a dramatic increasein the activity of the high-affinity system.These results are similar to those reported

for the uptake of a variety of solutes in micro-organisms where the activity of a high-affinitysystem either appears de novo or is increasedafter a period of starvation for the solute. Suchreports suggest a mechanism by which an or-ganism might adapt to different external soluteconcentrations. Namely, all uptake of the sol-ute above that carried out by the low-affinity

system present at its constitutive level is pro-vided by the high-affinity system, derepressedto an appropriate extent. Quantitative evidencein support of such a control mechanism islacking. We chose to test this hypothesis bystudying the regulation of phosphate uptakeactivity in N. crassa growing exponentially atdifferent phosphate concentrations. An essen-tial part of the study has been the developmentof experimental techniques to describe accu-rately the uptake characteristics of the fungusat the instant of harvest. In this paper wereport these techniques together with a de-tailed account of the uptake characteristics ofgerminating conidia (germlings) grown in me-dium containing phosphate at either a high orlow concentration. In the accompanying paper(1), we examine the behavior of germlingsgrown at concentrations between these twoextremes and discuss the extent to which thekinetic parameters of the uptake systems canaccount for the observed phosphate uptakerates of growing germlings.

MATERIALS AND METHODSOur general method has been to grow N. crassa

conidia for 2.5 h in a growth medium containingthe appropriate concentration of phosphate and then

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to transfer the germlings rapidly to fresh growthmedium with 32Pi at different concentrations tofollow uptake. The technique of preincubating cellsfor a period in a simple buffer solution before assay,which has previously been used in phosphate uptakestudies of N. crassa (16, 19), has been avoided. Wesaw no advantage of this technique for our studiesand considered that such manipulations might alterthe uptake characteristics from those existing dur-ing growth.Where appropriate, values are given as the mean

+1 standard deviation.Inoculum. The N. crassa wild-type strain, STA4

(St. Lawrence), was obtained from J. R. S. Fincham.Conidia were produced by growing the fungus onVogel's medium N (32) supplemented with sucrose(20 g/liter), agar (15 g/liter), and, in the case ofmost uptake experiments, 35SO42- (2 /Ci/ml). After6 days at 25°C, the conidia and aerial myceliumwere scraped from the agar surface and shaken inwater. The resulting suspension was filteredthrough glass wool to remove mycelial fragmentsand centrifuged, and the pellet of conidia was resus-pended in water. Appropriate volumes were thenadded to the different germination flasks of anexperiment to give a final concentration of 106conidia per ml. For technical reasons germinationflasks could not always be inoculated at the sametime, but storing the suspension for up to 3 h atroom temperature had no effect on the subsequentgermination or the uptake characteristics of thegermlings.Media for germination and uptake studies. The

base medium (BM) contained: KNO3, 2 mM;NH4NO3, 2 mM; (NH4)2SO4, 3 mM; MgSO4, 2 mM;Ca(NO3)2, 2 mM; citrate, 10 mM (1 M sodium citratebuffer, pH 6.4, 10 ml/liter); sucrose, 58 mM; ferricmonosodium ethylenediaminetetraacetate, 54 tLM;H3BO: , 46 AM; MnCl2, 9 AM; ZnCl2, 0.73 AM;CuSO4, 0.32 ,M; (NH4)6Mo7O04, 0.032 tkM; d-biotin,0.02 AM. BM was prepared at 2.5 times finalstrength, with the pH adjusted to 6.4. Phosphatesolutions were prepared at twice final strength fromKH2PO4 adjusted with KOH, such that when mixedwith BM and diluted the final pH was 6.4. BM andphosphate solutions were autoclaved separately(121°C, 15 min). Radioactive phosphate and cyclo-heximide (final concentration, 14 ,M; Upjohn Co.,Kalamazoo, Mich.) were not autoclaved and wereadded directly to the appropriate sterilized solu-tions.

Germination and growth. Conidia were germi-nated by incubation at 30°C for 2.5 h in 100 to 500ml of medium in a flask of four to five times themedium volume on a reciprocating shaker (thrust,3 cm; 90 rpm). The medium was either BM + 10mM phosphate (10 mM germlings) or BM + 50 4Mphosphate (50 ALM germlings). During incubationthe pH of the medium remained between 6.3 and 6.4.

Uptake assays. Samples of 2.5-h germlings wereharvested from the growth medium by vacuumfiltration onto a cellulose nitrate filter (pore size,1.2 Am). The pad of germlings was washed thor-oughly with BM minus phosphate and dropped into

resuspension solution (see below). The germlingswere dislodged from the filter by swirling the flask,and the filter was then removed. In some experi-ments the resuspension solution was the final assaysolution and contained BM + cycloheximide + 32p;in the same volume as the original germling sample.In most experiments the resuspension solution wastwice final strength (BM + cycloheximide) at halfthe original sample volume. Subsamples (5 to 15ml) were then removed and added to an equalvolume of the appropriate 32P, solution at twicestrength, to give correct final concentrations ofgermlings and medium. If germlings were left intwice strength (BM + cycloheximide) for longerthan about 5 min, the subsequent phosphate uptakerate was slightly reduced, and so germlings werenever left in this resuspension solution for longerthan 3 min. Samples (1 ml) were removed at inter-vals and filtered by suction through glass-fiberfilters (Whatman GF/A or GF/C). Each filter waswashed four to five times with 10-ml volumes of 1mM potassium phosphate buffer (pH 6.4) at roomtemperature (21 to 23°C) and then placed in ascintillation vial for radioactivity measurement.

Cycloheximide is known to inhibit growth (12)and prevent protein synthesis in N. crassa (11).This compound was included in assay media toprevent possible changes occurring in uptake char-acteristics due to the synthesis of new uptake activ-ity during the course of the assay.

For inhibitor studies (see Table 1), germlings weresuspended in BM + cycloheximide + 2p; for 10min, and then inhibitor was added. The pH of theinhibitors was adjusted to keep the final mediumpH at about 6.4. Nystatin (Mycostatin; E. R. Squibb& Sons, Princeton, N.J.) and 11-deoxycorticosterone(Sigma Chemical Co., St. Louis, Mo.) were dissolvedin ethanol before addition. The final ethanol concen-tration was 1%, and by itself this did not affectphosphate uptake. For the 0OC treatment, germlingssuspended in twice strength (BM -t- cycloheximide)were chilled on ice before addition of chilled, twice-strength 32p; solution.

Radiotracer. Radioactive sulfate and phosphatewere obtained from the Radiochemical Centre,Amersham, England. We did not encounter thedifficulties with using 32p, reported by Lowendorf etal. (19) in a similar study. One benefit of using 2p;was that a S2P/355 double-labeling technique couldbe used.

Measurement of radioactivity. Glass-fiber filtersamples were prepared for counting by adding 1 mlof water plus 9 ml of scintillation fluid (based onthe 2:1 toluene-Triton X-100 mixture of Pattersonand Greene [23b). Aqueous samples were made to 1ml with water, and scintillation fluid was added.Vials were capped, shaken thoroughly on a Vortexmixer, and left overnight. This prolonged soakingwas necessary to obtain reproducible counting be-havior of the samples. The vials were shaken againand counted by liquid scintillation spectrometry(Packard model 3320), using the channels ratiomethod for double-label counting (9). Quenchedstandards were prepared for each experiment, and

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PHOSPHATE UPTAKE IN FUNGI 513

the data were quench-corrected using computer-generated curves. To maintain similar accuracy forall 35S measurements, the 32p overlap in any samplewas not permitted to exceed 20% of the total 35Scounts. This was achieved by choosing appropriatespecific activities for the 32p, solutions.

Use of 35S labeling. Inclusion of 35SO42- in theagar medium when growing the fungus providedconidia that were radioactively labeled with 35S.The 35S content of the germlings in a given experi-ment (about 6 x 105 cpm/mg, dry weight) was thenused to correct for slight differences in sample size.The 32p counts in each sample were adjusted by thefactor required to normalize the 35S counts in thatsample to the mean 35S sample count for the experi-ment. In practice, corrections of up to about 10%were required.

Calculation of initial uptake rates. Samplingtimes were spaced such that at least five sampleswere taken during the linear period of uptake. Inall instances, sampling was completed by 16 min.Uptake rates were calculated from lines fitted byleast-squares analysis. Although it was not possibleto determine the exact moment when germlingsmade effective contact with the 32p,, because of thetime taken for mixing, most calculated uptake linespassed through or close to the origin. In the case of10 mM germlings assayed at high-phosphate con-centrations, lines usually intercepted the ordinateabove the origin (Fig. 1A. The concentration ofphosphate in the uptake medium was taken as theconcentration at the midpoint of the linear uptakeperiod, thereby correcting for loss during this pe-riod. No correction was needed for phosphate solu-tions greater than 100 ,gM.

Uptake rates were first calculated on a per-milli-liter basis. It was not practicable to measure germ-ling dry weight in every experiment because of thelarge volumes of culture needed to obtain accurat'weights. Standard uptake rate values on a per-gram(dry weight) basis were measured directly for 10mM germlings assayed at 1 mM phosphate (2.08,umol/g [dry weight] per min) and for 50 p.M germ-lings assayed at 50 p.M phosphate (2.99 ,umol/g [dryweight] per min). Similar assays were included ineach experiment, and the ratio of the uptake ratesof these assays (on a per-milliliter basis) to thestandard uptake rate values was then used to con-vert the other measurements.

For comparative purposes we suggest that ouruptake rate data may be approximately convertedinto other units as follows: to convert to uptake permilliliter (106 germlings), assume 106 2.5-h-oldgermlings weigh 38.6 /.g; to convert to uptake perunit of cell water, assume the ratio of intracellularwater to dry weight is 2.54 (31) or 2.3 (26); toconvert to uptake per unit of surface area, assumethe ratio of surface area to dry weight is 4.6 m2/g;to convert to uptake per unit of fresh weight, as-sume the ratio of dry weight to fresh weight is 0.186.

Derivation of kinetic parameters. Phosphate up-take in N. crassa has been analyzed by assumingthe simultaneous operation of two uptake systemseach obeying Michaelis-Menten kinetics. Thus,

Vmax(LA)CS Vmax(HA)CSU= ~+

K,,j,LA) + s K,,i(HA) + S

where v is uptake rate; s is phosphate concentration;VMa.r,LA) and V,mia.1iA) are the maximum uptake ratesof the low-affinity and high-affinity systems, respec-tively; and Kmn LA) and K,,,h(fA) are the phosphateconcentrations that give rise to half-maximum up-take rates for each system.When uptake measurements were made over a

wide phosphate concentration range, estimates ofthe kinetic parameters, Vrnarl,A), V1r1alVHA), K,n(LA)and Km(,HA), were derived by fitting the double-hy-perbola equation to the data, using a computer-based method. Data were partitioned into two sub-sets, with each being repetitively solved for a singlehyperbola, using the direct linear plot method (5,6), after subtracting the calculated contribution ofthe hyperbola corresponding to the other subset. Inall cases the first subset contained data from phos-phate concentrations up to and including 10 ,uM.Measurements at higher concentrations comprisedthe second subset. Iteration of the fitting methodwas continued until successive estimates of each ofthe parameters differed by less than 0.1%. Themerits of this method are discussed elsewhere(D. J. W. Burns and S. A. Tucker, Eur. J. Biochem.,in press).When uptake was followed over a restricted con-

centration range, the relationship between uptakerate and phosphate concentration was essentiallylinear on a Hofstee plot (10) and thus appeared toobey simple Michaelis-Menten kinetics. The twokinetic parameters of this "single" system wereestimated by using a computer version of the directlinear plot method.

Parameters derived by single-hyperbola analysisare termed one-system estimates, whereas thosederived from double-hyperbola analysis are termedtwo-system estimates.

RESULTSInitial kinetic analysis of low- and high-

affinity systems. In initial experiments wesought to find growth conditions, differing onlyin the external phosphate concentration, inwhich N. crassa was able to grow exponentiallywith either the low- or high-affinity systembeing primarily responsible for phosphate up-take. A 10 mM phosphate concentration waschosen as a maximum, since it would ensurevirtual saturation of the constitutive low-affin-ity system [reported K,fl(LA) of 470 p.M at pH6.43; 19]. Similarly, a 50 ,uM phosphate concen-tration was chosen as a minimum since theactivity of the low-affinity system presumablywould be restricted (approximately 10% ofVmax,LA)), whereas that of the high-affinity sys-tem would be saturated [reported K,,, HA) ofabout 3 ,uM; 18]. Growth, measured as dry-weight increase, was exponential by 2.5 h and

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remained identical for 50 AM and 10 mM germ-lings over the first 6 h (1).When 10 mM germlings were examined at

phosphate concentrations of 400 ,uM and above,uptake was linear with time (Fig. 1A). A Hof-stee plot of the data gave a straight line (Fig.1B), suggesting that uptake of phosphate fromthese solutions was due primarily to a single

A [Pi],mM50 * 3.2

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TIME (minutes)

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~00)

-5

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1

1 2 3 4 5UPTAKE RATE ( pmol/g d.w. /min

[PHOSPHATa] (mM)FIG. 1. Phosphate uptake by 10 mM germlings

over a narrow concentration range (400 AM to 3.2mM). (A) Uptake with respect to time at differentphosphate concentrations. (B) Hofstee plot of data.The kinetic parameters for a single hyperbolic func-tion fitted to the data are: K,, 843 MM; Vm,,x, 3.64,mollg (dry weight [d.w.]} per min. This function is

given by the solid line.

uptake system obeying Michaelis-Menten ki-netics. The mean estimated one-system kineticparameters of the single system were: K,,,, 754+98 uM; Vtnax 3.59 + 0.26 Amol/g (dry weight)per min (seven experiments). From the similar-ity in the respective K,,, values, it was clearthat this system corresponded to the low-affin-ity system previously described (19).Uptake by 50 A.tM germlings over the range

1 to 50 ,uM phosphate was also linear withtime (Fig. 2A). The data obtained from 1 to 10,M concentrations gave a linear Hofstee plot(Fig. 2B), indicating the predominant operationat dilute concentrations of a single uptakesystem obeying Michaelis-Menten kinetics.The mean estimated one-system kinetic param-eters of this system were: K,,,, 3.01 - 0.29 AM;Vmax,,S 2.59 + 0.17 g.mol/g (dry weight) per min(seven experiments). It was concluded that thissystem corresponded to the high-affinity sys-tem of Lowendorf et al. (18). The uptake rateat 50 ,uM phosphate was higher than thatexpected from the operation of the high-affinitysystem alone (see Fig. 2B) and suggested thatthere was a second uptake system contributingsignificantly to uptake at this phosphate con-centration.Metabolic properties of the uptake systems.

Properties of the two systems were examinedfurther by following uptake by germlings at aphosphate concentration at which uptakewould be due largely to one of the two systems.Phosphate uptake by both systems was almostabolished by the respiratory inhibitors cyanideand azide and by lowering the temperature to0°C (Table 1). Arsenate, which competes withphosphate in a number of biological systemsincluding phosphate uptake in yeast (25), ap-proximately halved the uptake rate whenadded in amounts equimolar to the phosphatepresent in the assay. Phosphate uptake wasalso affected by inhibitors of membrane func-tion. The addition of nystatin, known to affectpermeability of N. crassa cells (14), not onlyprevented further accumulation but also al-lowed leakage of accumulated radioactivityback into the medium. The steroid 11-deoxycor-ticosterone, which is suggested to be a generaluptake inhibitor in N. crassa (17, 30), alsoprevented uptake and allowed leakage.

Efflux studies. Although in some uptakesystems it is found that significant efflux of anion occurs during uptake of that ion, we havenot been able to demonstrate significant effluxof phosphate under our experimental condi-tions. In one experiment, 2.25-h 10 mM and 50,uM germlings were exposed, in the presence ofcycloheximide, for 0.5 h to a 32p; solution at

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PHOSPHATE UPTAKE IN FUNGI 515

25

¢ 20-600)E 15Z

L"

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TIME (minutes)

3c

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0

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200 400 600 800 1000

UPTAKE RATE ( pmol/g d.w./min[PHOSPHATE] ( mm)

FIG. 2. Phosphate uptake by 50 WIM germlingsover a narrow concentration range (1 to 50 1iM). (A)Uptake with respect to time at different phosphateconcentrations. (B) Hofstee plot of data, plus threefurther uptake rate determinations at 10 MM. Thekinetic parameters for a single hyperbolic functionfitted to the data (excluding the 50 p.M value) are:

Ki,} 2.76 IAM; Vmax, 2.60 pLmol/g (dry weight [d.w.])per min. This function is given by the solid line.

either 1 mM or 50 gM, respectively. They were

then harvested and resuspended in mediumlacking phosphate or medium containing non-

radioactive phosphate at 50 ,uM or 1, 10, or 200mM. The radioactivity present in the germlings1.8 h after suspension was between 94 and110% of the initial value. In another experi-ment, to avoid any effect of harvesting and

resuspension, a "swamping" amount of nonra-dioactive phosphate was added during thecourse of radioactive phosphate uptake. Detect-able uptake of 32p, ceased immediately, but noloss of accumulated radioactivity was observedsubsequently (Fig. 3).

Detailed kinetic analysis of the uptake sys-tems. The results of Lowendorf et al. (18, 19)and of some of our early kinetic experimentssuggested that N. crassa germlings containedmore than one uptake system. This was inves-tigated further by following uptake over widephosphate concentration ranges for each of thetwo types of germlings. These ranges (1 to3,200 ,M for 10 mM germlings, 1 to 1,600 ,Mfor 50 ,uM germlings) were chosen, on the basisof preliminary experiments, to span the K,,,regions of both systems in each case. Uptakewas followed at 15 or 16 different concentrationswithin each range in three independent exper-iments. The data obtained for both types ofgermlings showed a curve when displayed on aHofstee plot (Fig. 4 and 5). Using double hyper-bola analysis, we derived two-system parame-ter estimates for the systems from these data.The curved lines shown in Fig. 4 and 5 werecalculated by using the derived parameter val-ues. These calculated curves pass close to theexperimental data points, but it is difficult tojudge the goodness of fit visually. A betterassessment can be obtained from a direct com-parison of the experimental data and the cor-responding calculated values. Such a compari-son showed that most pairs of values agreed towithin 4%, with the worst agreement being

TABLE 1. Effect of inhibitors and temperature onthe phosphate uptake systems ofN. crassa

Uptake as % control"

Treatmenta 10 mM germ- 50 AM germ-lings in 1 lings in 50mM phos- ,M phos-phate' phated

NaN3 .................. 3 3NaCN ................. 1 52,4-Dinitrophenol .......... 5 0Na2HAsO4 ................. 40 65Nystatin ................. -11 -2211-Deoxycorticosterone ..... -9 -80°C .................. 7 3

a Inhibitors were present at 1 mM, exceptNa2HAsO4 (50 AM in 50 ,M phosphate uptaketreatment) and nystatin (7.5 ,g/nm).

I Negative values indicate leakage of accumu-lated radioactivity. All uptake and leakage rateswere essentially linear for 30 min.

c Low-affinity system contribution, 86% (Fig. 6).d High-affinity system contribution, 75% (Fig. 6).

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60

40

20

E80

60

40

20

5 10 15 20 25

TIME (minutes)

FIG. 3. Lack of efflux of previously accumulated32pi or other 32P-labeled compounds in the presenceof excess unlabeled phosphate. 10 mMgermlings (A)or 50 pM germlings (B) were resuspended in BM +

cycloheximide + 32Pi at either 1 mM (A) or 20 pM(B). At times marked by arrows, 6 ml of the suspen-sion was removed and added to 0.5 ml of 1 Mnonradioactive phosphate.

found for the 50 yM values (differences of 7.1and 8.9% for Fig. 4 and 5, respectively). Theaverage parameter estimates from the experi-ments (Table 2) show that K,WHA, is the onlyparameter not significantly different from itscounterpart in the other type of germling.From these parameter values the uptake

rates of the germlings at the phosphate concen-

tration of each growth medium can be calcu-lated. The rate for 10 mM germlings is 3.37,mol/g (dry weight) per min, and that for 50AM germlings (at the 2.5-h phosphate concen-

tration of 44 ,M) is 2.88 ,umol/g (dry weight)per min.

Relative contributions of the systems touptake at different phosphate concentrations.Using the kinetic parameters of Table 2, we

have calculated the relative contributions ofeach system to uptake over the concentrationrange 1 ,M to 10 mM (Fig. 6). It can be seen

that for 50 AxM germlings the two systemscontribute equally to uptake at 210 ,uM,whereas for 10 mM germlings equal contribu-tion occurs at 88 ,M.

DISCUSSIONBiochemical properties of the uptake sys-

tems. Preliminary kinetic assays indicated that

phosphate uptake by 10 mM germlings duringgrowth was due primarily to a low-affinitysystem, whereas that of 50 ,uM germlings wasdue primarily to a high-affinity system (Fig. 1and 2). The different phosphate uptake behav-ior of the two types of germlings resulted froma physiological adaptation to the phosphateconcentration in the medium rather than fromchanges in the growth rate, since dry matterincreases were identical (1). The activity ofboth systems showed a similar dependence oncontinued metabolic energy production and onmembrane integrity (Table 1). Lowendorf etal. (19) showed an energy dependence for thelow-affinity system but did not report a similarstudy for the high-affinity system present inphosphate-starved cells (18). The energy re-quirement suggests that all phosphate uptakeinvolves movement against an electrochemicalgradient. This is supported by the observationsthat N. crassa hyphae are negatively chargedwith respect to the surrounding medium (29)and that inorganic phosphate levels in 2.5-hgermlings are between 5 and 10 mM (unpub-lished observations).We found no detectable efflux of phosphate

or phosphorus compounds irrespective of which

E3

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11

20 40 60 80UPTAKE RATE pmol/g d.w./min

CPHOSPHATE] mM

FIG. 4. Hofstee plot for uptake data obtained over

a wide phosphate concentration range (1 ,M to 3.2mM} using 10 mM germlings. The data were ana-

lyzed to give the kinetic parameters of the two com-

ponent uptake systems. The high-affinity system (K.,3.19 MM; V,,,ax, 0.26 ,mol/g [dry weight, d.w.] per

min) is shown by dotted line H, and the low-affinitysystem (K,,, 1 ,204 pM; V1,(J, X 3.61 /i mol/g [d w. / permin) is shown by dotted line L. The solid line is thecalculated sum of these two systems. The inset ex-

pands the portion of the plot close to the ordinate.

A

B!' * .~~~~~~~

tB X~~~~~~~~

<,....~~~~~

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PHOSPHATE UPTAKE IN FUNGI 517

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of

D 4

200 400 600 800UPTAKE RATE pmol/g d.w./min

[HOSPHATE] mM

FIG. 5. Hofstee plot for uptake data obtained overa wide phosphate concentration range (1 ,uM to 1.6mM) using 50 p-M germlings. The data were ana-lyzed to give the kinetic parameters of the two com-ponent systems. The high-affinity system (K,,, 2.31MM; V,,,,,,, 2.16 ,mol/g [dry weight, d.w.] per min)is shown by dotted line H, and the low-affinitysystem (K,,,, 366 ,uM; V,60r, 6.66 pimollg [d.w.] permin) is shown by dotted line L. The solid line is thecalculated sum of these two systems. The inset ex-pands the portion of the graph close to the ordinate.

TABLE 2. Comparison of two-system kineticparameter estimates of the phosphate uptake systems

of10 mM and 50 MM germlings a

Vm<= (gmol/gSystem K,, (AuM) [dry wt] per

min)

Low-afflnity10 mM germlings ........ 1,029 153 3.41 ± 0.2450 ,M germlings ........ 370 25 6.33 ± 0.32Difference ......... .... S S

High-affinity10 mM germlings ........ 2.85 ± 0.31 0.28 ± 0.0550 gM germlings ........ 2.43 ± 0.10 2.33 ± 0.15Difference ............. NS S

Analyzed assuming that the two systems contributesignificantly to uptake over the entire phosphate concentra-tion range. Values are based on three independent experi-ments.

b Significant (S) or nonsignificant (NS) at the 1% level,using the t test.

system was mainly responsible for uptake (Fig.3). In contrast, Lowendorf et al. (19) foundefflux of both phosphate (up to 20 to 30% of theuptake rate) and other phosphorus compoundsduring uptake. We suggest that the effluxobserved by these workers may be an artifactresulting from some abnormal alteration to theplasma membrane. This could result from the

z

0o

z0

u

0z

w

-4

1o0-6 lo-5 40°X -3 no-2PHOSPHATE IN ASSAY MEDIUM (M)

FIG. 6. Diagram showing the relatiue contribu-tions of the high- and low-affinity systems in 10 mMand 50 MM germlings to uptake over a wide phos-phate concentration range. Curves were calculatedwith the parameter values listed in Table 2.

assay system they used, as this involved resus-pending mycelium for 25 min in a nongrowthbuffer before adding radioactive phosphate.The occurrence of efflux complicates the deri-vation of kinetic parameters for influx (21) andmay account, in part, for the differences be-tween our parameter estimates and theirs (seebelow).

Kinetic interpretation over a wide concen-tration range. The markedly curved Hofsteeplots for both 10 mM and 50 ,uM germlings,when uptake was measured over a wide concen-tration range (Fig. 4 and 5), are not consistentwith the operation of a single Michaelis-Men-ten uptake system, but can be adequately ex-plained by two uptake systems operating si-multaneously across the plasma membrane. Inour analysis of the uptake data, we allowed forthe possibility that each kinetic parameter ofthe uptake systems might vary according tothe physiological state of the fungus. For 50,uM germlings the values of Vmax(L A) andVm,,a.HA, were increased about 1.9 and 8.3 times,respectively, over the corresponding values in10 mM germlings (Table 2). The relative in-crease in uptake activity of each system causedby these changes would, in the absence of K,,Mchanges, apply equally to all phosphate concen-trations. However, whereas no difference wasfound between the K,,,mHA) values of the two setsof germlings, the K,,,(I A) value of 50 ,uM germ-lings was only 0.36 times that of 10 mM germ-lings. The effect of this change is to make thelow-affinity system in the 50 ,uM germlingsrelatively more efficient at taking up phosphatefrom dilute solutions.

Despite the different experimental condi-tions, some comparisons can be made with theresults of Lowendorf and his colleagues (18-20). Although these workers analyzed their

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518 BURNS AND BEEVER

data in terms of two uptake systems, theyassumed that the KM,, values of the two uptakesystems would be unaltered in fungus of differ-ent physiological status. This assumptionmeant that a reduction in K,,,A), such as we

have found to be associated with a loweredexternal phosphate concentration, could nothave been detected. Our K,, (LA) of 1,029 ,tM atpH 6.4 for 10 mM germlings is similar to theirK,,,,LA) value of 470 ,tM at the same pH obtainedfor mycelium grown at 37 mM phosphate.These workers reported that K,,,LA) values were

markedly influenced by assay pH. Slight dif-ferences in pH can be eliminated as a possiblecause of the differences in Km,(A, that we havefound, because our uptake assays for the twogermling types were carried out in identicalsolutions prepared at the same time. Our Km(HA)values of 2.43 to 2.85 ,aM at pH 6.4 are similarto the values of 2.2 to 2.6 ,uM at pH 5.8 foundby Lowendorf et al. (18, 20), who also reportedthat Km(HA) values were relatively unaffectedby pH. These workers found that the majorchange after a period of phosphate starvationwas a large increase in Vrnax(HA), a result con-

sistent with our finding that Vmar values are

higher in 50 .tM germlings than in 10 mMgermlings.

In the present work we have used the widelyadopted dual-system interpretation to explainthe nonconformity of the data with simpleMichaelis-Menten kinetics. However, complexkinetics of a similar nature have also beenexplained on the basis of a single uptake sys-

tem with properties of negative cooperativity(7, 22). Criteria for distinguishing betweenthese two interpretations have been discussedby Glover et al. (7). The strongest criterion is

the existence of mutants that lack one of thetwo systems. In the case of N. crassa, the nuc-

1 and nuc-2 mutants have been shown to give

phosphate uptake kinetics consistent with theoperation of a single low-affinity system (16,20). Furthermore, metabolic perturbations mayreveal two systems through preferential effectson one of the components. In N. crassa, phos-phorus limitation has differential effects on thekinetic parameters of the two systems. Also,the effect of pH on the systems is markedlydifferent (see above). For these reasons, andbecause of its simplicity, we have chosen thedual-system approach but recognize thatmodels based on negative cooperativity are notunequivocally excluded.

Relevance to uptake studies in general.Some of the results of the present study havegeneral significance in uptake studies. Whenuptake is followed over a relatively narrow

TABLE 3. Comparison of one- and two-systemkinetic parameter estimates of the phosphate uptake

systems of50 iM germlings(1

System K (,M V.., ( mol/g [drywt] per min)

Low-affinityOne-system 276 14 8.98 + 0.19Two-system 370 + 25 6.33 + 0.32Differenceb S S

High-affinityOne-system 2.77 + 0.08 2.55 + 0.19Two-system 2.43 + 0.10 2.33 + 0.15Differenceb S NS

a The same data from three experiments wereanalyzed by one- and two-system analysis. For two-system analysis it was assumed that both systemscontributed significantly to uptake over the entireconcentration range. For one-system analysis thecontribution of the high-affinity system to uptakeat high concentrations, and that of the low-affinitysystem at low concentrations, was ignored. Thus,the high-affinity system analysis was based onmeasurements from 1 to 10 kkM, and the low-affinitysystem analysis was based on measurements above400 AM.

b Significant (S) or nonsignificant (NS) at the 1%level, using the t test.

concentration range, data may be obtained thatappear to obey simple Michaelis-Menten kinet-ics (e.g., Fig. 1 and 2). By assuming that onlyone system is operating, the kinetic parameterscan be estimated, but it is important to realizethat such one-system estimates are only ofqualitative significance until it is shown thatthese kinetics are adhered to over a wide con-centration range, or that the influence of otheruptake systems is virtually negligible. For ex-ample, in 10 mM germlings the contribution ofthe high-affinity system to uptake in the K,, (I,A)region is very small, and there is no significantdifference between one- and two-system esti-mates for the two uptake systems (data notgiven), but this clearly is not the case for 50,lM germlings (Table 3). Thus, in situationswhere two uptake systems are present, param-eter estimates must be determined by double-hyperbola analysis.

ACKNOWLEDGMENTS

We thank Philippa Clark and Beth Dye for their excel-lent technical assistance.

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