Expression of Kinase-Dependent Glucose Uptake in Saccharomyces

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1 JOURNAL OF BACTERIOLOGY, Sept. 1984, p ol. 159, No /84/ $02.00/0 Copyright 1984, American Society for Microbiology Expression of Kinase-Dependent Glucose Uptake in Saccharomyces cerevisiae LINDA F. BISSON AND DAN G. FRAENKEL* Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts Received 17 February 1984/Accepted 4 June 1984 There are both low- and high-affinity mechanisms for uptake of glucose in Saccharomyces cerevisiae; highaffinity uptake somehow depends on the presence of hexose kinases (L. F. Bisson and D. G. Fraenkel, Proc. Natl. Acad. Sci. U.S.A. 80: , 1983; L. F. Bisson and D. G. Fraenkel, J. Bacteriol. 155: , 1983). We report here on the effect of culture conditions on the level of high-affinity uptake. The high-affinity component was low during growth in high concentrations of glucose (100 mm), increased as glucose was exhausted from the medium, and decreased again during prolonged incubation in the stationary phase. The higher level of uptake was found in growth on low concentrations of glucose (0.5 mm) and in growth on normal concentrations of galactose, lactate plus glycerol, or ethanol. These results suggest that some component of high-affinity uptake is repressible by glucose. A shift from medium with 100 mm glucose to medium with 5 mm glucose resulted in up to a 10-fold increase in the level of high-affinity uptake within 90 min; the increase did not occur in the presence of cycloheximide or 2,4-dinitrophenol or in buffer alone with low glucose, suggesting that protein synthesis or energy metabolism (or both) was required. Reimposition of the high glucose concentration caused loss of high-affinity uptake, a process not prevented by cycloheximide. The use of hexokinase single-gene mutants showed that the derepression of high-affinity uptake was not clearly correlated with changes in levels of the kinases themselves. These results place the phenomenon of high- and low-affinity uptake in a physiological context, in that high-affinity uptake seems to be expressed best in conditions where it might be needed. Apparent similarities between glucose uptake in yeast and animal cells are noted. We have previously reported (1) that the kinetics of uptake of glucose and fructose by Saccharomyces cerevisiae are influenced by the kinases for these sugars. Thus, mutant strains (hxkl hxk2) unable to phosphorylate fructose because they lack both hexokinase A (P1) and B (P2) took up fructose with the low-affinity Km of ca. 50 mm, whereas strains with one or both of these kinases also showed a high-affinity uptake component with a Km of ca. 5 mm. For glucose, which can also be phosphorylated by a third enzyme, glucokinase, a triple mutant strain (hxkl hxk2 glk) had only low-affinity uptake with a Km of ca. mm, whereas strains with any one of the three kinases also showed high-affinity uptake with a Km of 1 to 2 mm. The same phenomenon of kinase dependence was also observed with the nonmetabolized analog 6-deoxy-D-glucose, i.e., both low- and high-affinity uptake components when one of the three kinases was present, but only lowaffinity uptake in their absence (2). This result seems to show that the kinase-dependent high-affinity uptake is not merely a consequence of phosphorylation and metabolism of the substrate, but reflects some other role for the kinases in the uptake process. Neither the mechanism nor the function of kinase-dependent uptake is understood. One indication of function would be to find conditions affecting the expression of high-affinity uptake. Although the qualitative presence of high-affinity glucose uptake was observed in the wild-type strain grown either on glucose or on noncarbohydrate carbon sources (1), the cells were always obtained from growing cultures at an absorbancy at 580 nm (A580) of about 8. In the present report we have extended studies of the wild-type strain to the several phases of batch growth on glucose as well as to other * Corresponding author media. The main findings are that high-affinity glucose uptake is apparently a derepressible function, and that the derepression is not primarily a reflection of the levels of the kinases themselves. Situations causing loss of high-affinity glucose uptake are also described. MATERIALS AND METHODS Materials. Reagents were obtained from the following sources: D-[U-14C]glucose and D-[U-14C]fructose were from ICN Pharmaceuticals, Inc.; cycloheximide was from CalBiochem; 2,4-dinitrophenol, sodium azide, iodoacetic acid, tunicamycin, peroxidase-glucose oxidase, and o-dianisidine were from Sigma Chemical Co.; media constituents were from Difco Laboratories. All other chemicals were reagent grade. Strains. S. cerevisiae strains DFY1 (D585-11C) (a lysi MAL SUC) and the mutant lacking all three glucose phosphorylating enzymes, DFY437 (ot lysi leu2 hxkl hxk2 glk), have been described previously (1). The single gene hexokinase mutants DFY449 (hxkl leu2 trpl) and DFY450 (hxk2 lysi), carrying the same mutant alleles as DFY437, were kindly supplied by R. B. Walsh. Growth and uptake assay. Cells were grown at 30 C on a gyratory shaker in a buffered medium (ph 6.5) containing yeast extract, tryptone, and a major carbon source as indicated. Culture samples were filtered, washed, and suspended, and uptake (5 s) was assessed, all as described previously (1). Medium shift. Cultures of (usually) 0 ml at an A580 of about 1 (grown from an A580 of ) were filtered, washed three times in fresh medium without the added carbon source (or for shifts to buffer [0.1 M potassium phosphate, ph 6.5], washed with this buffer), and suspended to the original volume in medium with the indicated carbon source (or in buffer). During a subsequent incubation at

2 1014 BISSON AND FRAENKEL 30 C, samples were taken for the usual assay of sugar uptake. Kinase activity and glucose determination. Preparation of crude extracts and assay of kinase activities was as described previously (2). For glucose determination with glucose oxidase (Sigma), culture samples were first filtered by using a syringe and Gelman 0.45-,um filter unit. According to this assay, growth medium without added glucose contained 0.08 mm glucose. RESULTS ariation in uptake characteristics during batch culture. Uptake experiments were done with cells from the wild-type strain DFY1 as harvested at four different times from rich medium initially containing 2% glucose (Fig. 1). At least 10 divisions occurred before sample A (early logarithmic phase), and glucose was exhausted (Fig. 1, inset) some time between samples B (late logarithmic phase) and C (early stationary phase). For sample B, as reported earlier (cells from growth on glucose at an A580 of 8, glucose still present in the culture), two uptake components were observed with high affinity (the shallow slope) and low affinity (the steeper slope). However, the curves were different for the other samples. There was apparently less high-affinity uptake when the cells were harvested early with glucose still present in high concentration (sample A) than later (samples B and C), and the amount of this component declined again after extended incubation in the stationary phase (sample D). On the other hand, low-affinity uptake was evident in both samples from o ~ ~ ~ ~ ~ ~ o t A~~~~ detegthon 2%gucoe.henitalnm+ A B C l 10 C AA DA (hrso rime C B /S5 FIG. 1. Eadie-Hofstee plot of glucose uptake in samples from different times in growth on 2% glucose. The initial inoculum, according to dilution, had an A580 of Symbols: (0) A, early logarithmic phase (A580 Of 1.1); (0) B, late logarithmic phase (A580 Of 7.8); (A) C, early stationary phase (A580 of 16); (A) D, late stationary phase (4 days, A580 of 41). The insert shows the growth and glucose in the medium. J. BACTERIOL. TABLE 1. Hexose uptake in cells grown on different carbon sources Uptake (nmol/min per mg [wet wt] of cells)b Expt Carbon source A 580 at (initial % concn) harvest Glucose Fructose (0.5 (2.0 mm) mm) 1 Glucose (2) 1.1 (85)C 1.6 _d 7.8 (17) (NDG) (NDG) Glucose (2) Fructose (2) Glucose (0.02) 0.06 (1.0) (0.33) (0.11) (NDG) Glucose (0.1) 0.13 (4.8) (2.8) (1.5) (0.14) (NDG) Fructose (0.1) Ethanol (2) Lactate-glycerol (2/2) Galactose (2) a In all experiments the initial absorbance was sufficiently low to allow approximately 15 doublings to occur before the first sample was taken. b Uptake values are ±% with three to six experiments for each determination. Wet weight/dry weight ratios do not vary much throughout growth. C alues within parentheses are glucose concentration (millimolar) in the medium at the time of harvest. NDG, No detectable glucose remaining in the medium. d -, Not measured. growth on glucose (A and B), but decreased upon glucose exhaustion. For glucose, uptake at 0.5 mm is an approximate measure of the high-affinity system, since this concentration is about 1/2 of the high-affinity Km and 1/30 of the low-affinity Km. By this measure, the amount of high-affinity uptake seemed to increase by a factor of two- to threefold as glucose concentration diminished (Table 1, experiment 1). A similar phenomenon was also observed for uptake of fructose by glucose-grown cells (Table 1, experiment 2); 2 mm is a convenient concentration to measure high-affinity uptake of fructose. (We have already reported [1] that competition studies show both high- and low-affinity uptake systems to act on both glucose and fructose.) Growth on 2% fructose itself, with harvest at a low A580, gave the same level of highaffinity fructose uptake as growth on high glucose (Table 1, experiment 3). When glucose was present initially at low concentration (0.02% is ca. 1 mm), but the cells were harvested at such low density that glucose was still present, then characteristically high levels of high-affinity glucose uptake were found even with the earliest samples (Table 1, experiment 4). With growth on 0.1% glucose, the earliest sample showed a relatively low level of high-affinity uptake, but as glucose concentration fell the rate attained the higher level before an A580 of 1 (Table 1, experiment 5). Growth on low fructose concentration with early harvest also showed a high level of high-affinity uptake (Table 1, experiment 6).

3 OL. 159, 1984 KINASE-DEPENDENT GLUCOSE UPTAKE IN S. CEREISIAE 1015 When ethanol, glycerol plus lactate, or galactose was substituted for glucose or fructose, and the cells were sampled at low absorbancies so that the substrates would still be present near their original high concentrations, high values for high-affinity uptake were seen, similar to those from the cells in the late logarithmic phase on glucose (Table 1, experiments 7 through 9). Together, these results suggest that high-affinity uptake of glucose and fructose may be a "derepressible" function. In addition, there seems to be at least one other type of control, for its level was low in late-stationary-phase cultures in all the media (Table 1). The overall range of expression in the various conditions was about 10-fold. Derepression after transfer. When cells growing on 2% glucose at low density were collected by filtration, washed, and incubated in medium with 0.1% glucose, increases of 5- to 10-fold in high-affinity uptake occurred over the next 90 min (Fig. 2; Table 2, experiment 1). The derepression was marginal when buffer rather than medium was used (Table 2, experiment 2) and was prevented by cycloheximide, dinitrophenol, and azide (partially), but not by tunicamycin (Table 2, experiment 3). A similar transfer experiment with cells grown in galactose (and hence already derepressed) with 90-min incubation with either 0.1% galactose or 0.1% glucose gave no increase in the rate of high-affinity glucose uptake (Table 2, experiment 4). Conditions causing loss of high-affinity uptake. When 2% glucose was restored to the cultures derepressed in 0.1% glucose for 90 min, high-affinity uptake decreased to the original level in the subsequent 90 min; the decrease did occur in the presence of cycloheximide and also occurred in buffer (Fig. 3; Table 2, experiment 5). Iodoacetic acid is known to decrease affinity for glucose uptake (6, 24). The effect was also seen in the present system with the wild-type strain; a 1-h treatment with 5 mm iodoacetic acid caused a striking loss of high-affinity uptake (Fig. 4). It is not known whether the effects of high glucose (Fig. 3) and iodoacetic acid (Fig. 4) involve the same mechansim. Both treatments seem to leave low-affinity uptake intact. The insensitivity of low-affinity uptake to iodoacetic acid treatment is also shown by its lack of effect on glucose uptake in a triple kinase mutant strain DFY437, which has only low-affinity uptake (Fig. 4). 60 TABLE 2. Effect of transfer to different concentration of hexosea Time of Uptake of Culture of incubation bfr 0.5 lcs mm Expt origin (initial % Transferred before glucose concn) sampling (nmol/min for uptake per mg (min) [wet wt]) 1 Glucose (2) Not transferred % glucose % glucose % glucose % glucose % glucose % glucose Glucose (2) 2.0% glucose % glucose % glucose in M potassium phosphate 3 Glucose (2) 0.1% glucose 0 2.2b 0.1% glucose b 0.1% glucose plus NaN3 0.1% glucose plus dinitrophenol 0.1% glucose plus cycloheximide 0.1% glucose plus tunicamycin 4 Galactose (2) Not transferred % galactose % glucose Glucose (0.1), 2.0% glucose min 2.0% glucose % glucose % glucose plus cycloheximide 2.0% glucose in M potassium phosphate a In experiments 1 through 4, cells were taken from growth (A580 of 0.6 to 1.5) in medium with the indicated carbon sources, washed, and transferred at 0 min to fresh medium (or, experiment 2 entry 3, 0.1 M potassium phosphate, ph. 6.5) and incubated for the time specified, and culture samples were assayed for glucose uptake as usual. In experiment 3 the added agents were NaN3 (5mM), dinitrophenol (5 mm), cycloheximide (,ug/ml), and tunicamycin (10,ug/ml). (Controls for the latter experiments were to allow 90 min of derepression in the absence of the added agent and then add it for min before sampling for uptake; such treatment did not affect uptake (data not shown). However, 60 min of incubation in buffer in presence of dinitrophenol and sodium azide together did decrease high-affinity uptake by ca. 50%.) In experiment 5, cells from 2% glucose were first incubated in medium with 0.1% glucose for 90 min (as in experiments 1 through 3); 2% glucose was then added (0 min) and incubation was continued as indicated. baverage of four cultures la~~~l/ /s FIG. 2. Glucose uptake in cells shifted or not shifted from 2 to 0.1% (110 to 5 mm) glucose. Cells were grown in 2% glucose to an A580 of 1.3, harvested, and suspended at 0 min in fresh medium with either 2 or 0.1% glucose and reincubated. Uptake was assessed in samples taken during the reincubation. Symbols: (0) 2% glucose, 0 min; (O) 2% glucose, 90 min; (A) 0.1% glucose, 0 min; (0) 0.1% glucose, 35 min; (A) 0.1% glucose, 90 min. Is there a correlation between high-affinity uptake and level of hexokinases? Since high-affinity uptake seems to somehow depend on the presence of the cognate kinase, and since it is known that the expression of kinases depends to some degree on metabolic conditions (hexokinase P2 is "constitutive" and hexokinase P1 is repressible [11]), it was possible that the apparent derepression of high-affinity uptake might directly reflect changes in the level of the kinases during the various regimes. We tested this possibility for fructose uptake, since only hexokinases A (P1) and B (P2) are involved, and an indication of their individual levels is given by assay of fructose/ glucose phosphorylation ratios (the values being ca. 1 for

4 1016 BISSON AND FRAENKEL J. BACTERIOL. v/s FIG. 3. Glucose uptake in derepressed cells after readdition of 2% glucose. Cells from 2% glucose (0) were first shifted to 0.1% glucose for 90 min (0), 2% glucose was added, and the incubation was continued for (A) 45 min or (A) 90 min. Incubation for 180 min in 0.1% glucose gave the same curve as (0). hexokinase B and ca. 3 for hexokinase A). The wild-type strain DFY1 and two single gene mutant strains DFY449 (hxkl) and DFY450 (hxk2) were grown on 2% glucose and then incubated for 60 min with 0.1 or 2% glucose. Fructose uptake curves were then obtained (Fig. 5), and fructose/ glucose phosphorylation ratios were measured in extracts (Table 3). In both the wild-type strain and the hxkl mutant hexokinase B was the main isoenzyme in cells from the culture with 2% glucose. Its amount did not change much during the hour of incubation with 0.1% glucose; neither did the amount of hexokinase A in the wild-type strain. Nonetheless, in both strains the incubation in 0.1% glucose caused a marked increase in the rate of high-affinity fructose uptake (Fig. 5). For the hxk2 mutant strain, high-affinity fructose uptake was substantially higher in the 2% glucose culture than in the other two strains from that condition, but phosphorylation activity, which was clearly hexokinase A, was less. In this strain the treatment with 0.1% glucose increased both uptake and hexokinase A by a factor of about /S FIG. 4. Glucose uptake in cultures treated with iodoacetic acid. Cells were grown to an A580 of 8 (DFY1 in glucose and DFY437 in galactose-containing medium), 5 mm iodoacetic acid was added or not added, and samples were assessed for glucose uptake after a further 60 min of incubation. Symbols: (0) DFY1, no addition; (0) DFY1 plus iodoacetic acid; (A) DFY437, no addition; (A) DFY437 plus iodoacetic acid. /S FIG. 5. Fructose uptake in the wild type and single-gene hexokinase mutants shifted from 2 to 0.1 or 2% glucose for 60 min. A, DFY1 (wild type) (HXKI HXK2) taken at an A580 of 1.7; B, DFY449 (hxkl) taken at an A580 of 4; C, DFY450 (hxk2) taken at an A580 of 4. Symbols: (0) cells incubated with 2% glucose; (0) cells incubated with 0.1% glucose. These results, although not completely disproving it, do not support the idea that the component of high-affinity uptake whose induction or derepression is occurring in the present experiments is one of the kinases itself. The increased high-affinity uptake in the hxk2 mutant even in cells grown in 2% glucose may be another example of the known effect of hxk2 mutation on catabolite derepression (9, ). However, even in this strain incubation in medium with 0.1% glucose gave further derepression. DISCUSSION The main findings reported here are that (i) at least one component of high-affinity glucose uptake seems to be subject to derepression, (ii) high-affinity uptake is lost during incubation in high concentrations of glucose and is also lost in the stationary phase, and (iii) low-affinity uptake seems to be present maximally in conditions of growth in high concentrations of glucose. With respect to repressibility, the main conclusion to be drawn might be that it seems to accord with the physiology, in that high-affinity uptake is not expressed well in conditions where it might not be needed, i.e., growth in high concentrations of glucose. The expression of highaffinity uptake in low concentrations of glucose has obvious utility, and its equally good expression in derepressed conditions on other carbon sources might only reflect the fact that it is derepressible. This report does not directly address several obvious questions with respect to glucose uptake in yeast, but the findings bear on these questions. (i) Are the two types of TABLE 3. Levels of hexose phosphorylating activities after shift to 2 or 0.1% glucose' Strainucose in concn Strain Min Fructose phosphorylation Glucose phosphorylation preincubation (U/mg of protein) (U/mg of protein) DFY1 (wild type) DFY449 (hxkl) DFY450 (hxk2) a The same cultures as described in Fig. 5 activity. were assayed for enzyme

5 OL. 159, 1984 KINASE-DEPENDENT GLUCOSE UPTAKE IN S. CEREISIAE 1017 uptake, high affinity and low affinity, different systems or different fucntional forms of the same system? The present results would fit either possibility. (ii) Is uptake energy linked? Considerable earlier work accords with at least lowaffinity uptake being via facilitated diffusion (6, 13, 17). High-affinity uptake is also likely to be facilitated diffusion, as shown by lack of accumulation of 6-deoxyglucose above external level even when it employs high-affinity uptake (2). Also, a glucokinase clone (pglk) showed not only enhanced rates of 6-deoxyglucose influx, but also a more rapid rate of efflux, consistent with facilitated diffusion (2). Furthermore, metabolic inhibitors such as dinitrophenol showed no effect on 6-deoxyglucose uptake (data not presented). However, the question is difficult to resolve for glucose itself, which is rapidly phosphorylated after, or even concomitant with, entry. Nonetheless, neither of the two apparent uptake systems for glucose in S. cerevisiae resembles the derepressible high-affinity glucose uptake system of Neurospora crassa, which depends on proton motive force and is concentrative (22, 23). (iii) How does one account for the apparent need for glucose phosphorylating enzymes in high-affinity uptake? We have speculated (1) that this finding might either reflect an interaction of the kinases with the putative membrane carrier(s) or some enzymatic activity of the kinases (other than glucose phosphorylation) indirectly affecting uptake Km. The present results, that there are metabolic effects on expression of high-affinity uptake, lead one to consider a third type of explanation: that the lack of kinases might prevent derepression, rather than functioning, of the system. It is clear that the lack of kinases does not cause a general inability to derepress repressible functions in yeast-quite the contrary (9, )-but it is conceivable that the kinases have a role in derepression of a component in transport. In this regard the key question from the present work is, just what component is being derepressed? Since it seems unlikely to be one of the kinases, it is tempting to inquire whether it might be the membrane carrier itself. Finally, it should be mentioned that in some respects glucose uptake in yeast resembles glucose uptake in higher animal cells. In higher cells, glucose uptake is thought to involve facilitated diffusion (reviewed in reference 12); in some cells an apparent derepression occurs in glucose starvation (18, 25), and glucose also causes loss of uptake capacity (4, 5). (The latter phenomenon is also known for galactose transport in yeast [19].) By analogy with the mammalian systems, one also wonders whether any of the present findings for yeast might involve the movement of glucose carriers between compartments (16). With respect to kinase involvement in glucose uptake, it is also worth considering whether there might be an analogous phenomenon in higher cells. Hexokinase binding to mitochondria has been studied extensively (3, 10, 15, 21), but there are also reports of its binding to the plasma membrane (7, 8, 14). ACKNOWLEDGMENTS This work was supported by grant PCM from the National Science Foundation. LITERATURE CITED 1. Bisson, L. F., and D. G. Fraenkel Involvement of kinases in glucose and fructose uptake by Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 80: Bisson, L. F., and D. G. Fraenkel Transport of 6- deoxyglucose in Saccharomyces cerevisiae. J. Bacteriol. 155: Bustamante, E., and P. L. Pederson High aerobic glycolysis of rat hepatoma cells in culture: Role of mitochondrial hexokinase. Proc. Natl. Acad. 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