Glucose Transport in a Kinaseless Saccharomyces cerevisiae Mutant

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1 JOURNAL OF BACTERIOLOGY, July 1987, p /87/ $2./ Copyright 1987, American Society for Microbiology Vol. 169, No. 7 Glucose Transport in a Kinaseless Saccharomyces cerevisiae Mutant JAY M. LANG AND VINCENT P. CIRILLO* Department ofbiochemistry, State University of New York, Stony Brook, New York Received 3 December 1986/Accepted 3 March 1987 Wild-type Saccharomyces cerevisiae organisms contan three kinases which catalyze the phosphorylation of glucose: two hexo e isozymes (PI and PII) and one ghucokinase. Glucose transport measurements for triple-kinaseless mutants, which lack all three of these kinases, confirm that the kinases are involved in the low apparent K. transport process observed in metabolizing cells. Thus kinase-positive cells containg one or more of the three kinases exhibit biphasic transport kinetics with a low apparent K,,, (1 to 2 mm) and high apparent Km (4 to 5 mm) component. Triple-kinaseless cells, however, exhibit only the high apparent Km component of kinase-positive cells (6 mm). Kinetic analysis of glucose transport in the triple-kinaseless cells shows that glucose is transported by a facilitated diffusion process which exhibits trans-stimulated equilibrium exchange and influx counterfiow. Monosaccharide transport in bakers' yeast (Saccharomyces cerevisiae) exhibits different characteristics under different metabolic conditions. Metabolizable monosaccharides (glucose, mannose, and fructose) are transported by low apparent Km (1 to 3 mm) and high apparent Km (2 to 1 mm) processes under metabolizing conditions (2, 28) but only by the high apparent Km process under nonmetabolizing conditions (i.e., in iodoacetate-inhibited cells [34, 36] and in kinaseless mutants [2, 35]). Studies with nonmetabolized glucose analogs (4, 7, 28, 29) and with glucose and fructose under nonmetabolizing conditions (6, 35) have shown that the high apparent Km process occurs by carrier-mediated facilitated diffusion. On the other hand, the low apparent Km process has been shown to involve sugar kinases (e.g., ATP:sugar phosphotransferases). Wild-type strains contain three cognate kinases for the phosphorylation of glucose, two hexokinase isozymes (PI and PII) and one glucokinase. Wild-type strains containing all three kinases, as well as strains containing any one of the kinases, exhibit biphasic kinetics with a high and low apparent Km component. Triple-kinaseless mutants, which lack all three of the kinases (2), exhibit only monophasic kinetics, corresponding to the high apparent Km component of kinase-positive strains. Transformation of the triple-kinaseless mutants with plasmids containing the cloned genes for any of the three cognate kinases restores both glucose metabolism and biphasic kinetics with high and low apparent Km components (2). The precise role of the kinases in the low apparent Km transport process is not clear, since recent studies have shown that kinases also affect the transport of the nonphosphorylated glucose analog 6-deoxy-D-glucose. Thus in kinase-positive cells, 6-deoxy-D-glucose is transported with biphasic kinetics with high and low apparent Km components, while in triple-kinaseless cells, it is transported only by the high apparent Km component (3). However, whether 6-deoxy-Dglucose is transported by kinase-positive or kinase-negative cells, it is transported by facilitated diffusion (3, 25), characteristic of nonmetabolized sugars. Most authors have proposed that both high and low apparent Km transport processes involve the same carrier in two different affinity states (8, 18). Sols et al. (3) and Serrano and DelaFuente (28) proposed that the change is * Corresponding author mediated by sugar metabolites and that the involvement of the kinases in the regulation is indirect. Van Steveninck and colleagues proposed that the change is the result of direct kinase-carrier interaction which mediates a low apparent Km vectorial phosphorylation (27, 34-36) analogous to the bacterial phosphotransferase system (27). Support for this mechanism comes from recent pulse-labeling studies with 2-deoxy-D-glucose (9, 22). Although the more recent experiments (9, 22) were carried out in a manner that avoided the experimental errors which were shown by Michaljanicova and Kotyk (16) to invalidate earlier pulse-labeling experiments (34), the presence of multiple metabolic pools makes the interpretation of pulse-labeling experiments in whole cells very difficult. Contrary to the view that the same carrier is involved in both high and low apparent Km processes, Lobo and Maitra (21) proposed that the kinase-mediated process represents a separate phosphorylation pathway which is independent of the carrier-mediated process. They based this conclusion on the fact that among deoxy- D-glucose resistant isolates, none were transport defective; 871 proved to be kinase defective and 3 were due to other enzymatic defects. However, given the multiplicity of sugar transport systems by which 2dG could enter the yeast cell (17), their interpretation may prove too narrow. In an attempt to better understand the mechanism(s) of glucose transport in S. cerevisiae, we have begun a program of isolating and characterizing the glucose transport systems in reconstituted system(s). We have recently succeeded in preparing isolated plasma membrane vesicles (1) and reconstituted proteoliposomes (11) which exhibit glucosefacilitated diffusion activity. To determine whether the facilitated diffusion activity observed in vesicles and proteoliposomes is unchanged from that of whole cells, it is necessary to characterize the cellular facilitated diffusion process more fully. In this paper we report on this characterization in triple-kinaseless cells which lack the kinasedependent pathway. MATERIALS AND METHODS Strains and growth conditions. The strains of S. cerevisiae used in this study were kindly provided by P. K. Maitra and Z. Lobo, TATA Institute, Bombay, India. The HSC strain is the parental wild-type strain, containing both hexokinase isozymes, PI and PII, and glucokinase (MATa adel trpl his2 met4 HXKI HXK2 GLKI); strain D-38 contains only

2 VOL. 169, 1987 GLUCOSE TRANSPORT IN KINASELESS S. CEREVISIAE 2933 TABLE 1. Comparison of Km5 for sugar transport in mutant and wild-type strains Km (mm)a Strain Kinase genotype Glucose Fructose Low High Low High HSC HXKI HXK2 GLKI 2.2 (2) 5 (2) 4.6 (2) 44 (2) D38 hxkl hxk2 GLKI 1.4 (4) 42 (2) NDb 56 (2) D38.3 hxkl hxk2 glkl ND 51(6) ND 66 (2) a Numbers in parentheses indicate number of experiments. b ND, None detected. glucokinase (hxkl hxk2 GLKI); and strain D-38.3 lacks all three kinases (hxkl hxk2 glkl) (Yeast Genetic Stock Center, University of California, Berkeley, Calif.) The genotypes with respect to kinase are listed in Table 1. The wild-type and glucokinase-containing strains were maintained on YPG or YPGal 1% agar slants (containing 1% Difco yeast extract, 2% Bacto-Peptone [Difco Laboratories, Detroit, Mich.], and 2% glucose or 2% galactose, respectively). Cells used for transport studies were grown for 24 to 48 h at 3 C in a rotary shaker in 5-ml flasks containing 2 ml of YPGal, YPE, or YPGly liquid medium. These media substitute the 2% galactose with 2% ethanol (YPE) or 2% glycerol (YPGly). The cells were harvested by centrifugation, washed twice in distilled water, suspended to 4% (wet wt/vol), and kept at ice bath temperature until used. If cell suspensions of the kinaseless cells are stored overnight at C, they must be washed before use, since free glucose (i.e., glucose oxidasepositive material) accumulates in the external medium. The source of the glucose is not known; it might arise by trehalose hydrolysis. Transport experiments. Cell incubations were carried out at 23 C in 15-ml Corex centrifuge tubes usually containing 5% cell suspensions (5 mg (wet weight) per ml) in distilled water. The suspensions were mixed vigorously with a magnetic stirrer. The sugar concentrations used are specified in the individual experiments described below. Samples (usually.1 ml, containing 5 mg of cells) were removed at appropriate intervals and transferred to 5 ml of ice-cold distilled water over glass fiber filters (Reeve Angel 934 AH). The suspension was filtered by suction and washed with two 5-ml portions of ice-cold distilled water. The filters with the washed cells were transferred to scintillation vials. The radioactivity was measured in 5 ml of ACS counting fluid (Amersham Corp., Arlington Heights, Ill.) with a 333 Tri-Carb liquid scintillation spectrometer (Packard Instrument Co., Inc., Rockville, Md.). The intracellular sugar concentration was expressed as nanomoles per milligram (wet weight) of yeast cells or as nanomoles per microliter cell water. The latter was calculated by assuming that 1 mg (wet weight) of cells is equivalent to.5,ul of cell water (7). For conversion to other units, the following relations hold: 1 mg wet weight =.2 mg dry weight =.1 mg of protein. The initial velocity of sugar transport was measured in triplicate experiments during the linear phase of uptake; samples were taken at 15-s intervals for 45 s for nonmetabolized sugar and at 5-s intervals for a total of 15 s for metabolized sugar. For metabolized sugars (for the experiments described in Fig. 1 and Table 2), the cell density was reduced to.5 mg (wet weight) per ml. The initial velocity was expressed as nanomoles per milligram (Wet weight) of cells per minute at 23 C. The following nomenclature was used to describe the types of transport experiments involved (1, 31): zero trans influx for sugar uptake by cells not previously loaded with sugar; zero trans efflux for efflux from cells previously loaded with sugar into a sugar-free medium; equilibrium exchange influx for uptake of labeled sugar by cells previously equilibrated with unlabeled sugar at the same concentration; influx counterflow for uptake of labeled sugar by cells previously equilibrated with unlabeled sugar at a concentration higher than that used for uptake; efflux counterflow for the expulsion from cells of previously equilibrated labeled sugar induced by the addition of a competing sugar to the external medium. The Vmax and apparent Km from transport experiments were determined graphically from Eadie and Hofstee plots (14, 38). The Vma,, and apparent Km for the two components of the biphasic kinetics exhibited by metabolizing cells were determined graphically by the method of Rosenthal (26; see also reference 23). The assumption that the nonlinear kinetics represent the sum of two processes is justified by genetic evidence with kinaseless yeast mutants (2, 3). Materials. 3H- and 14C-labeled sugars were purchased from Amersham Corp., and New England Nuclear Corp., Boston, Mass. The ACS liquid scintillation fluid was from Amersham. The glass fiber filters, (Reeve Angel 934 AH) were from Arthur H. Thomas, Philadelphia, Pa. Constituents for growth media were from Difco Laboratories. All other chemical reagents were of the highest commercial grade available. RESULTS Glucose uptake by triple-kinaseless cells. Bisson and Fraenkel (2) have shown that Eadie-Hofstee plots of glucose uptake by cells containing any one of the cognate kinases show two components, corresponding to a high and low apparent Km process, whereas similar plots for triplekinaseless cells show only one component, which corresponds to the high apparent Km process of kinase-positive cells. Their observations have been confirmed in our laboratory and are illustrated in Fig. 1A. The two components of glucose uptake for wild-type cells correspond to apparent Kms of 2.5 and 65 mm. The apparent Km for the singlecomponent process for triple-kinaseless cells (Fig. 1B) is 6 mm, which corresponds to the high apparent Km process of the kinase-positive cells. The time course of D-glucose uptake by triple-kinaseless mutant cells is shown in Fig. 2; the intracellular concentration is expressed as the ratio of intracellular to extracellular concentration. The uptake data show that the rate at which the intracellular concentration reaches the steady state is markedly higher for 5 mm than for 1 mm glucose. Furthermore, the data of Fig. 2 show that irrespective of the extracellular glucose concentration, glucose is not accumulated against a concentration gradient. In fact, for all concentrations used (which includes a greater range than shown in Fig. 2), glucose equilibrates to about 6% of the calculated cell water. The markedly higher rate of equilibration of cells incubated with 5 mm versus 1 mm glucose, as well as the fact that glucose is not concentrated against a gradient, is characteristic of a facilitated diffusion process and has been observed for all the nonmetabolized monosaccharides taken up by S. cerevisiae (2-9, 15-19, 22, 25, 28-36). Equilibrium exchange and counterflow in triple-kinaseless cells. Facilitated diffusion processes are characterized by the phenomena of equilibrium exchange and counterflow (1, 15, 19, 32). These phenomena have been demonstrated for glucose uptake into triple-kinaseless mutant cells and are

3 2934 LANG AND CIRILLO presented in the following figures. Equilibrium exchange is demonstrated by a comparison of the uptake of labeled glucose from an external concentration of 1 mm into cells loaded by preincubation with 1 mm unlabeled sugar for 9 min and uptake into unloaded cells (Fig. 3). The trans stimulation of the uptake of labeled sugar under equilibriumn exchange conditions (Fig. 3) has been seen with external glucose concentrations from 1 to 1 mm (data not shown). Influx counterflow was induced by transferrng cells equilibrated with 25 mm unlabeled glucose to a medium containing labeled sugar at a concentration of 5 mm (Fig. 4). The characteristic overshoot of influx counterflow is clearly observed; the overshoot maximum was reached within 2 min, and the intracellular concentration of labeled glucose fell to the equilibrium value (i.e., C/IC = 1) after 3 min. Identical counterflow characteristics are observed irrespective of the growth substrate. (Note that in these experiments, the results are expressed as the concentration of label and not of total sugar.) The effect of temperature on the influx counterflow of glucose has been found to be similar to that reported earlier for galactose transport by the inducible galactose transport system of S. cerevisiae (19). Thus at 3 C the time course of influx counterflow was so rapid that by 1 min the overshoot maximum had already occurred, and only the downward portion of the counterflow curve is observed (Fig. 5). At C, a temperature at which zero trans influx is completely blocked, influx counterflow still took place, although at a lower rate; the overshoot maximum was not reached even after 3 min. A similar difference in the effect of temperature on zero trans versus influx counterflow has been observed for transport in S. cerevisiae (19), Escherichia coli (37) and erythrocytes (13, 2). V 5- A V/S V/S FIG. 1. Determination of Vmax and apparent Km for glucose uptake by wild-type and triple-kinaseless yeast cells. Eadie-Hofstee plot of [14C]glucose uptake from external glucose concentrations varying from.6 to 2 mm. Uptake by wild-type cells (A) was measured after a 1-s incubation of a.4% (wet wt/vol) cell suspension at 23 C;.1-ml samples (4. mg [wet weight] of cells) were removed, filtered over glass fiber filters, and counted in a scintillation counter. Uptake by triple-kinaseless cells (B) was measured after a 1- to 3-s incubation of a 4% cell suspension at 23 C;.1-ml samples (4 mg [wet weight]) were treated as above. The dashed line represents the high apparent Km component calculated for wild-type cells ii panel A. V.4 C /Co., MINUTES 1 mm FIG. 2. Time course of glucose uptake by triple-kinaseless cells. Cells (5 mg [wet weight] per ml) were incubated with the indicated concentrations of D-[14Clglucose at 23 C;.1-ml samples were removed at the indicated intervals, filtered, washed, and counted. The results are expressed as the ratio of intracellular (C,) to extracellular (CO) glucose concentration. The intracellular glucose concentration was calculated on the assumption that 1 mg (wet weight) of yeast cells contains.5,ul of cell water. Kinetic characteristics of glucose transport in kinaseless cells. The previous experiments show that in triplekinaseless cells, glucose is transported by a facilitated diffusion process. The kinetic characterization of facilitated diffusion processes requires the determination of the Vmax W llj -J C) 3o TIME (min) J. BACTERIOL. FIG. 3. Zero trans influx in triple-kinaseless mutant cells. Cells (5 mg [wet weight] per ml) were incubated with 1 mm labeled D-glucose (.2,uCi of ['4C]glucose per,umol) at 23 C;.1-ml samples (5 mg [wet weight] of cells) were removed at the indicated intervals, filtered, washed, and counted. For equilibrium exchange, a 5% cell suspensidn was incubated with 1 mm unlabeled glucose for 9 min at 3 C. Labeled glucose was added to a final specific activity of.2 ±Ci/p.mol; samples were removed, washed, and counted as indicated above. The dashed line indicates the intracellular glucose concentration for equilibration with the extracellular medium.

4 VOL. 169, 1987 GLUCOSE TRANSPORT IN KINASELESS S. CEREVISIAE 3 OC 1._ _ cr C,/C 2- I MINUTES FIG. 4. Influx counterflow in triple-kinaseless mutant cells grown on different substrates. Cells at 4 mg (wet weight) per ml were incubated for 9 niin at 23 C in 1 mm unlabeled D-glucose. Influx counterflow was initiated by dilution of a.1-ml sample of the unwashed cell suspension in 1.9 ml of a solution containing carrierfree D-[14C]glucose; the D-glucose concentration after dilution was 5 mm, and the concentration of labeled sugar was 2.5,Ci/ml. Samples (4 mg [wet weight]) were removed and processed as usual. The cells were grown on the following substrates 2% galactose (, ), 2% glycerol (A, A) and 2% ethanol ([). and the Km of glucose transport under three fundamental conditions: zero trans influx, zero trans efflux, and equilibrium exchange. Only the Vm, and apparent Km for zero trans influx and equilibrium exchange could be determined in an apparently unambiguous manner; the values are presented in Table 2. The ratio of apparent Km to Vmax for the two processes seem reasonably close, as expected for a simple carrier-mediated process (1, 32). If the uptake were indeed a simple carriermediated process, the ratio of apparent Km to Vn, for zero trans efflux should also be the same. However, despite many efforts, we have not been able to measure the parameters for zero trans efflux; the efflux process is anomalous, It is too fast to measure at the same temperature at which zero trans influx and equilibrium exchange are easily measurable. The loaded sugar is lost in two phases, of which the first is too fast to measure and the second is far too slow to be consistent with the operation of a simple carrier process. This anomalous behavior may be due to the accumulation of metabolic products during the preloading procedure. Thinlayer chromatography of cell extracts from preloaded cells showed a product which may be gluconate. We have tried to eliminate the problem by reducing the time of preloading, I- z w I I C i TIME (min) FIG. 5. Effect of temperature on zero trans influx and influx counterflow. For zero trans influx (), the 4% cell suspensions were incubated at either 3 or C as shown. The external glucose concentration was 1 mm; the concentration of label was.1,ci/ml. For influx counterflow (), the cells were incubated for 9 min at 3 C with 1 mm unlabeled D-glucose. Influx counterflow was initiated by dilution in 1 mm labeled D-glucose at either 3 or C. Sam,pling was carried out as usual. The data are expressed as the ratio of the concentration of labeled sugar (not total sugar) in the cell water (Ci) and the external medium. but without success. If the cells are loaded sufficiently to show influx counterflow, efflux is anomalous. Fructose uptake by wild-type cells and kinaseless mutants. Previous reports (2, 8) have described the dependence of low apparent Km fructose transport on the presence of the hexokinases in bakers' yeast as being similar to the dependence of low apparent Km glucose transport on its cognate kinases. This dependence is shown in Table 1, in which the apparent Kms for glucose and fructose are presented for wild-type cells (HSC), glucokinase-positive-hexokinasenegative mutants (D38), and triple-kinaseless mutants (D38.3). (Note that fructose is not a substrate of glucokinase.) As for glucose, fructose uptake shows high and low apparent Km components in cells containing hexokinase, but only one component, the high apparent Km process, in hexokinaseless mutants. We have also demonstrated that fructose uptake in hexokinaseless cells shows the same features of influx TABLE 2. Kinetic characteristics of glucose transport by the kinaseless mutant Km Transport mode (mm) (nmol/mg[wet KI/V. wt] per min) Zero trans influx Equilibrium exchange

5 2936 LANG AND CIRILLO counterflow in triple-kinaseless cells as previously described for glucose (data not shown). DISCUSSION The results of the present study confirm the involvement of the cognate kinases in the low apparent Km transport process for glucose and fructose. Thus in strains containing their cognate kinases, glucose and fructose are transported by a high and low apparent Km process but in strains lacking the appropriate kinases, they are transported only by the high apparent Km process. To evaluate the several hypotheses which have been proposed concerning the possible relationship between the high and low apparent Km process, we decided that it was necessary to characterize the high apparent Km process. We have chosen the low-affinity process of triple-kinaseless cells. The process of glucose transport in triple-kinaseless cells has most of the features of a carrier-mediated, facilitated diffusion process; these are energy-independent, nonconcentrative uptake; saturation kinetics for zero trans influx and equilibrium exchange; and counterflow. However, we cannot characterize the system as a simple carrier-mediated process, because of the anomalous efflux behavior. When glucose-loaded cells were diluted into sugar-free medium, sugar was lost so rapidly that we could not measure efflux kinetics accurately or reproducibly. A simple carrier process should exhibit symmetrical behavior below the apparent Km, for which the rate constant for both influx and efflux is given by the Vmj,/apparent Km ratio (1, 32). We have not been able to determine this owing to the anomalous efflux behavior. Whether this means that the mechanism is more complex than a simple carrier or is due to the accumulation of glucose oxidation products formed during the preloading procedure is not clear. Although these accumulation products do not prevent the measurement of equilibrium exchange and counterflow, which also occur with cells preloaded with glucose, they may affect the efflux process. A further indication that the mechanism is more complex than that of a simple carrier-mediated, facilitated diffusion is the inability of computer simulations to account for the kinetics and the extent of the counterflow overshoot seen in experiments such as those shown in Fig. 3. Using the Stein and Lieb (32) or Baker and Widdas (1) equation and the Vmax/apparent Km ratios from Table 2, we could find no value for the Vmax for zero trans efflux which could predict the observed rates or extent of the overshoot. The accumulated oxidation products may also be the cause of this anomalous behavior. However, the simple carrier model may simply be inadequate to account for glucose transport in yeast cells; it is inadequate to account for glucose transport in erythrocytes (for a review, see reference 3). The effect of temperature on counterflow kinetics shown in Fig. 5 is a reflection of the differential effects of temperature on equilibrium exchange and net efflux of the intracellular sugar load. The rapid course of counterflow at 3 C is the result of a high rate of both equilibrium exchange and net efflux. The time course of counterflow at C reflects the fact that at C net efflux is negligible, whereas equilibrium exchange occurs at a significant rate. The greater inhibitory effect of temperature on net efflux than on equilibrium exchange (and thus influx counterflow) is characteristic of sugar transport in such diverse systems as galactose transport in S. cerevisiae (19) and erythrocytes (13, 2) and lactose transport in E. coli (37). Although the present studies and those of Bisson and Fraenkel (2, 3) show that the kinases are involved in the low apparent Km mechanism of glucose transport, they do not distinguish among the several hypotheses which have been proposed for the relationship between the low and high apparent Km processes. The proposal that the carrier involved in the high apparent Km facilitated diffusion process is also involved in the low apparent Km process will be tested by using reconstituted membrane vesicles (1-12), which transport glucose with characteristics similar to that of the high apparent Km process described in the present communication (24). We are now in a position to ask whether the reconstituted high apparent Km carrier can be converted to a low apparent Km form by combination with either the appropriate kinases or sugar metabolites, as predicted by the hypotheses which propose that a common carrier is involved in both low and high apparent Km processes. We expect that these techniques, together with further genetic techniques including cloning of the carrier genes (3a, 33), will lead to a resolution of this controversy. ACKNOWLEDGMENTS J. BACTERIOL. We are happy to express our appreciation to Alexis Franzusoff for many useful discussions and suggestions during the course of this investigation and for his comments on the manuscript. We also thank Antonio H. Romano for making available to us the results of his experiments with 6-deoxy-D-glucose before they were published and to Linda Bisson and Dan Fraenkel for sharing their results on triple mutants before they were published; they played an important role in our experiments. We are happy to acknowledge the excellent technical assistance of Jane Uman and Sheng-Ping Hwang. We acknowledge the National Science Foundation (grant PCM-8-557) and the National Institutes of Health (Public Health Service grant A1262) for support of this work. LITERATURE CITED 1. Baker, G. F., and W. F. 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