uptake of sucrose by saccharomyces cerevisiae

ARCHIWS OF BIOCHEMISTRY AND BIOPHYSICS Vol. 216, No. 2, July, pp. 652-660, 1982

Uptake of Sucrose by Saccharomyces cerevisiae’

EUGENIO SANTOS; LUIS RODRIGUEZ, M. VICTORIA ELORZA, AND RAFAEL SENTANDREU’

Dqn.wtamt?nto de Micmbih&, Jnstituto de Mzimbih Biquimica, CSIC, Fdtades de CM ?/ Farmacia, Universided de Solamanu~, Spain

Received November 19,1981, and in revised form March 12,1982

Sucrose transport has been shown to occur in several Sue- and Sue+ Sacc~romgm cerevisiae strains as an energy-dependent process. Assay conditions have been estab- lished to avoid both extra- and intracellular hydrolysis of the disaccharide thus allowing the identification of sucrose as such inside the cell immediately after the uptake; acid pH values (4.0-5.0) were optimal for transport although significant uptake was also detected at neutral PH. Transport of sucrose was not dependent on ATP and seemed to be driven by protonmotive force supplied by the electrochemical gradient of protons across the plasma membrane. The actual symport of protons along with sucrose was directly detected by continuous pH measurement of the reaction mixtures and the initial rate of proton movement in the symport process was determined. KC1 inhibited transport of sucrose suggesting that exit of K+ ions might well be involved in main- taining the electroneutrality of the process. On the other hand, NaCl stimulated transport by 50% in our experimental conditions. The specificity of sucrose transport was also tested using different disaccharides.

Several constitutive (1) and inducible (2-4) systems for sugar transport have already been described in Saccharom~ces cerevisiae. Unlike other yeast (5, 6), in which monosaccharide transport has been described as an energy-dependent process tightly associated to respiration, the transport of hexoses in X cerevisiae has been shown to be equilibrative and carried out by mediated diffusion (1, 2, 7). Re- garding other saccharides, only maltose (8) and a-methylglucoside (3,9) have been unequivocally characterized as being actively transported into the cells.

1 This work was supported in part by Grant 2088 from Comision Asesora de Investigation Cientifica y Tecnica of Spain.

2To whom correspondence should be sent: Labo- ratory of Cellular and Molecular Biology, NCI, NIH, Building 37, Room lAO7, Bethesda, Md. 20205.

8 Present address: Departamento de Microbiologia, Facultad de Farmacia, Universidad de Valencia Blase0 Ibanez 13, Valencia 10, Spain.

Disaccharides, such as trehalose and sucrose, in spite of an early report by Avigad (lo), were generally assumed to be hydrolytically split before entering the cells (4, 11, 12). However, uptake of trehalose by an active process has been reported recently and here we also show that sucrose is actively transported without molecular change by different genetic strains (Sue- and SW+) of S. cerevisiae.

Regarding the energy source for transport, there is now an important body of evidence that chemiosmotic phenomena play a central role in the process. The existence of proton symport in the transfer of solutes into cells of microorganisms has been shown to occur not only in bacteria (14), but also in lower eucaryotic systems such as Chknwlla wt.&ark (15), Neurospora crassa (16), and different species of yeast (6, 17, 18). The participation in sugar transport of the membrane potential across the plasma membrane has also

0003-9861/82/080652-09$02.00/O Copyright 0 1982 by Academic Press. Inc. All righta of reproduction in any form reserved.

652

TRANSPORT OF SUCROSE IN YEAST 653

been directly shown in some of the above organisms (16, 18).

Here, we present for the first time experimental evidence for the occurrence of a proton/sucrose symport system in Sue+ and Sue- strains of S. cerevisiae. A partial characterization of this transport system is also presented.

EXPERIMENTAL PROCEDURES

0rganisms and growth conditions The strains A364A (Sue+, MaI+), ts? (St&, Mal+), C65 (Sue-, Mal+), and Xi1 (Sue-, Mal-) of Succharomyces cerevisiae obtained from Dr. L. H. Hartwell of the University of Washington, Seattle, were used in this work.

The cells were maintained in slants of YM-1 medium (19) solidified with 1.5% agar, and propagated in the same liquid medium supplemented with 2% glucose to produce catabolic repression of invertase. The cells were collected at the early exponential phase by centrifugation. The cells were washed twice in 0.1 M tartaric acid adjusted to pH 4.2 with tris(hydroxymethyl)aminomethane (Tris) and resus- pended in the same buffer at a concentration of 100 mg dry wt./ml unless otherwise specified in the text. This suspension was used directly in the experiments.

Mea.wrements of uptake. Unless otherwise specified, experiments were carried out at 15°C in media buffered with 0.1 M tartaric acid adjusted to pH 4.2 with Tris. The reaction mixture contained, in a final volume of 50 pl, 4 mg dry weight cells, 2 rnxu each of glucose and fructose and 10 mM [U-“C$nrcrose at the specific activity indicated where appropriate. The suspension was incubated for 10 s (or the period of time indicated in each experiment) and the uptake halted by dilution with 10 ml of chilled buffer. The cells were collected on glass-fiber filters (Whatman GF/C,25 mm) and washed with 20 ml of the same ice-cold buffer. Controls were run in parallel but boiled cells were used. The radioactivity retained on the filters was counted, without drying, in Tritosol (20) in a liquid scintillation Spectrometer Packard Model Tri-Carb 3320.

Meusurtment of pH changes. Yeast were incubated in water at room temperature in a final volume of 2 ml and the pH was recorded by using a combination electrode (Radiometer GK 2401 C) attached to a Ra- diometer expanded scale pH meter. The pH changes were registered in a recorder with full scale adjusted to 0.5 pH units.

Chromatography of ceU extmcm. Cells collected on glass fiber filters were extracted with ethanol at 37°C for 60 min in screw-capped tubes. Aliquots of the extract were then applied to Whatman No. 1 paper and the chromatography was run in ethyl acetate: pyridine:water (8:2:1, by volume) for 24 h. Sugar spots

were visualized either by chemical development (21) or by counting the radioactivity on strips of the chromatogram in a toluene scintillation cocktail.

Chemicals [U-‘4C]Sucrose was obtained from the Radiochemical Centre, Amersham. Its chromato- graphic analysis revealed more than 99% purity, with less than 0.55% [“Clglucose and 0.23% [‘“Clfructose contaminating the original product. An- tymicin, 2-deoxyglucose (2-DOG),’ 2,4-dinitrophenol (DNP), iodoacetamide, invertase, and fructose were from Sigma. Glucose and sucrose were obtained from Merck. Amphotericin B and nystatin were the gen- erous gift of Dr. J. F. Martin (Department of Micro- biology, Salamanca). All other reagents were of an- alytical grade.

RESULTS

Sucrose Uptake and pH Dependmug of the Process in Sue+ and Sue- Strains of s. cwevisiae

Invertase-catalyzed hydrolysis of radioactive sucrose and subsequent uptake by the cell of radioactive glucose and fructose was the main difficulty in analyzing sucrose transport in yeast. In order to avoid this difficulty, the cells were grown in the presence of high concentration of glucose to repress synthesis of external invertase. In addition, sucrose transport was assayed in the presence of nonradioactive glucose and fructose in concentrations sufficient to dilute any radioactive monosaccharide arising from splitting of sucrose.

The incorporation of sucrose was dependent on pH. When the assays were carried out at pH 7.5, an increasing, time-dependent incorporation of radioactive sucrose into Sue- and Sue+ strains of S. cereGsiae was detected (Fig. 1). In addition, when the experiments were carried out at pH 4.2, a two- to threefold increase in the radioactivity incorporated was found in all the strains tested (Fig. 2 shows results obtained for strain A364A). Paper chromatography analysis of ethanol extracts of the cells showed that most of the radioactivity was actually incorporated in a material that cochromatographed with sucrose. Treatment with invertase and re- chromatography of this radioactive ma-

’ Abbreviations used: DOG, deoxyglucose; DNP, dinitrophenol.

654 SANTOS ET AL.

terial yielded two new spots which comi- grated with glucose and fructose standards (Fig. 3). When the eluted material was treated as above but invertase was omitted, the radioactive material ran as sucrose.

After short incubation times (40 s and less) most of the radioactivity incorporated remains unchanged as sucrose, but as the incubation progresses, part of the radioactivity remains at the origin of the chromatogram, possibly as phosphate de- rivatives of monosaccharides arising from internal hydrolysis of the sucrose incorporated (data not shown).

The results described above suggest that there is a transport system in S. cerevz%ue that pumps sucrose into the cells. In order to characterize some of the properties of this transport system, the strain A364A was used throughout the rest of the work, unless otherwise specified.

Kinetic Characterization of the Transport Sgd43m

(a) Time course of incorpm-atiun and initial rate of uptake. In order to obtain a meaningful, nonunderestimated measurement of the initial rate of sucrose incorporation by measuring radioactivity retained in filters, it was necessary to avoid nonspecific losses of radioactivity as sugar

:hc. 30 60 90 TIME (SECONDS)

FIG. 1. Incorporation of sucrose by different strains of S: cerevisiae. The experiments were carried out as described under Experimental Procedures in 50 mM Tris-HCl buffer, pH 7.5. Sucrose incorporated by cells (0); control with heated cells (0). The specific activity of [v-‘Quicrose was 0.2 gCi/rmol.

28 I , I , ,

24-

TIME (seconds)

FIG. 2. Uptake of sucrose by S. caevisiae A364A. Uptake was assayed at room temperature as described under Experimental Procedures using either 50 mM Tris-HCl buffer, pH 7.4 (open symbols) or 0.1 M tartaric acid adjusted to pH 4.2 with Tris (solid symbols). Sucrose incorporation at pH 4.2 (O), controls with heated cells at pH 4.2 (A), sucrose incorporation at pH 7.4 (0), controls at pH 7.4 (A). Specific activity of [U-“C]sucrose was as in Fig. 1.

phosphates or as 14C02 or r4C]ethanol which might be produced when the incubations were carried out for prolonged time at room temperature. In order to cope with those difficulties, the rate of incorporation of radioactive sucrose was measured by incubating cells at pH 4.2 under the conditions previously described, but at 15°C and reducing the incubation time to lo-15 s. Increasing incorporation was obtained for at least 40 min, but the rate of [14C]sucrose incorporation decreased grad- ually and only the initial 10-s part of the curve (Fig. 4) was used to calculate the initial rate of uptake. An initial rate of 2.07 + 0.09 nmol/min/mg dry wt was calculated from six different experiments.

The possibility of an uphill transport of sucrose could not be tested directly be- cause the actual steady-state concentration gradient of sucrose could not be measured due to fast metabolization of the internalized sucrose that prevented measurement of the internal concentration of this sugar. However, an internal concentration higher than the external 10 mM could be calculated after 30 min of incubation assuming that all the radioactivity incorporated were sucrose.


01 10 20 30

DISTANCE FROM THE ORIGIN (cm)

FIG. 3. Distribution of radioactivity in paper chro- matograms of extracts from S cerevisiae A364A. Cells (30 mg dry wt) were incubated with glucose, fructose, and [u-“c]sucrose (3.75 pCi) under the conditions described in the text, for 60 s at room temperature. Cells were extracted with ethanol and the extract was chromatographed as described. The chromatogram (3 cm wide) was cut into l-cm strips and the radioactivity determined. The spot which comi- grated with the sucrose standard was eluted by soak- ing the paper strips (fractions 8-13, frame B) in 5 ml of distilled water overnight at 37°C. The solution was lyophilized and redissolved in 300 ~1 of distilled water. 200 pl of the solution was mixed with 20 ~1 of a 10 mg/ml solution of invertase and incubated for 15 min at room temperature. The remaining 100 ~1 were treated in the same way except that invertase was omitted (not shown in figure). Both treated and un- treated aliquots were concentrated and chromatographed as described. (A) Sugar standards (S, sucrose; G, glucose; F, fructose). (B) Ethanolic extract. (C) Material eluted from fractions 8-13 (frame B) treated with invertase.

(b) Saturation kinetics. Study of saturation of the system, under the conditions previously described, showed a value of about 6 mM for the half-saturation con- stant (KJ and 2.0 nmol/min/mg dry wt for J max (the maximum transport rate). It should be noted that these constants do not have the significance of K, and V as there is probably more than one system involved in the transport of sucrose. This could reasonably be deduced when either the Lineweaver-Burk (not shown) or Ea- die-Hofstee plots (Fig. 5) obtained from the saturation curve were studied. The lat- ter plot as in the case of trehalose (13) reveals a sigmoid pattern at concentrations lower than 2 mM of sucrose and at

least two different kinetics components with very different apparent affinities, at higher concentration of substrate. The point of inflexion showing the separation between both components appears around 13 mM sucrose. Thus, it seems that a sim- ple Michaelis-Menten kinetics does not apply for this transport system. Similar complex kinetic situations showing high- and low-affinity components have been described before for xilose transport in yeast (18), 6 deoxyglucose in Chlorellu vul- gar& (22), and lactose transport in Esch- erichia coli membrane vesicles (23).

Eflect of Inhibitors of Energy Metabolism on Sucrose Transport

In order to get some insight into the source of energy for transport, the effect of various inhibitors on the sucrose uptake was studied. First, conditions that affect the ATP levels in the cell did not seem to affect the transport of sucrose. Thus, the shortage of the ATP supply from glyco- lytic pathways caused by iodoacetamide (10 mM), did not decrease the incorporation of the disaccharide. Treatment of the cells with antimycin and 2-deoxyglucose

10 20 30 40

TIME (seconds)

FIG. 4. Time course of sucrose uptake in S cerevisiae A364A. 4 mg (dry wt) cells were incubated at 15°C in a 50-~1 mixture containing 0.1 M tartaric acid adjusted to pH 4.2 with Tris, 10 mM lJl-i’C$ucrose (sp act 1 pCi/pmol) and 2 mrd each of glucose and fructose. After different times, the cells were filtered, washed, and the radioactivity was counted as described under Experimental Procedures.

656 SANTOS ET AL.

under conditions that produce a 50- to lOO- fold reduction in the ATP content of the cells (8) did not decrease the uptake of sucrose either. Furthermore, the incubation of cells in the presence of KCN for 20 min before the transport assay resulted in a maximum inhibition of 20% when the concentration was raised to 10 mM (Fig. 6). Thus, it seems that the oxidative phosphorylation is not immediately linked to sucrose transport and may be concluded that ATP is not the form of energy that directly powers sucrose uptake.

By contrast, an almost complete transport inhibition was found when the uncouplers sodium aside and 2,4-dinitrophenol, were used (Fig. 6). In fact, an inhibition of 60 and 80% of sucrose uptake was detected at a concentration of 10 mM of these drugs. This inhibition cannot be accounted for by the action of the drugs on the mi- tochondrial phosphorylation, as only a small reduction of ATP levels (less than 30%) was detected under these conditions. These drugs are known not to affect significantly the ATP levels in yeast (8, 24,

‘,i 0.05 0.1 0.15

v&ucnoa

FIG. 5. Saturation kinetics of sucrose transport. 4 mg (dry wt) cells were incubated at room temperature (20°C) in a 55-~1 mixture containing 0.1 M tartaric acid adjusted to pH 4.2 with Tris, 2 mM each of glucose and fructose, and an increasing concentration of [U-“C]sucrose (sp act 2.6 pCi/pmol). After 20 s the cells were filtered, washed, and counted as described. An Eadie-Hoffstee plot is presented here. No single line appeared, and at least two crossing ones could be inferred from the plot.

I I I 10-4 10-S 10-2

INHlBlTOR CONCENTRATION (M)

FIG. 6. Effect of uncouplers, KCN and NaF, on sucrose transport. Incubations were done at 20°C under standard conditions as described previously with the inhibitors added to the concentrations specified in the figure. DNP was added in ethanol and the ade- quate control was run in this case. DNP (0); NaNs (A); NaF (0); KCN (0).

our observations), but on the contrary, produce leakiness to protons across the plasma membrane, thus dissipating the proton gradient across it (8, 24, 25). This dissipating action seems to be the most reasonable cause for the inhibition of transport, suggesting that the driving force for sucrose transport is indeed the electrochemical gradient of protons across the yeast plasma membrane.

This view was reinforced when the effect on transport of the polyene antibiotics amphotericin B and nystatin was tested. In both cases, more than 70% inhibition on the transport was found at concentrations of 1 Fg antibioticimg dry wt cells (Fig. 7). It is known (29) that the primary effect caused in yeast by such low concentration of polyene antibiotics is an increase in the proton permeability of the plasma membrane and no disruption of the permeability barrier or leakage of internal metabolites occurs under these conditions.

The inhibition of transport found when NaF was added to the reaction mixture (Fig. 6) remains unexplained since no uncoupling effect has so far been associated with this compound, despite the fact that it is known to be an unspecific inhibitor of a variety of enzymes.


pR INHIBITOR/mg DRY WEIGHT CELLS

FIG. ‘7. Effect of polyene antibiotics on sucrose transport. The antibiotics, solubilized in dimethyl sulfoxide, were added to standard incubation mixture at the concentration described in the figure. Points represent percentage of control run only in the presence of dimethyl sulfoxide. Amphotericin (0); nystatin (0).

Changes of pH Associated with Sucrose Transport

A direct observation of proton uptake along with sucrose incorporation would be a proof of the above-mentioned view that the electrochemical gradient of protons is driving the transport of sucrose in S. cer- etiae. In order to monitor changes of proton concentration in the external medium, a direct measurement of pH was carried out following addition of sucrose to an unbuffered cell suspension.

S. cerewisiae Xl4 (Sue-, Mal-) was used in these experiments so that neither glucose nor fructose had to be added to the reaction, thus avoiding nonspecific proton movements due to the possible uptake of these sugars.

Figure 8 shows the time course of pH measurements in a typical experiment carried out with this strain. These experiments were done under conditions under which no intracellular ATP would inter- fere with the process: antimycin, 2-deoxyglucose, and iodoacetamide were added to the cell suspension. Shortly after sucrose addition, an alkalinization of the medium took place that lasted for about 90 a. New additions of sucrose after short periods of time, promoted further alkalinizations al-

addition (not shown). Measurement of the initial rate of proton uptake from the slope of the initial portion of the curve gives a value of about 240 nmol H+/min for sucrose uptake. Although this value is not directly comparable to the initial rate calculated in Section a, as different strains and different conditions (no fructose or glucose in the incubation) were used, the proximity in values points to a stoichiometry of 1:l at the pH at which the measurements were done.

Efect of Potassium and Sodium Salts on Sucrose Transport

In order to get an insight into the ionic movements involved in the maintenance of the electroneutrality during sucrose uptake, the effect of Na+ and K+ on it was studied. Sucrose transport was strongly inhibited in the presence of KC1 whereas sodium chloride produced the opposite effect, giving rise to a 50% increase over the controls (Table I).

IOOnmol HCI

FIG. 8. pH changes associated with sucrose transport. 166 mg (dry wt) S. ccrewkiac X-14 cells were incubated in 2 ml of water containing 5 gg antimycin/ mg dry wt, 6 mM 2-DOG, 10 mM iodoacetamide, and 15 mM MgCl,. The incubation was mixed with a mag- netic stirrer at room temperature. The initial mixture showed a pH between 6.0 and 6.5 in all the experiments. The pH was lowered to 4.0-4.5 by pulses of HCl and after that a continuous recording of pH was carried out. The buffering capacity of the cell suspension was such that addition of 500 nmol of HCl produced a change of 0.1 pH units. Sucrose was added to a final concentration of 15 rnM when indicated. A combination pH electrode connected to a Radiometer, expanded scale pH meter, and a recorder were used

though to a lesser extent than the first throughout.

658 SANTOS ET AL.

Eflect of Other LXsaccharides cm Sucrose TABLE I

Transport

The effect on sucrose transport of the disaccharides maltose, trehalose, and melibiose was also studied in order to deter- mine the specificity of the transport system. While trehalose produced a slight stimulation (by 30%), melibiose showed no effect and maltose inhibited sucrose by a maximum of 40% at concentrations higher than 10 mM. This result suggests the existence of some interaction between the transport systems of sucrose, maltose, and trehalose, disaccharides for which an active uptake has been postulated (8, 13, and this manuscript).

EFFECT OF SODIUM AND POTASSIUM SALTS ON SUCROSE TRANSPORT

cpm

Control (no addition) 6796 100

0.5 M NaCl 10061 148.0 0.25 M NaCl 10608 156.1 0.5 M KCI 2735 40.2 0.25 M KC1 3055 44.9

DISCUSSION

Note. The standard incubation mixture was supplemented with KC1 or NaCl, at the concentrations described, just prior to addition of the mixture containing the radioactive sucrose (specific activity 1 &i/pmol). Time of incorporation of radioactivity was 15 9.

Contradictory results have been reported in the past (4, 10) concerning the occurrence of sucrose transport in yeast. However, a specific system for sucrose transport in Saccharom~ces was likely to exist in order to explain different experimental observations. First, this sugar has been shown to be involved in regulation of metabolism in Saochorom~ces curl&r- gensis, as sucrose added to the medium (and not glucose or fructose) inactivated the internal enzyme fructose diphospha- tase (26). Furthermore, the presence of sucrose in the cytosol would provide a role for the cytosolic invertase (27, 28). The data presented in this paper provide con- clusive evidence showing that the molecule of sucrose is in fact transported into the cells, the molecule remaining unchanged while being transported and shortly after uptake.

acidic pH values, which are known to be optimal for yeast growth. This adds a physiological significance to the sucrose uptake as supporter of growth.

When sucrose uptake was assayed under optimal conditions of time and temperature, the initial rate of transport and the concentration of substrate needed to sat- urate the system seemed to be in the same range as those reported for maltose or trehalose transport (8, 13). A remarkable point is that under standard conditions, the initial rate of sucrose uptake is ap- proximately the same in S. corevisiae A364A,(Suc+, Mal+) and S. cerevtiae 65A (Sue-, Mal+) suggesting that the ability to transport sucrose is not a genetic trait directly related to the presence of external invertase.

Transport of sucrose by the cells was demonstrated by carrying out the experiment in the presence of high concentration of glucose and fructose to repress synthesis of external invertase and to dilute radioactive glucose and fructose split from radioactive sucrose. Under these conditions, uptake of sucrose in Sue- and Sue+ strains of S. corevisiae took place and the disaccharide was found in the ethanol extracts of the cells. It is also important to point out that the system works best at

The study of the effect of different inhibitors revealed that the transport is an energy-linked process not directly dependent on ATP but on ApH+, the electrochemical gradient of protons across the yeast plasma membrane. Pretreatment of the cells with iodoacetamide, a drug which inhibits glycolysis and subsequent substrate phosphorylation, or antimycin and 2-deoxyglucose, under conditions which produce an almost complete depletion of ATP in the cells (8), did not affect the transport of sucrose. Conversely, a marked

Percentage of control


inhibition of its transport could be detected in the presence of uncouplers which collapse the gradient of protons without affecting significantly ATP levels (8, 24, 25) or the polyene antibiotics amphoteri- tin B and nystatin, which are known to produce a primary uncoupling effect at the low concentrations (1 pg/mg yeast) used in this study (29).

The effect of uncouplers on sucrose transport suggested the existence of H+/ sucrose symport. The direct proof of this would be the detection of the entry of protons along with sucrose by means of continuous measurement of the pH of the incubation mixture. S cerewisiae X-14 (Sue-, Mall) which was used in these experiments showed a high buffering capacity, possibly due to the presence of large amounts of phosphate groups in the man- nan of cell wall but despite this we detected an input of protons along with sucrose. The initial rates of uptake showed an apparent H+/sucrose stoichiometry of 1:l at the acid pH (4-5) at which the experiments were carried out.

Slayman and Slayman (16) reported the stoichiometry of H+/glucose transport in A? ~cz.ssu to be 0.8-1.4; Komor and Tanner (15) measured the stoichiometry of H+/6- DOG and H+/glucose symport in C. vu& guri.s as 1; Htifer and Misra (18) also reported a 1:l ratio for H+/monosaccharide transport in Rhodotorula gracilis and Ser- rano (8) gave a 1:l ratio for maltose/H+ symport in S. cerewisiae. Nevertheless, in spite of this tendency to find a fixed stoichiometry for H+/sugar symport, it now seems that these values depend largely on the external pH and increase to 2 or more as external pH increases (30).

The effect of the monovalent cations K+ and Na+ on transport provided some insight into the ionic movements subsequent to H+ and sucrose symport. The inhibition by K+ strongly suggests that this cation might well be acting as a counter ion to maintain electroneutrality in physiological conditions. On the other hand, the stimulation of sucrose transport in the presence of external Na+ suggests the presence of a Na+/H+ antiporter at the level of the plasma membrane which would

convert the imposed Na+ gradient into a gradient of protons increasing the preex- isting ApH+ already present under our experimental conditions (pH 4.2).

Regarding the specificity of the system for sucrose transport, the data currently available show a much closer similarity to the maltose transport than to the trehalose system in S. cerevisiae. Several observations support this view: In addition to being (i) inhibited by maltose, the sucrose transport is also (ii) dependent on the electrochemical gradient of protons; (iii) as in the transport of maltose, NaF exerts an as yet unexplained inhibitory effect; and (iv) even though Serrano (8) does not mention it in his paper, it can be observed in his data that Na+ also stim- ulates maltose transport, although to a lesser extent than in the case of sucrose. However, the facts that (a) the maltose inhibition of sucrose transport is not concentration dependent, (b) sucrose transport need not be induced as is maltose, and (c) the stimulation by Na+ is quantita- tively much more important in the case of sucrose transport than in maltose suggest that maltose and sucrose are in fact transported by different systems, although related to one another.

At this point of the discussion, it is worthwhile to note that (i) the anomalous kinetics, and (ii) the studies of specificity strongly suggest that more than one system are involved in transport of sucrose. It is possible that specific system(s) for sucrose working along with nonspecific ones, give rise to such a complex pattern. In any event, the electrochemical gradient of protons provides the energy for transport. This gradient of protons could be made up in yeast either by the action of the plasma membrane ATPase (24,31) or by the extrusion of organic acids (32) which occurs actively under metabolic conditions. Protons extruded by either method would be subsequently symported along with sucrose and the exit of K+ would maintain electroneutrality. The presence of Na/H+ antiporter would en- hance the ArH+ whenever high concentration of Na+ would be present externally to the cell.

SANTOS ET AL.

ACKNOWLEDGMENTS

We thank Dr. V. Notario and Dr. J. Burgillo for stimulating discussions and help with the manuscript.

REFERENCES

1. HEREDIA, C. F., SOLS, A., AND DE LA FUENTE, G. (1968) Eur. J. Biochem 5,321-329.

2. Kuo, S. C., CHRISTENSEN, M. S., AND CIRILLO, v. P. (1970) J. Ba&?ri& 103,671-678.

3. OKADA, H., AND HALVORSON, H. 0. (1962) Biochim Biophgg Acta 82,538-546.

4. DE LA FUENTE, G., AND SOLS, A. (1962) Biochim Biophys A&z 56,49-62.

5. KOTYK, A., AND HOFER, M. (1965) B&him Bio- phys. Actn 102.410-422.

6. DEAK, T. (1978) Arch. iificrobiol 116,205-211. 7. KOTYK, A. (1973) Third International Specialized

Symposium on Yeast, Helsinki, Part II, pp. 103-127.

8. SERRANO, R. (1977) Eur. J. Biochem 80,97-102. 9. OKADA, H., AND HALVORSON, H. 0. (1963) J. Box-

h-id 86,966-970. 10. AVIGAD, G. (1960) Biochim Biophys. Ada 40,124-

134. 11. MITWSHEVA, N. M., AND UGOLEV, A. M. (1970)

Doll. Akad Hank SSSR 195,506. 12. JANDA, S., AND HEDENSTROM (1974) Arch. Mti

bioL 101,273-280. 13. KOTYK, A., AND MICHALJANICOVA, D. (1979) J.

Gen Microbid 110,323-332. 14. HAROLD, F. M. (1974) Ann N. Y: Ad Sci 227,

297-311. 15. KOMOR, E., AND TANNER, W. (1974) Eur. J. Bab

&em 44.219-223.

16. SLAYMAN, C. L., AND SLAYMAN, C. W. (1974) Proc Nat. Ad Sk USA. 71,1935-1939.

17. SEASTON, A., LUKSON, C., AND EDDY, A. A. (1973) Bidwm. J. 134,1031-1043.

18. HOOFER, H., AND MISRA, P. C. (1978) Bidem J. 172, 15-22.

19. HARTWELL, L. H. (1937) J. Bacterid 93, 1662- 1670.

20. FRICKE, J. (1975) And Biochem 63,555~558. 21. TREVELYAN, W. E., PROCTER, D. P., AND HARRI-

SON, J. S. (1950) Nature (London) 166,444-445. 22. KOMOR, E., AND TANNER, W. (1975) Plmta 123,

195-198. 23. ROBERTSON, D., KACZOROWSKI, G., GARCIA, M. L.,

AND KABACK, H. R. (1980) Biochemistry 19, 5692-5702.

24. SERRANO, R. (1980) Eur. J Bidwm 105,419~424. 25. RIEMERSMA, J. C., AND ABSBACH, E. J. J. (1974)

Biochem Biophj+s. Acta 339.274-284. 26. SCHAMHART, D. H. J., VAN DEN HEIJKANT,

M. P. M., AND VAN DE POLL, K. W. (1977) J. Bacterid 130,526-528.

27. RODRIGUEZ, L., Rurz, T., VILLANUEVA, J. R., AND SENTANDREU, R. (1978) Currem Microbial 1, 41-44.

28. HOLBEIN, B. E., AND KIDBY, D. K. (1979) Caned J. Microbid 25,528-534.

29. PALACIOS, J., AND SERRANO, R. (1978) FEBS Lett 91,198-201.

30. RAMOS, S., AND KABACK, H. R. (1977) Biochem- i&y 16,4271-4275.

31. WILLSKY, G. R. (1979) J. Bid Chem 254, 3326- 3332.

32. SIGLER, K., WURST, M., AND KNTKOVA (1978) VIth International Specialized Symposium on Yea&, Montpellier s 1 x 15.

uptake of sucrose by saccharomyces cerevisiae

Documents