The uptake of 6-deoxyglucose was measured in wild-type Saccharomyces .... Since plots of 1/V versus ... of 6- deoxyglucose by cells grown in glycerol plus.
Vol. 155, No. 3
JOURNAL OF BACTERIOLOGY, Sept. 1983, p. 995-1000
0021-9193/83/090995-06$02.00/0 Copyright © 1983, American Society for Microbiology
Transport of 6-Deoxyglucose in Saccharomyces cerevisiae LINDA F. BISSON AND DAN G. FRAENKEL* and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 of Microbiology Department
Received 4 March 1983/Accepted 16 June 1983
The uptake of 6-deoxyglucose was measured in wild-type Saccharomyces cerevisiae, in a double mutant strain lacking activity for hexokinases A and B (hxkl hxk2), in a triple mutant strain lacking activity for both hexokinases and glucokinase (hxkl hxk2 glk), and in the triple mutant with high levels of activity of single kinases restored by introduction of the cloned genes. In the wild-type strain, uptake of the glucose analog showed two components, with Km values of ca. 20 mM ("high affinity") and 250 mM ("low affinity"), respectively. The double mutant also had high- and low-affinity components, but the triple mutant showed only low-affinity uptake. Reintroduction of the single kinases to the triple mutant restored high-affinity uptake. (Other experiments on 6-deoxyglucose uptake are also presented, including the apparent use of the galactose transport system when induced.) These results show that the recent implication of the kinases in transport of glucose (L. F. Bisson and D. G. Fraenkel, Proc. Natl. Acad. Sci. U.S.A. 80:1730-1734, 1983) applies equally to the nonmetabolized analog 6-deoxyglucose and suggests that the role of the kinases in transport is not merely a consequence of metabolism of the transported compound.
Although it is generally agreed that the uptake of glucose in Saccharomyces cerevisiae is not concentrative, there has been controversy over the nature of the transport system, for the results of three types of studies have been difficult to reconcile. Experiments primarily using glucose analogs and showing their equilibration and competition for entry seem to accord with facilitated diffusion (3, 6, 9), but experiments using metabolic inhibitors, such as iodacetic acid, have indicated that affinity for uptake may depend on metabolism (2, 13). Several studies have shown that the phosphorylated sugar may be found internally before the nonphosphorylated sugar is found (4, 7, 11). We have recently shown (1) that S. cerevisiae has at least two different types of transport system for glucose and fructose, distinguishable on the basis of apparent affinity, their Km values for glucose being ca. 2 mM ("high affinity") and 15 mM ("low affinity"). Furthermore, highaffinity transport depended on the ability to phosphorylate the sugar. There are three known enzymes for glucose phosphorylation in the 6 position, hexokinase A (P-I), hexokinase B (PII), and glucokinase; the first two enzymes also act on fructose. Strains without the first two enzymes lacked high-affinity uptake for fructose but retained it for glucose, whereas strains lacking all three enzymes had no high-affinity uptake for glucose either. The presence of multiple
transport systems might, in fact, account for some of the earlier and seemingly contradictory results. Thus, for example, the apparent metabolic effects on affinity might reflect physiological control of the kinase-dependent uptake, and results indicative of vectorial phosphorylation might primarily reflect entrance via the highaffinity systems in the low-substrate concentra-
tions of those experiments. Nonetheless, even if there are two types of uptake, one depending on the kinases and the other not, their mechanisms remain to be explained-particularly, the nature of kinase-dependent uptake. Two quite different speculative models (1) would be (i) that it is indeed metabolism of the substrate, by the kinases or subsequently, which accounts for kinase dependence or (ii) that the necessity for the kinases does not depend on their phosphorylating activity for the transported substrates, but on some other property, for example, phosphorylation of a different metabolite or perhaps a physical interaction with a cytoplasmic membrane hexose carrier affecting uptake. The purpose of the present work was to study the uptake of 6-deoxyglucose, which cannot be phosphorylated in the 6 position by the kinases. If 6-deoxyglucose is an appropriate analog of glucose, then a finding of both highand low-affinity components for its uptake, as well as dependence of the former component on the kinases, would favor model ii over model i. 995
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MATERIALS AND METHODS Materials. Reagents were obtained from the following sources: D-[U-14C]glucose, 6-deoxy-o-[G-3H]glucose, and D-[G-3H]galactose from New England Nuclear Corp.; 6-deoxy-D-glucose and benzamidine hydrochloride from Sigma Chemical Co.; glass microfiber and DE81 filter disks from Whatman Inc.; and medium constituents from Difco Laboratories. All other chemicals were reagent grade. Strains. The same strains were used as previously (1): wild-type strain DFY1 (D585-11C) (a lysi SUC MAL); the mutant strain lacking the two hexokinases but containing glucokinase, DFY436 (a lysi leu2 hxkl hxk2); the mutant strain also lacking glucokinase, DFY437 (a lysi leu2 hxkl hxk2 glkl); and DFY437 carrying plasmids pBW111, pBW112, and pBW113 for presumptive genes HXKI, HXK2, and GLK, respectively (14). Uptake of beld sugars. The method of uptake of radioactivity labeled sugars was as described previously (1), and, unless noted otherwise, incubation time was 5 s. The specific activity of the labeled 0-deoxy-Dglucose was ca. 10 ,uCi/,mol. For the sugar competition experiments, uptake was initiated by the addition of cells to the sugar mixture, and incubation time was also 5 s. Efflux of labeled sugars. For estimates of loss of sugars from cells, cells (0.1 ml) were incubated with a labeled substrate as for the uptake experiments for a period of 15 min. At the end of this time, 10 ml of 0.1 M potassium phosphate, pH 6.5, was added to the cell suspension and 1-ml samples were removed and treated as in the uptake experiments to assess the radioactivity remaining associated with the cells. Assays of kinase activity. Cells were grown as for the uptake experiments. Crude extracts for the determination of total glucose- and fructose-phosphorylating activity were prepared using glass beads as previously described (8), except that membrane debris was not removed from the extract by centrifugation and all buffers included 2 mM benzamidine hydrochloride as a protease inhibitor. The spectrophotometric assay based upon coupling of glucokinase or hexokinase activity to glcose-6phosphate dehydrogenase and phosphoglutose isomerase (for fructose) has been previously described (5). Protein was determined by the method of Bradford (la). For the determination of phosphorylation of 6deoxy-D-glucose, extracts were prepared as described above, except the extract buffer utilized was 50 mM triethanolamine (pH 7.4)-10 mM EDTA-2 mM 2mercaptoethanol-2 mM benzamidine hydrochloride. The extracts were incubated with labeled 0-deoxy-Dglucose (200 mM) (or with D-glucose [5 mM] (2 ,uCi/ pmol) and 1 mM ATP for 0, 1, 2, 5, 10, and 20 min at 30°C, in a final volume of 200 IlI. The reaction was stopped by placing the tubes in a boiling water bath for 2 min. Precipitated protein was cleared from the reaction mixture by centrifugation at 15,000 x gfor 15 min in an Eppendorf microfuge at 4°C. A sample of the supernatant (50 p,l) was spotted into DEAE filter disks (DE81), allowed to dry, and washed batchwise with several changes of deionized distilled water (500 ml). The filters were placed in scintillation vials, and the radioactivity was determined as for the uptake experiments.
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V/S (nmo/e/mli/rng wet wtl/(mM} FIG. 1. Eadie-Hofstee plot of the uptake of 6deoxy-D-glucose in lactate-glycerol-grown cells. Cells were grown to the log phase (absorbance at 580 nm of 5 to 8) in rich medium (1) containing 2% lactate and 2% glycerol. The dashed line represents a substrate concentration of 10 mM. Symbols: 0, DFY1; 0, DFY436; A DFY437. (Inset) 6-Deoxy-D-glucose uptake in galactose-grown DFY437.
RESULTS Uptake of 6-deoxyglucose by wild-type and kinase-defident srains. Since plots of 1/V versus 1/S emphasize lower substrate concentrations, in the present work, as before (1), we utilized plots of V versus V/S to reveal multicomponent transport. For 6-deoxyglucose uptake in the wild-type strain DFY1, there were uptake components of (at least) two different affinities, as reflected in the apparently biphasic plots in Fig. 1. Similar biphasic plots were observed for 6deoxyglucose uptake in strain DFY436, which lacks both hexokinases but contains glucokinase and grows on glucose. However, in the strain lacking all three kinases, DFY437, a linear plot was obtained, indicating only low-affinity uptake. In such plots, the slopes represent affinities, and we will provisionally refer to the shallow and steep components as high-affinity and lowaffinity uptake, respectively. Data from several experiments are collected in Table 1. In wildtype and double-kinase mutant strains the two Km values differed by a factor of ca. 10, being for the wild type 20 and 250 mM. For the triplekinase mutant the low-affinity uptake Km was ca. 500 mM. This result of high- and low-affinity uptake in strains with a kinase for glucose but only low affinity uptake in the strain without a kinase is analogous to that reported for glucose itself in the same set of strains, one difference
VOL. 155, 1983
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TABLE 1. Km values of 6-deoxyglucose uptake Strain
Km (mM)a
DFY1 ................. 20 t 8 (4); 250 ± 120 (4) DFY436 hxkl hxk2 ...... 9 ± 3 (3); 180 ± 70 (3) DFY437 hxkl hxk2 glk ................. 500 ± 270 (6) DFY437(pBW111) HXKI ............... 40 t 25 (3) DFY437(pBW112) HXK2 ............... 40 ± 25 (3) DFY437(pBW113) GLK ................ 30 ± 20 (3); 400 ± 250 (3) a Approximate Km values were determined from the quasi-linear portions of the curves in Fig. 3 and 4, with standard deviations, and number of experiments indicated in parentheses. When two values are given, they refer to the two slopes of the biphasic curves.
being that for glucose the two Km values were ca. 2 and 15 mM (1). The evident analogies between the characteristics of the uptake of 6-deoxyglucose and glucose do not, of course, establish that the two substances utilize the same uptake systems. Indeed, Kotyk et al. (10) showed that 6-deoxyglucose is apparently a substrate of the inducible galactose transport system, with a far lower Km value (3 mM) than its uptake by the constitutive glucose system (Km of 500 mM). Along the same lines, we observed (Fig. 1, insert) that 6-deoxyglucose uptake in the triple-kinase mutant strain differed when the cells had been grown on galactose instead of on glycerol plus lactate; instead of a straight line, the curve was biphasic, with Km components of ca. 30 and 100 mM. Competition studies with the triple-kinase mutant are shown in Fig. 2. The uptake of 6deoxyglucose by cells grown in glycerol plus lactate was insensitive to competition by galactose, whereas in galactose-grown cells the uptake of 6-deoxyglucose could be inhibited ca. 50%o by galactose, as though in the galactosegrown cells the galactose uptake system was responsible for about one half of 6-deoxyglucose uptake (Fig. 2A). Glucose competed equally well with 6-deoxyglucose uptake in both cases. Galactose caused little inhibition of glucose uptake, whereas glucose caused substantial inhibition of galactose uptake in galactose-grown cells '(Fig. 2B). The simplest interpretation of these results is that for cells not grown on galactose, 6deoxyglucose and glucose may use the same uptake systems, which are not employed very effectively by galactose. For cells grown on galactose, it appears as though the galactoseinducible uptake system can also be employed by 6-deoxyglucose, but not employed readily by glucose; nonetheless, the uptake of galactose or 6-deoxyglucose by the galactose system is readi-
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FIG. 2. Inhibition of sugar uptake in DFY437. (A) Inhibition of 6-deoxy-D-glucose uptake by glucose and galactose. Cells were grown to an absorbance at 580 nm of 10 in medium containing 2% galactose or 2% each of lactate and glycerol. The uptake of 6-deoxy-Dglucose (2 mM; 40 ,uCi/Lmol) in the presence of increasing concentrations of glucose or galactose was determined. Symbols: 0, galactose inhibition in lactate-glycerol-grown cells; 0, galactose inhibition in galactose-grown cells; A\, glucose inhibition in lactateglycerol-grown cells; A, glucose inhibition in galactose-grown cells. (B) Sugar inhibition in galactosegrown cells. Symbols: 0, galactose inhibition of glucose (1 mM; 2 ,uCi/,umol) uptake; 0, glucose inhibition of galactose (1 mM; 2 ,Ci/pmol) uptake.
ly competed by glucose. (A similar conclusion, that glucose effectively inhibited galactose entry by the galactose system, has been reached in other studies [M. Banieulos and D. G. Fraenkel, unpublished data].) It may also be noted that although Heredia et al. have shown that galactose can use the glucose uptake system (6), its Km was about 1 M, so this competition would not have been observed at the highest concentrations employed in the experiments shown in Fig. 2 (50 mM). The above results show, therefore, that although the uptake of 6-deoxyglucose is somewhat complicated by its apparent employment of the galactose transport system (when induced), 6-deoxyglucose does seem to act as an analog of glucose and to employ both high- and low-
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TABLE 2. Activities of glucose and fructose phosphorylation in the strainsa Strain
DFY1 (wild type) DFY436 hxkl hxk2 DFY437 hxkl hxk2 glk
Sp act (U/mg of protein) Glucose Fructose phosphorylation phosphorylation
1.1 0.12
