Regulation of Sulfate Transport Filamentous Fungi1 · concentration-dependent transport system ("permease"). Transport is unidirectional. In the presence of excess ex-ternal sulfate,

Plant Physiol. (1970) 46, 720-727

Regulation of Sulfate Transport in Filamentous Fungi1Received for publication February 23, 1970

GRETCHEN BRADFIELD, PAULA SOMERFIELD, TINA MEYN, MARILYN HOLBY, DONALD BABCOCK, DOROTHYBRADLEY, AND IRWIN H. SEGEL'Department of Biochemistry and Biophysics, University of California, Davis, California 95616

ABSTRACT

Inorganic sulfate enters the mycelia of Aspergillusnidulans, Penicillium chrysogenum, and Penicillium nota-tum by a temperature-, energy-, pH-, ionic strength-, andconcentration-dependent transport system ("permease").Transport is unidirectional. In the presence of excess ex-ternal sulfate, ATP sulfurylase-negative mutants will ac-cumulate inorganic sulfate intracellularly to a level ofabout 0.04 M. The intracellular sulfate can be retainedagainst a concentration gradient. Retention is not energy-dependent, nor is there any exchange between intracellular(accumulated) and extracellular sulfate. The sulfate per-mease is under metabolic control. Sulfur starvation of highmethionine-grown mycelia results in about a 1009-fold in-crease in the specific sulfate transport activity at low ex-ternal sulfate concentrations. L-Methionine is a metabolicrepressor of the sulfate permease, while intracellular sul-fate and possibly L-cysteine (or a derivative of L-cysteine)are feedback inhibitors. Sulfate transport follows hyper-bolic saturation kinetics with a Michaelis constant (Km)value of 6 X 10-5 to 104 M and a Vmax (for maximally sulfur-starved mycelia) of about 5 micromoles per gram perminute. Refeeding sulfur-starved mycelia with sulfate orcysteine results in about a 10-fold decrease in the Vmax valuewith no marked change in the Km. Azide and dinitrophenolalso reduce the V.ax.

Yamamoto and Segel (17) described some of the characteristicsof a sulfate transport system ("permease") in Penicillium chryso-genum. Preliminary evidence suggested that the permease wasunder metabolic control, but the mode of regulation and theidentity of the effectors were not determined. While that work wasin progress, Scott and Spencer (8) described in a preliminary notesome of the characteristics of the sulfate permease of Aspergillusnidulans. Their results suggested that the A. nidulans sulfatepermease differed in many respects from that of P. chrysogenum.

' This research was supported by United States Public Health Serv-ice Research Grant GM-12292 and National Science FoundationResearch Grants GB-5376 and GB-7736 and was conducted over thepast 5 years by undergraduate students (first six authors) as part oftheir independent study or special summer research programs. Thefirst six authors were undergraduate research students in the Depart-ments of Biochemistry and Biophysics, Bacteriology, Family and Con-sumer Sciences, Biological Sciences, Biochemistry and Biophysics andChemistry, respectively.

2Address reprint requests to Dr. Irwin H. Segel.

The present work was undertaken in order to determine the modeof regulation of the sulfate permease in filamentous fungi, andalso to determine whether the differences between the A. nidulansand P. chrysogenum permeases are as marked as reported.

MATERIALS AND METHODS

Strains of Fungi. The organisms used for this study were Peni-cillium chrysogenum, strain PS-75 (wild-type), Penicillium nota-tum, strain CMI-38632, hereafter called 38632M (a white-sporedmutant lacking sulfate permease and at least one of the sulfate-activating enzymes), and strain CMI-38632R (a sulfate permease-positive revertant isolated in our laboratory from strain 38632M),Aspergillus nidulans (wild-type), strains iota and Si-2 (lacking atleast one sulfate-activating enzyme), strain eta (lacking PAPS'reductase), strain alpha (blocked somewhere between sulfite andcysteine, probably at O-acetylserine sulfhydrase), and strain"gamma" (lacking sulfate permease and probably PAPS reduc-tase as well). Strain gamma from the same source (CommonwealthMycological Institute) was reported to be sulfate permease-posi-tive and ATP sulfurylase-negative by Hussey et al. (5) and Spen-cer et al. (12). It seems quite likely that their strain is not identicalwith ours. The characteristics of our strain gamma are more likethose of mutant strains A and C described by these authors.Similarly, our strain iota lacks ATP-sulfurylase, while they re-ported iota to be APS-kinase-negative.Growth of Mycelia. Penicillia and Aspergilli mycelia for

permease studies were grown for 1 to 2 days on synthetic citrateNo. 3 medium (2) containing, as indicated, either 100 mg/liter(low level) or 1 g/liter (high level) of the desired sulfur source.The organisms were grown in submerged liquid culture in 500-mlErlenmeyer flasks containing 100 ml of medium. The flasks wereincubated at 25 C (Penicillia) or 32 to 34 C (Aspergilli) on arotary shaker operating at about 200 rpm and describing a 1-inchcircle. Only white, fine, filamentous ("hairy") mycelia were usedfor permease studies. The mycelia first produced from a sporeinoculum sometimes grew in the form of small pellets. Somestrains produced brownish pigmented hairy mycelia or pellets.Such cultures were transferred daily until all pellets and pig-mented mycelia were diluted out by the subsequent growth ofwhite, filamentous mycelia.Permease Assay. Sulfate permease activity was usually meas-

ured in 0.05 M K+-NH4+-phosphate buffer, pH 6.0, by assaymethod II as described earlier (2). For routine assays, 0.5 ml of10-2 M Na25O4 (specific radioactivity 5 X 105 to 106 cpm/,mole) was added to 50 ml of suspension containing 1 g wetweight mycelium. Transport rates were determined from at leastfour 10-ml aliquots taken at 30-sec intervals during the first 2 min

3Abbreviations: PAPS: 3'-phosphoadenosine 5'-phosphosulfate;APS: adenosine 5'-phosphosulfate; DNP: 2,4,-dinitrophenol; EDTA:sodium salts of ethylenediaminetetraacetic acid, pH 6.0; MES: 2-(N-morpholino)-ethane sulfonic acid.

720

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SULFATE TRANSPORT IN FUNGI

of incubation. All rates are reported on a dry weight basis. Unlessindicated otherwise, all transport assays were conducted at 25 C.

Chemicals. Carrier-free ISO42- was obtained from NuclearScience and Engineering Corporation and was added to un-labeled Na2SO4 to obtain solutions of the desired concentrationand specific radioactivity. All other chemicals were the purestavailable from Calbiochem, Sigma, and Mann. Cysteine-S-sulfate was synthesized by the method of Segel and Johnson (10).Unlabeled choline-O-sulfate was synthesized by the methoddescribed by Bellenger et al. (1).

RESULTS

Linearity of Sulfate Transport. Figure 1 shows that sulfatetransport is linear with respect to time for the duration of theassay. The intercept on the y axis probably represents 5SO42-nonspecifically occluded in the mycelial pad or adsorbed to thecell surface. Curves for mycelia incubated with 3SO42- at 2 Cshow the same intercept with no appreciable slope (uptake).Figure 2 shows that sulfate transport is also linear with respect tomycelial density up to 3.0 g wet weight per 50 ml of assay incu-bation medium. The sloping off at higher mycelial densities prob-ably results from quenching of the light emission during scintilla-tion counting, as well as from depletion of the external '5S042.For comparison, it may be noted that the usual mycelial densitiesobserved under our growth conditions range from 0.1 to 0.25 g

(5

LOI-

LJ

0

:E

z

0

LI)

INCUBATION TIME

-jI ~~~~~~~~~~~~~~~ID

w z

-J 0J5 Z

11 oJo- /40!a0 CK~~~~~~~~~~~~~~0a.~~~~~~~~~~~~~~~~~~~~~~~~~.

cz / X

0

o~~~~~~~~~~~~~~~~cz~~~~~~~~

' 0.05-20

CLn I-

p,)IU.)

0 1 2 3 4MYCELIUM PER ASSAY (G/50 ML)

FIG. 2. Linearity of sulfate transport by P. notatum strain 38632Ras a function of mycelial density. Rates were determined from foursamples taken at 30-sec intervals during the first 2 min of incubation.

75

C,)

I0

C.

uJ

0-

LI)

0

LI)

(MINUTES)

FIG. 1. Linearity of sulfate transport by A. nidulans, strain iota.The mycelia were grown in citrate No. 3 synthetic medium containing100 mg/liter of L-cysteic acid as sole sulfur source. After 2 days ofgrowth, the mycelia were filtered off, washed with deionized water,and resuspended at a density of 2 g wet weight per 100 ml in 0.05 M

K+-NH4+-phosphate buffer, pH 6.0. After 15 min of aeration, 1.0 mlof 10-2 M 35SO42 was added. Then 10-ml aliquots of the suspensionwere taken at 30-sec intervals and filtered rapidly with suction. Theresulting mycelial pads were immediately washed with ice-cold water,then peeled off the filter paper and resuspended in 7.0 ml of scintilla-tion fluid. After the radioactivity was counted, the contents of eachscintillation vial were filtered, washed with acetone, then water, andthe mycelia were dried overnight at 100 C. Each 10-ml aliquot con-tained approximately 12 mg dry weight mycelia. Sulfate transportedis shown on a dry weight basis.

wet weight per 50 ml (average inoculum size) up to 1 to 4 g wetweight per 50 ml (after 1 to 3 days aerobic, submerged growth).

Scott and Spencer (8) reported that sulfate transport in A.nidulans displayed three distinct phases: (a) an initial rapid up-take, followed by (b) an equally rapid but partial elimination ofthe accumulated sulfate, and then (c) a slower, but sustaineduptake that corresponded to fixation of the sulfur into organiccompounds. The authors stated that the onset of each phasedepended on the incubation temperature (8) and other undefinedconditions (12). The actual initial transport rates and the durationof each phase under any particular set of conditions were notreported. In an attempt to study this triphasic uptake pattern, weassayed sulfate transport in several A. nidulans strains undervarious incubation conditions. Some of our results are shown inFigure 3. It can be seen that at three different external sulfateconcentrations, and at two different incubation temperatures,none of the five strains tested showed a triphasic pattern. Theaddition of glucose to the assay buffer did not increase the initialtransport rate nor the total amount of 35SO42 accumulated.

Effect of pH. Figure 4 shows effect of pH on sulfate transportby A. nidulans, iota. Sulfate is a divalent ion throughout the pHrange tested. Consequently, any effect of external pH must havebeen on the cell surface or permease system itself.

Effect of Temperature. Figure 5 shows the effect of incubationtemperature on sulfate transport by A. nidulans, strain iota. Thehigh Q,o value (2-3 between 20 and 40 C) and the sharp declineabove 40 C is characteristic of a carrier-mediated, enzyme-likepermeation process.

Effect of Metabolic Inhibitors. As shown in Figure 6, DNP andazide (10-3 M) caused an immediate cessation of sulfate transportby A. nidulans. Similar results were obtained with P. chrysogenum,contrary to our preliminary finding that a preincubation periodwas necessary for DNP to act (17). Arsenate (10-3 M) was notinhibitory even when the mycelia was preincubated with theinhibitor for 15 min prior to adding the 3SO4. The rapid actionof DNP and azide and the lack of inhibition by arsenate suggest(a) that endogenous (cytoplasmic) ATP may be unavailable ornot involved in sulfate transport (6) and (b) energy productionand sulfate transport may be intimately coupled within the cell

Plant Physiol. Vol. 46, 1970 721



BRADFLELD ET AL.

INCUBATION TIME (MIN.)

I0

JC1,,w-j0

2:0

wI-0CY

zl4C-w

4-

UJ

x

_E

CY,

2:0

N.-

w-J0

0wI-00-z

w1--<U.

Ul)

PLant Physiol. Vol. 46, 1970

INCUBATION TIME (MIR)

INCUBATION TIME (MIN.) INCUBATION TIME (MIN.)

FIG. 3. Sulfate transport by various strains of A. nidulans at various external sulfate concentrations and incubation temperatures.

membrane. As suggested by Harris et al. (4), electron transportprocesses within the cell membrane may result in an "energized"membrane subunit. The potential energy of the energized subunitmay then be dissipated in ion transport without the intermediacyof ATP per se.

Effect of Other Sulfur Compounds and Analogues. Sulfatetransport by A. nidulans, strain Si-2, is inhibited by group VIanalogues of sulfate and by thiosulfate and dithionite, but not byany of the other common sulfur anions or common sulfur aminoacids. Tweedie and Segel (14) have shown that SeO42-, S2032-,

and MoO42- (above pH 5) are substrates of the sulfate permeasein P. notatum and A. nidulans.

In the usual experiment the potential inhibitor and the 35SOW-were added simultaneously to the mycelial suspension. In anothertype of experiment, transport was initiated by adding 10-4 M35SO42- at zero time, and 10-ml aliquots were taken as usual for1.5 min. At 1.75 min unlabeled Na2SO4, Na2S203, or Na2SeO4was added to a final concentration of 10-2 M. Samples were takenfor an additional 8 min. All three compounds caused an imme-diate halt in '5S accumulation (the Na2SO4 by dilution of the

722

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JNw-J0

2:

ow1--

0~C,)zC4

w!-4U.

cn

J

CD)

ICU)

-J0

x

2:k0LuI-

0a.CU)z.4I--

U,)




35SO42-, the other compounds by effective competition for thepermease). In no instance, however, was there an efflux of thepreviously accumulated "S. The results strongly suggest that sul-fate transport is unidirectional; i.e., the permease does notcatalyze an exchange between intracellular and extracellularsubstrates.

Effrect of Other Ions on Sulfate Transport. Sulfate transport byEDTA- and deionized water-washed mycelia were measured in0.05 M Mes-tris buffer, pH 6.0, containing various other anions

-JllJ

z-iC-)

I-

CY,

z

I-

-

CD)

a.

CO)

pH

FIG. 4. Effect of incubation pH on the sulfate Inidulans, strain iota. In one series of experiment!preincubated in 50 ml of 0.05 M buffers for 15 minsubstrate (0.5 ml of 10-2 M 35SO42). In another sethe mycelia were preincubated in 45 ml of 0.05 M Ebuffers (5 ml of 0.5 M) were added immediatelyThe buffers used were as follows: pH 3 to 5, citIphosphate; pH 8 to 9, tris; pH 9.5 to 10, glycine.

z

IL_ I4CDC,)w

4-

>0,,0.83

4.-

0Y 0.6C,)z

g2 0.4

E 0.2

C-)rw

a-

co)10 20 30

TEMPERATURE CC)0-

FIG. 5. Effect of incubation temperature on tirate of A. nidulans, strain iota. The washed mycein the prewarmed or precooled buffer and immesulfate transport at lr4 M external 35SO4F.

e-

X 8.0(2,

CO)w-J0

6.0

0wIy-00-

Ln 42.0

0

723

2 4 6 8 10 1;

INCUBATION TIME (MINUTES)FIG. 6. Effect of azide and 2 ,4-dinitrophenol on sulfate transport

by A. nidulans, strain Si-2. The mycelia were assayed under standardconditions. The additions were made after 1.5 niin, and the transportactivity was followed for an additional 8.5 min.

I \ and cations. Of all the metal ions tested (K+, Na+, NH4+, Ag+,L-1 Mg2+, Mn2+, Ca2+, Zn2+, Co2+, Cu2+, Hg2+, All+, Fe'+) none

10 stimulated transport and only Ag+, Cu2+, Hg2+, Fe3', and A13+inhibited significantly. Similarly, nitrate, acetate, citrate, andphosphate had no marked effect.

transport rate of A. None of the A. nidulans or P. notatum strains tested woulds, the mycelia were transport sulfate at measurable rates from 10-4 M solutions ofprior to adding the H235SO4 or Na235SO4 in deionized water. P. chrysogenum showed

riCl foof ex5 erimeThe about 10% of the maximal transport rate. The addition of anybefore the 35S04s2- one of several salts at 0.05 M allowed transport to proceed nor-

rate. pH 6.0 to 7.5 mally. Essentially identical effects were observed with KCI, NaCl,NH4C1, KNO3, Na acetate, MgCl2, CaCl2, MnCl2, ZnCl2, tris-Cl,Tris-Mes, Na2 EDTA, lysine HCI, arginine - HCI, histidine HCI,and DL-serine ethyl ester-HC1. Glucose, sucrose, mannitol, iso-electric (free base) glycine, serine, and alanine were ineffective.(The pH of each of the above suspensions was between 4.0 and7.6.) HCI at pH 1.9 and pH 2.8 was also ineffective, while 0.05 ML-2 ,4-diaminobutyric acid dihydrochloride (pH 2.0) was quiteeffective. HC1 at pH 4.4 was only about 10%/ as effective as 0.05 MDL-histidine -HCl (pH 4.2). The results suggest that the require-ment is either for an optimal ionic strength per se, or possibly forany net positively charged mobile ion (to balance the negativecharge on the sulfate), rather than for osmotic strength or for anyspecific metal ion. In contrast, it may be noted that the permeasesfor L-methionine (2) and choline-O-sulfate (1) (both zwitterions)show no marked ionic strength optimum.The KCI concentration dependence for 35SO42 transport was

measured at 10-4 M external 35SO42 as shown in Figure 7. Trans-port was 80 to 100% of maximal between 0.05 and 0.3 M KCI.Transport rates decreased markedly above and below these limits.

40 50 The high concentration requirements for the salt explains why notransport could be detected from 10-4 M solutions of Na235SO4.

he sulfate transport If the K+ serves to balance the negative charge on SO42 during~ia were suspended transport, then the relatively high concentration requirement isediately assayed for not surprising. The specificity and affinity of the permease are,

after all, for sulfate ions. A membrane-bound, binding protein

Plant Physiol. Vol. 46, 1970

/

./ sQ X

ADDITIONS

DNP

IAZIDE

2I_

I-

.

-)

0

I




probably is involved in concentrating sulfate ions at the cell sur-face (7). In order to provide an equivalent surface density ofmobile positive ions, a relatively high concentration of salt mustbe used. The mycelia may have specific binding proteins foreach of the positive ions also, but in metal ion-sufficient mycelia,these may be repressed to a low base level.

30

m 20-0

Q_-

<I

L) =-rIz

n-2

KCI (M}

FIG. 7. Effect of KCI concentration on sulfate transport by A.

nidulans, strain Si-2. The mycelia were suspended in deionized water

and aerated as usual. KCl (as a concentrated solution) was added

immediately before the 3SSO4F (10-4 M initial external concentration).

The subsequent transport rate was determined from four samples

taken within 2 min after adding the 3oSO4>.

Table I. Effiect of Externlal Sulfate Concentration on the Ultimate

Intracellular SulJate Pool

Low cysteic acid-grown mycelia of A. niEdulan7s, strain Si-2, were

incubated under standard assay conditions in 0.05 M K+-NH4+phosphate buffer, pH 6.0, containing 35SO42- at the indicated con-

centrations. The suspension contained approximately 0.29 g dry

weight of myceelia per 100 ml. The myceelial 35S and residual

medium 35SO42 were determined periodically over an 8-hr period.

The values recorded are those observed when no further change

could be detected and are based on mycelial 35S.

External Sulfate Concn

Initial

I1l10-7

10-6

10-4

2 X

3 X

4 X

5 X

10-410-4

l0-410-4

10-3

5 10-3

10-s

10--2

Final

M

ca. 0

ca.

ca.

CaI.ca. 0

ca. 0

ca. 0ca. 0

4.98 X 10-44.54 X 10-s

.3. .

Final (maximal) Intracellular Sulfate Pool

s,u01oles g

0.03450.3453.45

34.569103.5138172173175 (159)2173173

8.6 X 10-6

8.6 X 10-s

l 8.6 X 10-48.6 X 10-s

1.73 X 10-22.59 X lo-23.45 X Io-24.32 X 10-24.33 X 10-24.38 X l1-24.33 X l-24.33 X 10-2

1 Intracellular 35SO42- was calculated assuming 4 ml of intra-cellular water per g dry weight of mycelia.

2 Value calculated from '5SO42- that disappeared from themedium.

3 The difference between the initial and final external 35SO12-concentration was too small to determine accurately.

Table II. Retention of 35S by 30SO42>-preloaded MyceliumLow cysteic acid-grown mycelia of A. nidulans, strain iota,

were incubated in 1l-4 M 35S04> under standard assay conditions.The suspension contained approximately 0.20 g dry weight myce-lium per 100 ml. After 90 min the mycelia had accumulatedapproximately 45 ,moles/g. The mycelia were then removed fromthe incubation medium, washed, and resuspended at a density of20 g wet wt/liter in the various solutions indicated below. Allsuspensions except that indicated were aerated at 25 C. The my-celial 35S content was measured after 3 hr.

Suspension Conditions 'Mycelial 5S

Final (after 3 hr) in:Deionized water 40.9Buffer (0.05 M K+-NH4+-Phosphate, pH 6.0) 40.7+DNP (10-3 M) 42.2+azide (l0r-3 M) 42.5+L-methionine (l02 M) 48.6+L-cysteine (10-s M) 36.6+DL-homocysteine (10n2 M) 45.5+choline-O-Sulfate (l1 t M) 43.2+ Na2SO4 (102 M) 35.9+Na2SO3 (10-2 M) 44.4+Na2S203 (102 M) 41.5+actidione (l0- M) 46.7+mycostatin (Squibb), 500 units/ml 0.0+EDTA (10-2 M) 46.2

Buffer (0.05 M tris-citrate, pH 7.6) 44.2Buffer (0.05 M tris-citrate, pH 8.7) 35.8Buffer (0.05 M glycine-NaOH, pH 9.5) 35.1Buffer (0.05 M citrate-tris, pH 2.6) 0.3-8Buffer (0.05 M K+-NH4+-phosphate, pH 6.0) 47.8

at SCBuffer (0.05 M K+-NH4+-phosphate, pH 6.0) 43 .4anaerobically

Effect of External Sulfate on the Intracellular Sulfate Pool. Asshown in Table I, A. nidulans strain Si-2 will deplete 35SO42- from10-7 to 5 X 10-4 M solutions under standard assay conditions. Inthe presence of excess "SO42- (10-s M and greater) the myceliawill accumulate up to 175 ,umoles/g on a dry weight basis. If weassume a reasonable value of 4 ml of intracellular water per g dryweight (9), this corresponds to an intracellular concentration ofabout 0.04 M. It is apparent from the data presented in Table Ithat (a) the mycelia can retain at least 0.04 M intracellular sul-fate against an almost infinite concentration gradient, (b) sulfateis not partitioned according to a fixed distribution coefficient, and(c) there is an upper limit to the intracellular sulfate pool. Thus,in a sense, intracellular sulfate controls its own transport.

Retention of Intracellular Sulfate against a ConcentrationGradient. In another experiment the mycelia of strain iota werepreloaded with 35SO42-. The mycelia were then resuspended inthe various media listed in Table II. Almost all of the accumulatedsulfate was retained in all solutions except in the pH 2 buffer andin that containing mycostatin. The results confirm that underphysiological conditions (a) the mycelial membrane is essentiallyimpermeable to sulfate via free diffusion, (b) transport is uni-directional, and (c) the temperature, pH, ionic strength, andenergy dependence phenomena described earlier are charac-teristics of the transport process rather than properties related tomembrane stability. It is noteworthy that compounds that can"turn off" the permease in short term preincubations (e.g.,cysteine and homocysteine as described below), and compoundsthat yield mycelia that are maximally repressed or inhibited forthe sulfate permease when employed as sole sulfur sources (e.g.,L-methionine as described below) did not stimulate a leakage ofsulfate.

724 BRADFIELD ET AL.




Effect of Sulfur Source Used for Growth. The effect of differentsulfur sources used for growth on the subsequent sulfate transportactivity of the mycelia was determined. All sulfur sources wereprovided in excess of that required for growth. The results indi-cated very clearly that the sulfur source affects the specific trans-port activity of the mycelia. For example, the specific sulfatetransport rates of mycelia grown for 24 hr in medium containing1 g/liter L-methionine were usually less than 0.05 ,umole/g min.In contrast, mycelia grown on 1 g/liter L-cysteic acid, DL-homo-cysteic acid, taurine, or L-djenkolic acid transported sulfate atrates of 0.2 to 1.3 ,umoles/g-min. These results strongly suggestthat the sulfate permease is under metabolic control by one ormore intracellular sulfur compounds. Scott and Spencer (8)reported that no change in rate of transport was seen when cys-teine, methionine, taurine, djenkolic acid, or choline-O-sulfatewere used as sulfur sources for growth of A. nidulans. (The authorsdid not indicate whether the transport rates were equally highand maximal, or equally low and minimal on all sulfur sourcestested.) They concluded that the sulfate permease was not regu-lated by intracellular organic sulfur pools. Regardless of whetheror not the sulfur source made a difference, one can draw noconclusions concerning the nature of the regulators of the per-mease (organic versus inorganic) from this type of growth experi-ment. Fungi not only can produce organic sulfur compoundsfrom inorganic sulfur anions, but also can oxidize sulfur aminoacids to inorganic sulfate (9, 18).

Development of Sulfate Permease during Sulfur Starvation.Although high methionine-grown mycelia showed extremely lowtransport rates, the specific sulfate transport activity of strainseta, iota, Si-2, alpha, and 38632R increased markedly upon sulfurstarvation (to 1.4-2.3 ,umoles/g . min). The sulfate transportrates of strains "gamma" and 38632M remained at less than 0.05,umole/g -min. Little or no permease activity developed when themycelia were sulfur-starved in the absence of Mg24 and a metabo-lizable carbon source, or under anaerobic conditions. The resultsagain suggested that the sulfate permease is regulated by someintracellular sulfur-containing metabolite. In the presence of10-4 M actidione, an inhibitor of protein synthesis in fungi (13),none of the strains developed sulfate permease activity. Proteinsynthesis may be involved in the development process in severalways: (a) the increased permease activity may result from synthe-sis de novo of more permease (i.e., derepression), or (b) proteinsynthesis may provide a way of removing feedback inhibitors ofthe permease from the intracellular soluble pool (i.e., deinhibi-tion). We should also consider the possibility that protein synthe-sis per se is not important, but rather (c) cell growth, i.e., in-creased mycelial mass. An increase in mycelial mass during sulfurstarvation (at the expense of metabolizable endogenous sulfurreserves) would reduce the intracelllular concentration of per-mease regulators by dilution even if the regulators were notmetabolized.

Scott and Spencer (8) reported that the sulfate transport ac-tivity of A. nidulans showed no increase when a mutant, unable toactivate inorganic sulfate, was sulfur-starved. On the basis of thisobservation they concluded that intracellular sulfate was a regu-lator of its own permease. We agree with the conclusion but con-tend that the role of intracellular sulfate as a regulator cannot bedetermined by sulfur starvation experiments. The intracellularsulfate level would decrease upon sulfur starvation (by dilution)regardless of whether or not the cells can metabolize sulfate.Sulfur starvation under conditions of no increase in mycelial massis unlikely to effect an increase in permease activity regardless ofthe identity of the regulator. If the mycelia do not grow, thenthere is little reason why any intracellular sulfur compound(regulator) should be utilized. Some support for this suggestionis shown in Figure 8. We can see that the development of sulfatepermease activity depends on the cell density at which the myceliaare sulfur-starved. At low densities, both the mycelial mass and

permease activity increase significantly upon sulfur starvation.As the original suspension density increases, the subsequentgrowth and the final permease activity decrease.

Contrary to the results of Scott and Spencer (8), we found thathigh methionine-grown mycelia of strains iota, Si-2, and38632R could be derepressed (or deinhibited) by sulfur starvation.Cell-free extracts of all three strains show negligible ATP-sulfurylase activity as measured by the molybdate method (16),and negligible PAPS synthesis measured as charcoal-adsorbable35S from ATP and '5SO42 (Tweedie, J., and I. H., Segel, unpub-lished results).

Sulfur Starvation of Sulfate-loaded ATP-sulfurylase-negativeMutants. In another series of experiments the ATP-sulfurylase-negative mutants and two wild-type strains were grown in syn-thetic medium containing an excess of cysteic acid and an excessof inorganic sulfate. According to the results shown in Table I,the mutants grown in the presence of excess inorganic sulfatewould have accumulated about 0.04 M intracellular sulfate. Thesulfate transport activity of these sulfate-loaded mycelia, beforeand after subsequent sulfur starvation, is shown in Table III.The results leave no doubt that (a) intracellular inorganic sulfateis a regulator of its own permease, and (b) the depression ofsulfate permease activity by intracellular sulfate can be reversedduring sulfur starvation by a process that does not involve sulfateutilization. However, the results do not mean that sulfate is thesole regulator, nor that dilution of intracellular sulfate is the onlyexplanation for the development of sulfate permease activityupon sulfur starvation. IN

Reinhibition of the Sulfate Permease. A 2-hr preincubation ofsulfur-starved mycelia with any one of several sulfur compounds(at either 1 g/liter or 10- M) considerably reduced the sulfate

z

I0Cl

w-J0II-

t:

;7-CL)

c-

0a.Cl)z

I-

wa-Cl)

1.2-

0.61

0.4[

0.2[

U 2U 4.0 6.0 80 iwoMYCELIAL DENSITY DURING AERATION(GM WET WT/IOOrl S-FREE MEDIUM)

FIG. 8. Effect of mycelial density (strain iota) on the developmentof sulfate transport activity as a result of sulfur starvation. The my-celium was grown for 1 day in citrate No. 3 medium containing 1g/liter L-methionine as sole sulfur source. The mycelia were thensulfur-starved at the densities shown in sulfur-free citrate No. 3 me-dium. After 12 hr, the mycelia were assayed under standard condi-tions. The mycelia sulfur-starved at 0.5 to 2 g/100 ml nearly doubled inwet weight over the 12-hr period, while the mycelia starved at 4 to 7g/100 ml remained nearly constant in weight.





transport activity of the mycelia. The effective compounds in-clude Na2SO4, Na2S203, Na2SO3, L-cysteine, L-homocysteine, andL-cysteine-S-sulfate. The latter four compounds are not extra-cellular inhibitors of sulfate transport. The decreased transportactivity can be ascribed to either (a) the accumulation of an intra-cellular feedback inhibitor, or (b) the destruction of a permeasecomponent (or both). During the 2-hr preincubation period theincrease in mycelial mass was slight. The decrease, therefore,cannot be ascribed to repression. It is noteworthy that L-methio-nine, which was one of the most effective sulfur sources for grow-ing maximally repressed (or inhibited) mycelia, was relativelyineffective in reducing sulfate transport activity in the short pre-incubation experiments. The results suggest that L-methioninemay be a metabolic repressor of the sulfate permease, whileintracellular sulfate and cysteine (or compounds easily derivedfrom cysteine, e.g., sulfide) may be feedback inhibitors. (All ofthe other effective compounds can be easily converted intra-cellularly to either sulfate or cysteine.)

Kinetics of Sulfate Transport. The concentration dependence ofsulfate transport was measured over a wide range of extracellular35SO42-. The results for several strains and several culture condi-tions are shown in Figure 9. In maximally sulfur-starved myce-lia, transport obeyed hyperbolic saturation kinetics with a Kmof 6 X 10-5 to 10-4 M and a Vrnax of about 5 ,umoles/g.min.Mycelia grown on high sulfate medium had about the same Kmvalue, but the Vms1 value was reduced to 0.5 ,mole/g- min, orless. Essentially the same saturation curve was observed for sulfur-starved mycelia that were preloaded with unlabeled sulfatebefore assaying with 35SO42-. Similarly, DNP and azide caused amarked decrease in the Vma, with little or no change in the Kmvalue. Methionine-grown mycelia, on the other hand, hadbarely detectable sulfate transport activity which frequentlyappeared to be nonsaturable up to an external sulfate concentra-tion of 0.1 M. In some experiments, however, we did observe someindication of a saturation plateau in the region of 10-5 to 10-3 Mexternal 35SO42-. The results again suggest that methionine re-presses the synthesis of some component of the permease (per-haps a binding protein that functions to concentrate sulfate at thecell surface), while sulfate acts as a feedback inhibitor. The non-saturable uptake of 35SO42 by methionine-grown mycelia couldrepresent (a) a slow, nonspecific diffusion (perhaps into a fewdamaged cells), (b) transport of 35SO42- by some other permeasewith an extremely low affinity for sulfate (14), or (c) the direct

Table III. Effect of Sulfur Starvationi onl Sulfate TrantsportActivity of Mycelia

Each strain was grown for 2 days in citrate No. 3 medium con-taining L-cysteic acid (1 g/liter) (column 2), or L-Cysteic acid (1g/liter) plus Na2SO4 (1 g/liter) (column 3). The mycelia grown onL-cysteic acid plus sulfate were then sulfur-starved at a densityof 2 g wet weight per 100 ml for 14 hr and then assayed again(column 4). Sulfate transport was measured at 104 M '5SO42-.

Sulfate Transport Rate after MycelialGrowth under Following Conditions

Final WeightStrain - ______-of MyceliaCysteic acid after Sulfur

Cysteic Cysteic acid plus sulfate, Starvationacid plus sulfate then

sulfur-starved

lumoles/g-min g wet wt/100 ml

A. nidulans, wild 0.5 <0.05 2.68 4.1A. nidulans, Si-2 0.2 <0.05 1.38 4.8A. nidulans, iota 0.4 <0.05 1.64 4.3P. notatum, wild ... 0.03 3.20 4.8P. notatum, 38632R 1.2 0.05 2.28 3.8

Z

Ln 0

co :-

EXTRACELLULAR SO4 CONCENTRATION

FIG. 9. Concentration dependence of sulfate transport. All transportrates were determined from four samples taken within the first 2 minafter adding the 35S042.

reaction of external sulfate with an intramembrane permeasecomponent bypassing the repressed binding protein.

DISCUSSION

In spite of the wealth of information on the characteristics invivo of membrane transport systems in fungi, we still have nocomplete picture of their mechanism or mode of regulation. Thevarious permeases that we have studied (for sulfate, choline-O-sulfate [1], methionine [2], cystine [11], and L-a-amino acids [3])have several common properties: (a) they all develop in responseto a specific nutrient limitation; (b) the development is inhibitedby actidione; (c) the addition of actidione to cultures after aperiod of nutrient starvation results in a rapid decrease in pre-existing permease activity; (d) a specific permease activity can bemarkedly reduced by preincubating the nutrient-deficient my-celia with a substrate of that permease (without affecting otherpermeases); (e) transport of all the substrates is unidirectional;i.e., the permeases do not catalyze an exchange between internaland external substrate; and (f) all the permeases are highly tem-perature-dependent and are strongly inhibited by DNP and azide.They differ only in their response to ionic strength of the incuba-tion medium and in the degree to which they are present in nu-trient-suficient mycelia. Properties a, b, and c suggest that thedevelopment process involves synthesis de novo of a proteincomponent of the permease, and that this component has a rapidturnover rate (15). However, these observations can be equallywell explained in terms of an inhibition-deinhibition phenome-non. If the permease were subject to feedback inhibition by ametabolite whose major fate is incorporation into protein, thenanything that prevented protein synthesis would prevent removalof the metabolite from the soluble pool. The addition of a proteinsynthesis inhibitor to cells during the deinhibition process wouldhalt further removal of the inhibitor. If the degradation phase ofprotein turnover were not immediately halted, then the concen-tration of inhibitor in the soluble pool would increase again,leading to a net reduction in permease activity. Property d

726 BRADFIELD ET AL.




suggests that each permease is subject to inhibition by its in-tracellular substrate. However, this observation can also beexplained in another way, viz., that a high intracellular concen-tration of a permease substrate labilizes the permease and stimu-lates its destruction. It is clear that no definite conclusionsconcerning the mode of regulation can be reached until we havea way of quantitating the amount of "permease" independentlyof its activity in vivo.On the basis of the data presented in this paper and elsewhere,

we can formulate the following working hypothesis for the opera-tion and regulation of the sulfate permease: Sulfur starvationresults in both derepression of a binding protein or carrier com-ponent of the permease as well as deinhibition of existing per-mease (by utilization or dilution of the internal sulfate). At theexternal side of the membrane, a carrier combines with sulfatewith a binding constant ofabout 104 -1 (assuming that the Km ofthe transport system is identical to the dissociation constant forthe carrier-sulfate complex). The charged carrier is translocatedto the internal side of the membrane where its binding constant issignificantly reduced. The reduced binding constant could arisefrom a conformational change in the protein resulting fromphosphorylation by ATP. Alternately, the energy requirementmay be for the translocation process itself (of charged carrierfrom the external side to the internal side of the membrane, or ofuncharged carrier in the opposite direction). The different bindingconstant at the two sides of the membrane might then arise froma change in the hydrophobicity of the environment surroundingthe carrier-sulfate complex. The net result would be the move-ment of sulfate from the external medium into the cell, against aconcentration gradient at the expense of metabolic energy. Thelack of exchange can be explained if we assume that only theuncharged carrier can be translocated from the internal to theexternal side of the membrane (or only the charged carrier canbe moved from the external to internal sides). Consequently, ahigh intracellular sulfate concentration could immobilize thecarrier. For example, if the binding constant at the internal sideof the membrane were reduced by (e.g.) a factor of 10 or 100(from 104 to 103 or 102), then at 0.04 M intracellular sulfate, only1 out of 5 to 1 out of 41 carrier molecules would be free to moveto the external boundary. The Km of transport would be un-changed, but the Vmax value, which would be proportional to thenumber of uncharged carrier molecules at the external boundarywould be reduced to 2.5 to 20% of the original value. Experi-mentally, we find that preloading A. nidulans with unlabeledsulfate reduces subsequent transport of 35SO42 to 4 to 16% ofthe original rate. The decrease in Vm,,,, caused by DNP and azideis also consistent with the idea that energy is involved either intranslocating the carrier (charged or uncharged) or in releasingsulfate from the carrier, allowing the uncharged carrier to mi-grate to the external boundary.

The inhibition of sulfate transport by accumulated sulfite andcysteine is more difficult to explain. External sulfite and cysteineare not inhibitors of sulfate transport. Sulfite is probably oxidizedintracellularly to sulfate. Cysteine may be a feedback inhibitoroperating on the process that releases sulfate from the carrier atthe internal boundary. However, the possibility that both sulfiteand cysteine act nonspecifically must also be considered. Bothcompounds at high concentrations are toxic to many fungalstrains, probably because they reduce essential disulfide bonds.

Acknowledgments-We gratefully acknowledge the assistance of Miss Trudy Woodand Mrs. Cheryl Burton in several phases of this work.

LITERATURE CITED

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2. BENKO, P. V., T. C. WOOD, AND I. H. SEGEL. 1967. Specificity and regulation ofmethionine transport in the filamentous fungi. Arch. Biochem. Biophys. 122:783-804.

3. BENKO, P. V., T. C. WOOD, AND I. H. SEGEL. 1968. Multiplicity and regulation ofamino acid transport in Penicillium chrysogenum. Arch. Biochem. Biophys.129: 498-508.

4. HARRIS, R. A., J. T. PENNISTON, J. ASAI, AND D. E. GREEN. 1968. The conforma-tional basis of energy conservation in membrane systems. II. Correlation be-tween conformational change and functional states. Proc. Nat. Acad. Sci.U.S.A. 59: 830-837.

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8. Scorr, J. M. AND B. SPENCER. 1965. Sulfate transport in Aspergillus nidulansBiochem. J. 96: 78P.

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1 1. SKYE, G. E. AND I. H. SEGEL. 1970. Independent regulation of cysteine and cystinetransport in Penicillium chrysogenum. Arch. Biochem. Biophys. 138: 306-318.

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13. SUSSMAN, M. 1965. Inhibition by actidione of protein synthesis and UDP-galpolysaccharide transferase accumulation in Dictyostelium discoideum. Biochem.Biophys. Res. Commun. 18: 763-767.

14. TWEEDIE, J. AND I. H. SEGEL. 1970. Specificity of transport processes for sulfur,selenium, and molybdenum anions by filamentous fungi. Biochim. Biophys.Acta 196: 95-106.

15. WiLEY, W. R. AND W. H. MATCHErr. 1968. Tryptophan transport in Neurosporacrassa. J. Bacteriol. 95: 959-966.

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17. YAMAMOTO, L. A. AND I. H. SEGEL. 1966. The inorganic sulfate transport systemof Penkillium chrysogenum. Arch. Biochem. Biophys. 114: 523-538.

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Regulation of Sulfate Transport Filamentous Fungi1 · concentration-dependent transport system ("permease"). Transport is unidirectional. In the presence of excess ex-ternal sulfate,