New Jersey. the characteristics of glucose transport under. conditions which prevent its metabolism. yeast (Anheuser-Busch) was washed by repeated

Transcription

1 MECHANISM OF GLUCOSE TRANSPORT ACROSS THE YEAST CELL MEMBRANE VINCENT P. CIRILLO' Department of Microbiology, Seton Hall College of Medicine and Dentistry, Jersey City, New Jersey Received for publication April 16, 1962 ABSTRACT CIRILLO, VINCENT P. (Seton Hall College of Medicine and Dentistry, Jersey City, N.J.). Mechanism of glucose transport across the yeast cell membrane. J. Bacteriol. 84: The kinetics of D-glucose and L-sorbose transport was studied in Saccharomyces cerevisiae inhibited with iodoacetic acid under nitrogen to prevent glucose metabolism. D-Glucose was found to compete with L-sorbose for a common membrane transport system with an apparent affinity greater than 25 times that of sorbose. A comparison of the net rate of glucose and sorbose transport at 50 and 500 mm external concentration showed that glucose transport is greater than that of sorbose from the lower concentration, but sorbose transport is greater than glucose at the higher concentration. This reversal of transport rate of two sugars with markedly different affinities is predicted by the membrane carrier theory. A further prediction of carrier theory was confirmed by the demonstration that the rate of glucose transport into fructose-loaded cells is greater than into unloaded cells. The characteristics of the sugar-transport system in yeast have been receiving renewed attention in several laboratories (Cirillo, 1961c). Recent studies on the transport of nonmetabolizable sugars (Robertson and Halvorson, 1957; Burger, Hejmova, and Kleinzeller, 1959; Cirillo, 1961a, b; Kotyk, 1961; Avigad, 1960; Scharff, 1961) and certain metabolizable glycosides (De La Fuente and Sols, 1962) suggest that sugar transport across the yeast cell membrane is mediated by stereospecific membrane carriers. The present report presents further evidence in favor of the carrier hypothesis, and describes I U.S. Public Health Service Career Development Awardee. the characteristics of glucose transport under conditions which prevent its metabolism. MATERIALS AND METHODS Preparation of yeast cells. Commercial baker's yeast (Anheuser-Busch) was washed by repeated centrifugation in distilled water until the supernatant fluids were clear. The volume of the centrifuged yeast was determined after 5 min of centrifugation at 3,000 X g, and a 10% (v/v) suspension was prepared in distilled water. In most experiments, 2.0 ml of a 10% suspension were placed in 12-ml centrifuge tubes and centrifuged at 3,000 X g for 1 min, giving 0.2 ml of packed yeast per tube. Sugar-uptake procedure. Sugar-uptake experiments were begun by adding 5.0 ml of sugar solution to the packed yeast, which was then rapidly brought into suspension by stirring with two wooden applicator sticks. The tubes were shaken on an inclined platform in a temperaturecontrolled water bath. At the completion of the experimental period, the tubes were centrifuged at 3,000 X g for 1 to 2 min. The packed yeast was then washed by centrifugation with three 5-ml portions of 0.9% NaCl for 4 C. The cells were centrifuged for 1 min for each wash, the whole procedure taking about 5 to 6 min. Burger et al. (1959) showed that loss of intracellular sugar at 4 C is negligible. This was confirmed by the fact that intracellular sugar content determined in washed yeast and in unwashed yeast, corrected for extracellular space by using several nonpenetrating substances (Cirillo, 1961a), gave the same results. When nonmetabolizable sugars were to be analyzed, the washed cells were resuspended in 5.0 ml of distilled water and l)laced in a boilingwater bath for 20 min. If metabolizable sugars were being analyzed, the cells were washed with ice-cold NaCl containing 10-3 M iodoacetic acid (IAA); 1.0 ml of 95% ethanol was added to each 485

2 486 CIRILLO J. BACTERIOL. tube immediately after the last wash, while the cells were still at 4 C. If orthophosphate was to be measured, the washed cells were extracted for 1 hr with 5% trichloroacetic acid (TCA). In all cases, the extracts were separated from the cells by centrifugation, diluted appropriately, and assayed. Transport of free glucose under N2 and IAA. Glucose transport can be studied independently of its metabolism if its metabolism is blocked by IAA under nitrogen. The effectiveness of iodoacetate inhibition was confirmed as follows: (i) glycolysis, measured manometrically, was completely blocked; (ii) phosphate esterification, which accompanies glucose metabolism, was completely blocked; and (iii) no glucose disappearance could be measured (by glucostat assay) when 5% suspensions of yeast were incubated with 50,ug/ml of glucose for 30 min under N2 plus 10- M IAA. To determine whether glucose would be lost by metabolism during washing in 103 M IAA at 4 C, suspensions of yeast were incubated with 50,ug/ml of glucose in 10-3 M IAA at 4 C for 1 hr. At the end of 1 hr, less than 10% utilization occurred. (The washing period usually occupied less than 10 min.) Glucose loss during washing would, therefore, be negligible. Chemical analyses. Reducing sugars were determined by the Nelson method (Neish, 1952), glucose by the glucostat method (Worthington Chemical Corp., Freehold, N.J.), and ketoses by the alcoholic anthrone (Wise et al., 1955) or cysteine hydrochloride method (Dische and Devi, 1960). Inorganic phosphate was measured by the Fiske-SubbaRow method (Umbreit, Burris, and Stauffer, 1957). L-Sorbose and L- fucose were purchased from the Pfanstiehl Laboratories; D-glucose and D-fructose from Merck and Co. Calculation of intracellular sugar content. The intracellular sugar concentration was calculated from the sugar content per ml of packed cells, by correcting for the extracellular water and dry solids which together account for 53% of the packed-cell volume. The extracellular space was determined by the volume of distribution method with lactose as the sugar (Cirillo, 1961a). The intracellular water, therefore, represents 47 % of the packed volume. The sugar content per ml of intracellular water was calculated from the formula: mg of sugar/ml packed yeast 47 mg of sugar/ml intracellular water =~ 100 RESULTS AND DISCUSSION Sorbose and fucose transport under nitrogen plus IAA. Previous work had already shown that the transport of nonmetabolizable sugars continues under a nitrogen atmosphere in the absence of an exogenous source of energy, and is not associated with uptake of extracellular phosphate, which normally accompanies the uptake of metabolizable substrates (Cirillo, 1961a; Kotyk, 1961). Since yeast cells have little or no endogenous anaerobic metabolism, the continued uptake of nonmetabolizable sugars like sorbose and fucose, under nitrogen, indicates that sugar transport is independent of cell metabolism. However, to suppress marginal anaerobic metabolism which might contribute to sugar transport, the rate of sorbose and fucose transport was studied under nitrogen plus 10-3 M IAA. Sorbose and fucose transport continued under all conditions studied (Table 1). When sorbose uptake occurred in the absence of IAA, it was not accompanied by phosphate esterification (Table 2). The continued transport of sorbose and fucose under a nitrogen atmosphere and in the presence of 10- M IAA supports the earlier evidence against the participation of cell metabolism in the transport process. Glucose transport under N2 plus iodoacetic acid. Analyses of alcoholic extracts of the cells incubated with glucose plus IAA (Table 2) revealed the presence of free glucose. This, coupled with the demonstration that glucose metabolism was completely blocked by IAA under N2, showed that it is possible to study glucose transport in the absence of its metabolism. The occurrence of such transport is shown in Table 3. The absence of significant amounts of intracellular glucose in metabolizing cells is also indicated. Glucose transport followed at relatively short time intervals, as in Fig. 1, shows an initially rapid phase followed by a slower phase. The initial uptake is not due to adsorption, since it is absent at ice-bath temperatures. Relative affinity of glucose and sorbose for the transport system. The instantaneous rate of

3 VOL. 84, 1962 GLUCOSE TRANSPORT IN YEAST 487 TABLE 1. Sugar transport under N2 plus IAA* Sugar (mg/ml cell water) Expt Sugar Gas IAA 15 min 30 min 60 min 120 min 1 5% Sorbose Air % Sorbose Air 10-3 M % Sorbose N % Sorbose N2 10-3M % Sorbose Air % Sorbose N % Sorbose N M % Fucose N % Fucose N M * Packed yeast (0.2 ml) was suspended at 30 C in 2 ml of distilled water or 2 X 10-3 M IAA. In anaerobic experiments, the suspensions were then flushed with nitrogen for 3 min. The experiment was begun by addition of 2 ml of double-strength sugar solution. The incubation was stopped by centrifugation, and the cells were washed with 0.9% NaCl at 4 C. The cells were extracted with 5.0 ml of distilled water in a boiling-water bath for 15 min. Sorbose was analyzed for by the ketose method; fucose by the reducing-sugar method. TABLE 2. Phosphate esterification under nitrogen plus iodoacetic acid* Pi (mg/ml Sugar Gas IAA packed yeast) 1 None Air V%/ Sorbose Air % Glucose Air None N M % Sorbose N., 10-3 M % Glucose N M * Washed yeast (0.4 ml) was exposed to sugar as described in Table 1. The incubation was stopped by addition of arn equal volume of 10% TCA, and the cells were extracted for 1 hr at room temperature. The extracts were analyzed for orthophosphate. glucose transport as a function of external concentration is too rapid to be measured by the methods used. It is therefore not possible to determine the apparent Km by the standard Lineweaver-Burk plot. However, the fact that glucose inhibits sorbose transport and causes uphill sorbose efflux from sorbose-equilibrated cells (Cirillo, 1961b) suggests that glucose and sorbose share a common transport system. The apparent affinity of glucose for the transport system can, therefore, be determined by measuring inhibition of sorbose transport by varying concentrations of glucose. The concentration of glucose which results in 50% inhibition of sorbose TABI,E 3. Glucose transport under N2 plus IAA* Expt Glucose Gas IAA Glucose (mg/ml of cell water) min min min min 1 0 N N N2 10-M N M N2 10M3M * Yeast (0.2 ml) was mixed with sugar at 30 C as described in Table 1. The incubation was stopped by centrifugation, and the cells were washed with 10-3 M IAA in 0.9% NaCl at 4 C. After the final wash, the cells were extracted with 1 ml of 95% ethanol for 1 hr. The diluted extracts were analyzed by the glucostat method. transport, designated KI, will give the concentration of glucose which saturates the carriers by 50% provided the concentration of sorbose is far below the concentration which saturates the transport system. A reciprocal plot of the rate of sorbose transport (from 0.28 M solution) against varying external glucose concentrations is shown in Fig. 2. The apparent KI of glucose is about 11 X 10-3 M. Since the ratio of sorbose to glucose at the concentration of glucose which inhibits sorbose transport by 50% is 25: 1, the relative affinity of

4 488 CIRILLO J. BACTERIOL. 14- t 12 - cc - 1- w - E 8- "I - In 0 6- & 4- E 2- *- S-/ 0~~~~~3 external sugar concentration, the rate of glucose transport is slightly greater than that of sorbose (Fig. 3B). For both sugars, however, the rate at which the internal sugar approaches equilibrium with the external medium is greater at the lower concentration (Fig. 4). These inverse relationships, which at first sight seem surprising, are predicted by the kinetic equations of carrier transport developed by Le Fevre (1954), Widdas (1954), Rosenberg and Wilbrandt (1955), and V I.,I.,I.,.,I... -, I.I.,, I.,,,,, ,,,I 28 I 32.,. FIG. 1. Temperature effect on glucose uptake. Glucose uptake by 0.2 ml of cells exposed to 10% sugar in 10-3 M IAA + N2 was determined as in Table S cn 5.0-0m 0 U-1 x 3.0- U GLUCOSE CONCENTRATION ( X 10-3M) FIG. 2. Conmpetitive inhibition of sorbose uptake by glucose. The rate of sorbose uptake by 0.2 ml of yeast in 0.28 M sorbose under nitrogen plus IAA was measured over a 10-min interval without glucose or in the presence of varying concentrations of glucose. The reciprocal of the rate of sorbose transport is plotted against the glucose concentration. The intercept of the abscissa gives the apparent K1 of glucose. glucose for the carrier is more than 25 times greater than that of sorbose. Comparison of the kinetics of glucose and sorbose transport. A comparison of glucose and sorbose transport from 10 and 17% external sugar solutions is shown in Fig. 3 and 4, respectively. When the external sugar concentration is 10 %, the rate of sorbose transport is greater than that of glucose (Fig. 3A). On the other hand, at a 1% 5( c 4' w 31O- Sorbose -j A-10% 0r _ t 21O- 0 E E13 0- O- g ~~~Glucose w -J -i E c.) C 8-i T I FIG. 3. Glucose and sorbose transport under N M IAA at 30 C. (A) Relative rate of glucose and sorbose transport when the external medium contained 10c%e sutgar. (B) Rate at 1% external sugar. wcc z llj X 8-/% Glucose Solrbose 120 FIG. 4. Relative rate of sugar equilibration as a function of the external medium. The intracellular sugar content of the cells in Fig. 3 is plotted as the per cent of extracellular concentration.

5 VOL. 84, 1962 GLUCOSE TRANSPORT IN YEAST 489 Wilbrandt and Rosenberg (1961) for sugar transport in human erythrocytes. According to the carrier hypothesis, the cell membrane contains stereospecific carriers which can diffuse in both directions and at the same rate, whether combined or uncombined. The process is believed to involve three consecutive reactions: combination between the substrate and carrier to form a carrier-substrate complex, diffusion of the carrier-substrate complex across the cell membrane, and dissociation of the carriersubstrate complex into free substrate and carrier. Of these, the rate-limiting reaction is assumed to be the diffusion step. The rate of transport in either direction depends on two factors, a capacity factor and a saturation factor. The capacity factor (Tmax) is the maximal possible transport rate and is the product of the total concentration of carrier (Ct) and the permeability constant (D) of carrier diffusion through the cell membrane. The equation is Tmax = D* Ct (1) The fraction of the total carriers saturated by substrate is given by the Langmuir adsorption expression 0 S+Kt (2) where 0 is the fraction of saturation, S is the substrate concentration, and Kt the dissociation constant of carrier-substrate complex (i.e., the substrate concentration which gives 50% saturation). The rate of transport (T) in either direction would be the product of the capacity and saturation term T=Tmax (s2k) (3) The net rate of transport across the cell membrane would be the difference between independent but symmetrical influx and efflux reactions, each of which conforms to equation 3. The fundamental equation of membrane transport takes the form T = Tmax (So+K Si+K ) (4) where T is the net rate of transport, Tmax is the transport capacity at saturation, S. and Si the external and internal concentrations, and Kt the apparent dissociation constant of the sugarcarrier complex. This equation can be rewritten in another form ( (So- Si)Kt T = Trnax \(So + Kt) (Si + Kt),J (5) If one considers the extreme situation of low saturation, where the sugar concentrations are much smaller than Kt, the denominator of equation 5 becomes (Kt)2, and the equation reduces to T=Tm (So -Si) VKt At the opposite extreme of high saturation, where the sugar concentrations are much greater than Kt, the denominator of equation 5 becomes the product, (S.Si), and the equation reduces to T=Tmax (SS 1 Equations 6 and 7 show that the relationship between the rate of transport and the affinity (which is proportional to the reciprocal of the dissociation constant) is very different for low and high saturation. The equations predict that at low saturation the net rate of transport is directly proportional to affinity, while at high saturation the rate of transport is inversely proportional to affinity. The data of Fig. 3 show that the predictions are borne out for glucose and sorbose. Furthermore, equation 7 predicts that, at high saturation, the rate of transport is inversely proportional to the intracellular concentration (Si). Therefore, the rate of transport will slow down markedly as the internal concentration builds up, and may be barely detectable while the internal concentration is still far below the external. This prediction is also borne out at high glucose and sorbose concentrations (Fig. 4), since both glucose and sorbose transport fall to very low rates while the concentration difference between the medium and the cells is still great. A corollary prediction of the carrier hypothesis relates to the effect of competitive inhibitors of sugar transport which are themselves transported by the carrier. The equation describing the effect (6) (7)

6 490 CIRILJLO J. BACTERIOL. of an inhibitor on sugar transport is T = Tmax So ] ~(8) K So +Kt + Kt I Si+Kt+Kt )I where I and KI represent the concentration and the dissociation constant of the inhibitor, respectively. A preliminary study of the rate of glucose transport in the presence of varying concentrations of fructose has shown that fructose is a competitive inhibitor of glucose transport (unpublished data), and its affinity for the carrier is of the same order of magnitude as that of glucose. As a first approximation, then, Kt/K1 can be taken as unity, and equation 8 reduces to T = Tmax S+ S(9±Kt + () Comparison with equations 3 and 4 shows that the effect of the inhibitor is to decrease both the influx and the efflux processes. However, it is possible to inhibit influx and efflux differentially, depending on the relative concentrations of inhibitor and substrate inside and outside the cell. If the concentration of inhibitor relative to the substrate is greater on the outside of the cell than on the inside, influx is inhibited more than efflux. In this case the net movement of a previously equilibrated substrate would be out of the cell. This phenomenon of uphill efflux, called counterflow, has been described in yeast (Burger et al., 1959; Cirillo, 1961a), erythrocytes (Park et al., 1956; Rosenberg and Wilbrandt, 1958), and heart muscle (Park et al., 1959). If the concentration of inhibitor relative to the substrate is greater inside the cell than outside, there will be an acceleration of the net rate of influx of the substrate. This prediction was borne out by the following experiment. In this case, 0.2 ml of the cell preparation was equilibrated with 5 ml of 5% fructose under nitrogen plus IAA for 60 min; the intracellular fructose concentration reached 14.7 mg/ml of cell water. At this time, the cells were packed by centrifugation, washed, and resuspended in 5 ml of a mixture containing 5% glucose and 5% fructose. The course of glucose uptake is shown in Fig. 5, and is compared to uptake from a similar mixture by unloaded cells. cr: 18- -J -J C 14- "I 0uw: U, E L0 FIG. 5. Glucose cells and frucltose planation.) transport into fructose-loaded counterfiow. (See text for ex- Glucose transport into fructose-loaded cells was greatly accelerated. The observed acceleration is predicted from the fact that the concentration of fructose relative to glucose outside the cell was unity, whereas it was greater than unity inside the cell throughout the experimental period (compare top curve of Fig. 5 to lower curves). The tol) curve of Fig. 5 also shows that the intracellular fructose in the preloaded cells is leaving the cells against a concentration gradient during glucose influx. This fructose efflux against a concentration difference reflects the fact that for fructose the competition with glucose is in the opposite direction (i.e., greater on the outside than on the inside), and results in fructose counterflow. The conformance of glucose uptake with the predictions of the carrier hypothesis adds strong support to the general significance of this mechanism in sugar uptake in y-east. The recent evidence that carrier reactions are also involved in the sugar efflux reactions in Escherichia coli (Kepes, 1960; Rotman and Guzman, 1961) and in sugar transport in Tetrahymena (Cirillo, 1962) supports the conclusion that this mechanism is as fundamental in microorganisms as in higher forms. ACKNOWLEDGMENTS The competent technical assistance of Ljubinka Mirov and Marilyn Funkhouser is acknowledged

7 VOL. 84, 1l962 GLUCOSE TRANSPORT IN YEAST 491 with pleasure. I also wish to thank Joel Barkalow of Anheuser-Busch, Inc., for his generous supplies of baker's yeast. This investigation was supported by a research grant (8443) from the U.S. Public Health Service. LITERATURE CITED AVIGAD, A Accumulation of trehalose and sucrose in relation to the metabolism of a-glucosides in yeast of defined genotype. Biochim. et Biophys. Acta 40: BURGER, M., L. HEJMOVA, AND A. KLEINZELLER Transport of some monosaccharides into yeast cells. Biochem. J. 71: CIRILLO, V. P. 1961a. The transport of nonfermentable sugars across the yeast cell membrane, p In A. Kleinzeller and A. Kotyk [ed.], Membrane transport and metabolism. Academic Press, Inc., New York. CIRILLO, V. P. 1961b. The mechanism of sugar transport into the yeast cell. Trans. N.Y. Acad. Sci., Ser. II 23: CIRILLO, V. P. 1961c. Sugar transport in microorganisms. Ann. Rev. Microbiol. 15: CIRILLO, V. P Sugar transport in Tetrahymena. Bacteriol. Proc., p. 81. DE LA FUENTE, G., AND A. SOLS Transport of sugars in yeast. II. Mechanisms of utilization of disaccharides and related glycosides. Biochim. et Biophys. Acta 56: DISCHE, Z., AND A. DEVI A new colorimetric method for the determination of ketohexoses in the presence of aldoses, ketoheptoses and ketopentoses. Biochim. et Biophys. Acta 39: KEPES, A ]tudes cinetique sur la galactoside-permease d'escherichia coli. Biochim. et Biophys. Acta 40: KOTYK, A The effect of oxygen on transport phenomena in a respiration-deficient mutant of baker's yeast, p In A. Kleinzeller and A. Kotyk [ed.], Membrane transport metabolism. Academic Press, Inc., New York. I.E FEVRE, P The evidence for active transport of monosaccharides across the red cell membrane. Symposia Soc. Exptl. Biol. 8: NEISH, A. C Analytical methods for bacterial fermentations, 2nd revision. Rept. no , Natl. Council Canada. PARK, C. R., R. L. POST, C. F. KALMAN, J. H. WRIGHT, JR., L. H. JOHNSON, AND H. E. MORGAN The transport of glucose and other sugars across cell membrane transports involving insulin. Ciba Colloquia Endocrinol. 9: PARK, C. R., D. REINWEIN, M. J. HUNDERSON, E. CADENAS, AND H. E. MORGAN The action of insulin on the transport of glucose through the cell membrane. Am. J. Med. 26: ROBERTSON, J. J., AND H. 0. HALVORSON The components of maltozymase in yeast and their behavior during deadaptation. J. Bacteriol. 73: ROSENBERG, T., AND W. WILBRANDT The kinetics of membrane transport involving chemical reactions. Exptl. Cell Research 9: ROSENBERG, T., AND W. WILBRANDT Uphill transport induced by counterflow. J. Gen. Physiol. 41: ROTMAN, B., AND R. GU ZMAN Transport of galactose from the inside to the outside of Escherichia coli, p Pathologic- Biologie, Xe Congres International de Biologie Cellulaire. SCHARFF, T. (X Evidence for hexose transport in acetone-dried yeast. Arch. Biochem. Biophys. 95: UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUFFER Manometric techniques and tissue metabolism. Burgess Publishing Co., Minneapolis. WIDDAS, W. F Facilitated transfer of hexoses across the human erythrocyte membrane. J. Physiol. 125: WILBRANDT, W., AND T. ROSENBERG The concept of carrier transport and its corollaries in pharmacology. Pharmacol. Revs. 13: WISE, C. S., R. J. D)IMLER, H. A. DAVIS, AND C. E. RIST Determination of easily hydrolyzable fructose units in dextran preparations. Anal. Chem. 27:33-36.