- Email: [email protected]
Kinetic analysis of glucose transport in wild-type and transporter-deficient Saccharomyces cerevisiae strains under glucose repression and derepression
Journal of Biotechnology, 27 (1992) 47-57 © 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1656/92/$05.00
47
BIOTEC 00844
Kinetic analysis of glucose transport in wild-type and transporter-deficient Saccharomyces cerevisiae strains under glucose repression and derepression C. W r e d e a B. V61ker a, H. Kiintzel b and G.F. F u h r m a n n a a Department of Pharmacology and Toxicology, Philipps-Universitiit Marburg, Germany, and b Max-Planck-lnstitut fi~r Experimentelle Medizin, G6ttingen, Germany (Received 26 September 1991; accepted 15 December 1991)
Summary Glucose transport in Saccharomyces cerevisiae is under regulatory control by glucose. After transition from glucose repression to derepression, the affinity of facilitated glucose transport into the cells increases from K m values around 7 mM to 1-2 mM. This change is accompanied by a significant increase of Vm under these conditions. Published glucose-transport data obtained from the transporter-deficient single mutants snf3, hxt2 and the double mutant snf3,hxt2 (Kxuckeberg and Bisson, 1990) were evaluated by computer-assisted nonlinear regression analysis, which indicates that additional glucose transporters are encoded by other genes different from SNF3 and HXT2. Regulation of glucose transport under glucose repression and derepression requires the presence of the SNF3 gene.
Saccharomyces cerevisiae; Glucose transport; Mutants snf3, t~t2; Repression; Derepression
Introduction Glucose repression and derepression in Saccharomyces cerevisiae cells is a rather complex regulatory mechanism affecting the expression of many genes and cellular systems (reviewed by Fiechter et al., 1981, and Zimmermann, 1992, this issue). Correspondence to: G.F. Fuhrmann, Department of Pharmacology and Toxicology, Philipps-Universit~it Marburg, Karl-von-Frisch-Strasse, 3550 Marburg, Germany.
48 Glucose repression and derepression effects have been reported in glucose transport experiments in Saccharornyces ceret;isiae cells (Bisson and Fraenkel, 1984; Bisson et al., 1987; Bisson, 1988; Fuhrmann et al., 1989; Does and Bisson, 1989; Kruckeberg and Bisson, 1990; Fuhrmann and V61ker, 1992, this issue). However, there are main differences in evaluation and interpretation of the kinetics of glucose transport in Saccharornyces cerel.,isiae between the laboratory of Bisson and that of Fuhrmann. There are different working hypotheses concerning kinetics after repression and derepression of the glucose carrier. In many experiments on initial glucose uptake, analysed graphically by EadieHofstee plots, two pseudolinear slopes are obtained. In the laboratory of Bisson these two slopes are representative for two glucose-carrier systems, namely a 'high-affinity' system with a K m of 1 to 2 mM and a constitutive 'low-affinity' system with a K m of about 20 mM. In the laboratory of Fuhrmann evaluation of initial glucose uptake kinetics was done by computer-assisted nonlinear regression analysis (Fuhrmann and V61ker, 1992). By this analysis there are two transport forms apparent; one demonstrates Michaelis-Menten kinetics and the second can be described by a first-order term, which we interpret as simple diffusion. Our mathematical analysis demonstrates, that the graphical analysis used in nonlinear Eadie-Hofstee plots causes significant deviations from the true kinetics especially for the so-called 'low-affinity' system ( K m around 20 mM), which in our interpretation rather represents a diffusion. Expression of the SUC2 (invertase) gene is regulated only by glucose repression and is modulated over a greater than 200-fold range (Neigeborn and Carlson, 1984). The SUC2 gene encodes both intracellular and secreted forms of invertase; the latter enzyme is responsible for the extracellular hydrolysis of sucrose and the trisaccharide raffinose. Six mutants have been studied (snfl through snf6); the name snf (sucrose non fermenting) is used only loosely, since all these mutants exhibited decreased levels of secreted invertase activity, and many of them were able to grow on sucrose. Thus, the six genes (SNF1 through SNF6) are essential for regulation of SUC2 expression. A special interesting feature of the snf3 mutants was, that all these mutants were more defective in growth on sucrose and raffinose than would have been predicted from the presence of invertase activity. This fact led to the suggestion, that the snf3 mutants might be defective in uptake of glucose and fructose, which are released by the extracellular hydrolysis of sucrose and raffinose (Neigeborn and Carlson, 1984). Neigeborn et al. (1986) have cloned the SNF3 gene by complementation and demonstrated linkage of the cloned DNA to the chromosomal SNF3 locus. The gene encodes a 3-kb poly(A)-containing RNA, which was five-fold more abundant in cells deprived of glucose. The SNF3 gene was disrupted to create null mutations (snf3). Disruption resulted in growth phenotypes consistent with a defect in glucose uptake. Surprisingly, gene disruption did not cause aberrant regulation of SUC2 expression as was observed in the above missense mutations. Here we demonstrate characteristic changes in affinity and Vm under glucose repression and derepression. This regulation is impaired by eliminating the SNF3 function. Furthermore, we showed that after elimination of the SNF3 function
49 (our experiments and our mathematical analysis of data of the following authors) there is still glucose transport with K m values around 2 to 4 mM. This result is superficially in accordance with the presence of the so-called 'high-affinity' slope in Eadie-Hofstee plots for some snf3 mutants (strain with a genetic background DFY1 (Bisson, 1988) and strains LBY411 and LBY416 (Kruckeberg and Bisson, 1990)), but not for the snf3-72 mutant strain MCY657 (Bisson et al., 1987).
Materials and Methods
Strains and growth conditions.
The strains of Saccharomyces cerevisiae used in this study were: H 1022 (wild strain, obtained from E T H Zfirich) H K 2 MA Ta ura3 his3 H K 3 MATa / M A T a ura3 /ura3 his3~ + leu2 / + H K 60 MA Ta snf3 :: HIS3 ura3 his3 The wild strain H K 2 was used for disruption of the SNF3 gene. The cells were grown in batch cultures under aeration at 30°C. The composition of the growth medium was 2% peptone, 1% yeast extract and 2% carbohydrate. Growth phases were determined by counting the cells in a Neubauer-counting chamber.
Vesicle preparation. For vesicle preparation cells were harvested by centrifugation at the middle of the exponential growth phase. Plasma membrane vesicles were prepared as described by Kreutzfeldt and Fuhrmann (1984). Countertransport experiments. Countertransport experiments with 2% plasma membrane vesicles were carried out under ice bath conditions in 0.4 M KC1 solution adjusted to p H 4.5. As shown by Fuhrmann et al. (1976) the plasma membrane vesicles are only sealed between pH 4 and 5 and diffusion of glucose is negligibly small in countertransport experiments. The amount of glucose in vesicles was analysed by 14C labeled glucose. Separation of the vesicles was done by the Millipore filter technique (Fuhrmann et al., 1976 and Kreutzfeldt and Fuhrmann, 1984). The techniques and mathematics of countertransport are described in detail by Fuhrmann et al. (1989). The K m value was calculated according to Wilbrandt and Rosenberg (1961) at the tmax of countertransport by: K m = [S i -- ( R i / R o ) S o ] / [ ( R i / R o ) - 1] S i, S o and Ri, Ro are the concentrations of unlabeled and labeled glucose inside (i) and outside (o) the vesicles.
Initial glucose uptake experiments. Before measuring glucose uptake the cells were washed three times in ice-cold distilled water and three times in 100 mM sodium phosphate buffer, pH 6.5. The cytocrit was adjusted to 10% cells, Kinetic studies
50 for glucose transport were conducted in the concentration range of 0.5-200 mM (0.075 to 6/xCi /xmo1-1 D-[U-14C]glucose) and a cell density of 50 mg wet weight per ml. Glucose uptake was started by addition of 0.1 ml radioactively labeled glucose solution to 0.1 ml of 10% cells at 25°C. After exactly 5 s uptake was stopped by addition of 10 ml ice-cold 200 mM sorbitol solution, cells were filtered on a glass fiber filter (GF 92, Schleicher and Schuell, Dassel, F R G ) and rinsed with two 10 ml portions of ice-cold distilled water. The filters with the cells were immersed into 5 ml scintillation cocktail (Rotiscint eco plus, Roth, Karlsruhe, F R G ) and counted in a Beckman LS 6000IC scintillation counter.
Analysis of transport kinetics. Experimental data were analysed by computer assisted nonlinear regression analysis using GraphPAD software (Motulsky, 19851989). Materials. Peptone from casein and yeast extract were purchased from E. Merck, Darmstadt, FRG. D-[U-14C]Glucose, specific activity 300 mCi mmo1-1, was obtained from Amersham Buchler, Braunschweig, FRG. All other chemicals were of analytical grade quality.
Results
Glucose countertransport and efflux in plasma membrane vesicles prepared from repressed and derepressed cells The galactose transport system in Saccharomyces cerevisiae is very inefficient and, consequently, growth on galactose prevents glucose repression (Fiechter et al., 1981; Fuhrmann et al., 1989; Fuhrmann and V61ker, 1992). Therefore we used this sugar in our studies to derepress cells. Repressed cells were grown on high glucose concentrations and derepressed cells on galactose up to the middle of the exponential growth phase. The countertransport experiments in vesicles prepared from repressed and derepressed H K 2 cells are depicted in Fig. l a and b. As previously shown (Fuhrmann et al., 1989) in vesicles prepared from H 1022 cells, the vesicles prepared from repressed H K 2 cells also demonstrate a low countertransport maximum (tmax) of 3.1 _+ 0.08 SE ( R i / R o, Fig. la). In vesicles prepared from galactose derepressed cells tma x is significantly increased to a value of 5.5 + 0.49 SE (Ri/Ro, Fig. lb). The tm~ values are related to the affinity of the transport system. A high /max is proportional to high affinity and a low tmax to low affinity. A similar result is further obtained in countertransport experiments with vesicles prepared from another wild strain H K 3 (countertransport experiments not shown). Glucose countertransport and efflux in plasma membrane vesicles prepared from a snf3 mutant grown on high glucose or galactose The countertransport experiment with vesicles prepared from the snf3 mutant (strain H K 60), which was grown under repressed (glucose) and derepressed (galactose) conditions up to the middle of the exponential growth phase, is depicted in Fig. 2a and b. In contrast to the countertransport experiments in
51 I0
(a)
i I e o I
Ln" x
o-
II
0
. . . .
'
5
. . . . . . . . .
~0
'
.
.
.
.
.
.
15 20 minutes
I0
.
.
.
.
.
.
25
.
.
30
(b)
m i m
x
5
u
0
5
I0
~5 20 minutes
25
30
Fig. 1. Glucose countertransport and effiux in plasma membrane vesicles prepared from HK 2 cells. (a) The cells were grown to the middle of the exponential growth phase on glucose (2% in the medium) and in (b) on galactose (2% in the medium). R i / R o is the ratio of 14C-labeled glucose concentration inside to outside; S i is the inside concentration of unlabeled glucose. Mean values of 5 (a) and 4 (b) experiments _+SE.
vesicles from the strain H K 2 (Fig. la and b) there is no significant difference in tmax noticeable when the ceils were grown repressed and derepressed. In both cases a relatively high affinity can be assumed from the high tmax of 5.6 ± 0.18 SE and 6.38 + 0.40 SE (Fig. 2a and b). In Fig. 3 the K m values have been calculated from the corresponding tmax values and unlabeled glucose inside and outside concentrations (Si, S o) at the tmax. In vesicles p r e p a r e d from cells of H 1022, H K 3 and H K 2 the effects of glucose repression are clearly visible by the large changes in K m values from 8.8 ___0.8 SE, 7.5 + 0.2 SE and 6.9 _+ 1.6 SE m M to galactose derepressed K m values of 2.8 _+ 0.2 SE, 2.7 _+ 0.4 SE and 4.4 _+ 0.6 SE mM. In contrast to these results a high affinity is present under repressed and derepressed conditions in the snf3 mutant and the Krn values are not significantly different with 3.7 _+ 0.2 SE m M to 3.5 _+ 0.8 SE mM.
The effect of glucose repression and derepression in initial uptake experiments in H 1022 cells Since glucose transport, as shown by the countertransport experiments, occurs by facilitated diffusion (net flux = influx-effiux) it is necessary to keep the uptake
52 10
(a)
m i i o I
x
o
~
5
o
II
0
5
10
20
t5
25
30
minutes 10
(b)
i I m o I
unx
o
qm II
5
10
15 20 minutes
25
30
Fig. 2. (a and b) Glucose countertransport and effiux in plasma membrane vesicles prepared from a snf3 mutant. Conditions are given in Fig. la and b. Mean values of 4 experiments_+ SE.
]
repressed
10
sed
0 H t022
HK 3
HK 2
HK 60 (snf3-)
Fig. 3. Bar graph of K m values calculated from the countertransport maximum values (plasma membrane vesicles prepared from H 1022, HK 3, HK 2 and snf3 cells) according to Wilbrandt and Rosenberg (1961). Mean values _+SE of 4 to 5 experiments.
period short enough (5 s) in order to create no significant effiux (initial uptake experiments, Fuhrmann and V61ker, 1992). As already recalculated for the DFY1 strain (Fuhrmann and V61ker, 1992) in two independent experiments in 2%
53 TABLE 1 Nonlinear regression analysis of initial glucose uptake in H 1022 cells from early exponential, late exponential, early stationary and late stationary growth phases Strain H 1022
Condition early exp. late exp. early stat. late stat.
Km
Vm
4.03 + 0.53 1.91 +0.41 1.60± 0.26 2.49 + 0.33
5.85 + 0.51 17.69+ 1.91 27.83 _+2.22 18.41 _+1.44
Kd 0.027 + 0.004 0.031 +0.016 0.043 + 0.020 0.012 ± 0.011
in mM, Vm in nmol glucose mg I min-1 cells and Ka in/zl mg-1 rain i cells. Standard error (SE) is estimated by GraphPAD (Motulsky, 1985-1989).
Km
glucose grown cells the K m values decreased from 6.3 and 5.2 mM in the early exponential phase to high affinity values when glucose was used up to K m values of 2.4 and 1.2 mM. A similar result is obtained with the strain H 1022 (Table 1). The K m value decreased from 4 mM in the early exponential phase to 1.9 mM in the late exponential, to 1.6 mM in the early stationary and 2.5 mM in the late stationary phase. The increase in Vm during the first three phases and the decrease in the late stationary phase is typical for the regulatory behaviour of glucose in glucose transport of Saccharomyces cerevisiae (Fuhrmann and V61ker, 1992). In addition to Michaelis-Menten transport the kinetic analysis demonstrates a firstorder term, which we have assumed to be diffusion ( K d = diffusion constant).
The effect of glucose repression and derepression in initial uptake experiments in wild-type, hxt2 and snf3 strain cells Instead of graphical analysis of initial uptake experiments by Eadie-Hofstee plots, the experimental data of Kruckeberg and Bisson (1990) were analysed by computer-assisted nonlinear regression analysis. For this purpose their experimental data points were digitized with the help of a Hewlett-Packard-Plotter and the G r a p h P A D computer program (Motulsky, 1985-1989). Then V/S-values (velocity of transport/glucose concentration) in the Eadie-Hofstee plots were transformed to S values. By choosing adequate equations (Michaelis-Menten and first-order term) given in the computer program the V versus S values were fitted by nonlinear regression and a new curve was calculated and fitted to the data points. In Table 2 our recalculated values for Kin, Vm and K d of the experimental data of Kruckeberg and Bisson (1990) are given. The HXT2 ( h e x o s e transporter) gene of Saccharomyces cereuisiae was identified by Kruckeberg and Bisson (1990) on the basis of its ability to complement the defect in glucose transport of a snf3 mutant. In order to achieve derepression, the cells were shifted to low glucose concentrations of 0.05% for 90 min and for repression to 2% glucose for 150 min. The strain with the intact HXT2 and SNF3 genes behaved similarly in K m and Vm changes by derepression and repression as did DFY1, H 1022, HK 3 and HK 2 strains. The hxt2 mutant showed about normal changes in K m o n derepression and repression, but no significant increase in Vm on derepression, and the snf3 mutant had a decreased Vr, under derepression. The double mutant hxt2,snf3
54 TABLE 2 Nonlinear regression analysis of initial glucose uptake in strain LBY410 (HXT2 SNF3), LBY413 (hxt2 SNF3), LBY411 (HXT2 snf3) and LBY416 (hxt2 snf3) grown under low glucose (0.05%) and high glucose (2%). Strain
Condition
HXT2 SNF3 HXT2 SNF3
low glucose high glucose
hxt2 SNF3 hxt2 SNF3
Km
Vm
Kd
2.85 _+0.78 7.25 + 1.72
11.49 + 2.13 5.82 _+1.06
0.106 + 0.037 0.029 + 0.009
low glucose high glucose
3.34 +_0.22 13.34 + 2.30
6.74 + 0.29 7.93 + 1.16
0.008 _+0.002 0.013 + 0.007
HXT2 snf3 HXT2 snf3
low glucose high glucose
2.81 + 0.42 6.68 + 1.35
4.39 + 0.45 7.71 _+1.13
0.013 + 0.006 0.005 + 0.006
hxt2 snf3 hxt2 snf3
low glucose high glucose
1.88 + 0.49 2.37 _+0.36
2.48 _+0.43 3.87 + 0.43
0.024 + 0.010 0.065 + 0.010
The experimental data points were taken from Fig. 8A-D of Kruckeberg and Bisson (1990). Parameters as in Table 1. s h o w e d d e r e p r e s s e d a n d r e p r e s s e d a high affinity with K m values o f 1.9 and 2.4 m M a n d very low Vm values of 2.5 a n d 3.9 n m o l m g - ~ m i n - ~ cells. Thus, this d o u b l e m u t a n t still h a d glucose t r a n s p o r t activity, b u t was i m p a i r e d in glucose regulation.
Discussion T h e S N F 3 g e n e o f Saccharomyces cerevisiae has b e e n s e q u e n c e d by C e l e n z a et al. (1988). It c o d e s for a p r o t e i n of 97 k D a which is 28% h o m o l o g o u s to m a m m a l i a n glucose t r a n s p o r t e r s a n d to Escherichia coli a r a b i n o s e - H + a n d xyloseH + t r a n s p o r t e r s . T h e h y d r o p h o b i c i t y profile i n d i c a t e s twelve p u t a t i v e m e m b r a n e s p a n n i n g r e g i o n s similar to o t h e r sugar t r a n s p o r t e r s , a n d the g a l a c t o s i d a s e - a s s a y with SNF3-1acZ fusion p r o t e i n s u g g e s t e d a cell surface p r o t e i n , which is u n d e r glucose r e p r e s s i o n c o n t r o l (Bisson, 1988; C e l e n z a et al., 1988). A n o t h e r g e n e H X T 2 of Saccharomyces cerevisiae was i d e n t i f i e d by the ability of c o m p l e m e n t i n g t h e s o - c a l l e d ' h i g h - a f f i n i t y ' glucose t r a n s p o r t of snf3 m u t a n t s ( K r u c k e b e r g a n d Bisson, 1990). T h e h o m o l o g y o f the p r e d i c t e d a m i n o acid seq u e n c e to S N F 3 is 31% a n d to t h e g a l a c t o s e c a r r i e r gene, G A L 2 , 65%. T h e H X T 2 g e n e c o d e s for a p r o t e i n of 59.8 k D a with also twelve m e m b r a n e s p a n n i n g domains. T h e p o s s i b l e functions o f the S N F 3 a n d t h e H X T 2 gene p r o d u c t s in glucose t r a n s p o r t w e r e m a i n l y d e r i v e d f r o m e v a l u a t i o n o f kinetics by g r a p h i c a l analysis of n o n l i n e a r E a d i e - H o f s t e e plots (Bisson et al., 1987; Bisson, 1988; K r u c k e b e r g a n d Bisson, 1990). O n e m a i n i n d i c a t i o n for the c a r r i e r functions of t h e s e g e n e p r o d u c t s was t h e study of initial glucose u p t a k e in cells which w e r e grown u n d e r glucose r e p r e s s i o n ( 2 % glucose) a n d u n d e r glucose d e r e p r e s s i o n (0.05% glucose). T h e
55 results of Kruckeberg and Bisson (1990) are interpreted by the authors in the following way: " W h e n LBY410 (HXT2 SNF3; Fig. 8A) was grown on high glucose, only low-affinity transport (Km around 20 mM) was apparent, whereas a significant high-affinity transport system ( K m 1-2 mM) was evident after the cells were shifted to low glucose for 90 min, as expected. Transport by LBY413 (hxt2 SNF3; Fig. 8B) was not significantly different from that by the wild-type grown on high glucose. However, this strain lacked a major component of the high-affinity system after being shifted to low glucose. LBY411 (HXT2 snf3; Fig. 8C) was also deficient in a component of the high-affinity system when shifted to low glucose, as previously reported (Bisson et al., 1987). Furthermore, the residual high-affinity system of LBY411 was not significantly repressed in cells from high-glucose medium. In the double null strain LBY416 (hxt2 snf3; Fig. 8D), high-affinity transport was severely diminished. The presence of residual high-affinity transport in cells from high glucose was also seen in this strain. Patterns of transport kinetics identical to the pattern for LBY413 were observed for LBY406 (the original hxt2::LEU2 disruptant) and for LBY414, which is isogenic to LBY413. Similarly, other wild-type and snf3 strains gave results identical to those for the strains of the genotypes depicted in Fig. 8." We have also analysed, but by computer-assisted nonlinear regression analysis, the experimental data of Kruckeberg and Bisson (1990). As can be seen from our analysis (Table 2), there is no constitutive 'low-affinity' system with a K m value around 20 mM detectable in the strain LBY410 (HXT2 SNF3) as well as in the other mutants. Instead of this our mathematical analysis is consistent with a diffusion with the K d values given in the Table. The LBY410 (HXT2 SNF3) strain grown under glucose repression and derepression conditions showed K m values of 7.2 mM and 2.8 mM. This change in K m values is characteristic for glucose repression and derepression (Fuhrmann et al., 1989; Fuhrmann and V61ker, 1992; Fig. 1, Fig. 3 and Table 1). Also the increase in the Vm value by a significant factor of about 2 under derepression in comparison to repression is typical for many wild-type strains (example H 1022 Table 1, and reviewed by Fuhrmann and V61ker, 1992). This analysis clearly shows, that our method allows a precise description of the experimental data by the parameters given. As can be followed from the authors' description of their results in LBY410 (HXT2 SNF3) and other wild strains (Kruckeberg and Bisson, 1990) under glucose repression and derepression, there is no agreement with our analysis. The only qualitative description which might come close is that for the 'high-affinity' system ( K m 1-2 mM), but for example the K m changes under repression and derepression conditions were not calculated. Further the qualitative descriptions given by Kruckeberg and Bisson (1990) for the mutants hxt2 SNF3 (LBY413), HXT2 snf3 (LBY411) and the double mutant hxt2,snf3 (LBY416) in respect to the 'low-' and 'high-affinity' system are not reflected for the first and only superficially for the second system in our quantitative analysis (Table 2). The important facts described here are: In strain hxt2 SNF3 no significant increase in the Vm value under derepression occurs and for the
56 mutant HXT2 snf3 there is even a slight decrease in V,n noticeable under derepression. The double mutant hxt2 snf3 behaves quite unexpected by fact that the Vm value of 2.5 nmol mg 1 m i n - 1 is very low under derepression and does not further decrease under repression condition. As in the mutant HXT2 snf3 the reverse takes place: in both strains the Vm values increase on repression (see Table 2). The affinity, which remains a t K m values around 2 m M is further very remarkable in the mutant hxt2,snf3 and points to the presence of other high affinity transporters. The absence of typical changes in K m values are also documented in the snf3 strain investigated in Figs. 2 and 3. These results were obtained in countertransport experiments with plasma m e m b r a n e vesicles. Plasma m e m b r a n e vesicles from cells grown glucose repressed or galactose derepressed exhibited a relatively high affinity with K m values not significantly different, namely 3.7 and 3.5 mM. F u h r m a n n and V61ker (1992) also analysed data of another snf3-72 mutant strain MCY657 (Bisson et al., 1987, initial uptake experiments) under glucose repression and derepression and obtained no significant changes in Vm values, but K m changes of 7.3 to 4.0 mM. The wild-type SNF3 strain MCY638 (Bisson, 1988) demonstrated under glucose repression and derepression a significant 3-fold increase in Vm and the K m showed a slight decrease from 4.2 to 3.3 mM. Thus, from the glucose transport studies in snf3 mutants defects in regulation of glucose transport are obvious. Although the SNF3 and HXT2 gene products due to their primary structure (Celenza et al., 1988; Kruckeberg and Bisson, 1990) are probably involved in glucose transport, they remain to be identified kinetically as glucose transporters. The data of Bisson (1988) and Kruckeberg and Bisson (1990), as well as our mathematical analysis of them and our transport experiments suggest the presence of glucose transporters with K m values around 2 m M in strains lacking functional SNF3 a n d / o r HXT2 genes. On the other hand we could demonstrate that regulation of glucose transport capacity requires an intact SNF3 gene. The most important observation is that the typical increase in Vm on derepression is abolished in snf3 strains and that the characteristic affinity change under glucose repression and derepression can be absent.
Acknowledgments Supported by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie. It is a great pleasure to thank Professor K.J. Netter for his most valuable comments and suggestions.
References Bisson, L.F. and Fraenkel, D.G. (1984) Expression of kinase-dependent glucose uptake in Saccharomyces cerecisiae. J. Baeteriol. 159, 1013-1017.
57 Bisson, L.F., Neigeborn, L., Carlson, M. and Fraenkel, D.G. (1987) The SNF3 gene is required for high-affinity glucose transport in Saccharomyces cerevisiae. J. Bacteriol. 169, 1656-1662. Bisson, LF. (1988) High-affinity glucose transport in Saccharomyces cerevisiae is under general glucose repression control. J. Bacteriol. 170, 4838-4845. Celenza, J.L., Marshall-Carlson, L. and Carlson, M. (1988) The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein. Proc. Natl. Acad. Sci. USA 85, 2130-2134. Does, A.L. and Bisson, L.F. (1989) Comparison of glucose uptake kinetics in different yeasts. J. Bacteriol. 171, 1303-1308. Fiechter, A., Fuhrmann, G.F. and Kiippeli, O. (1981) Regulation of glucose metabolism in growing yeast cells. Adv. Microbiol. Physiol. 22, 123-183. Fuhrmann, G.F., Boehm, C. and Theuvenet, A,P.R. (1976) Sugar transport and potassium permeability in yeast plasma membrane vesicles. Biochim. Biophys. Acta 433, 583-596. Fuhrmann, G.F., V61ker, B., Sander, S. and Potthast, M. (1989) Kinetic analysis and simulation of glucose transport in plasma membrane vesicles of glucose-repressed and derepressed Saccharornyces ceret.,isiae cells. Experientia 45, 1018-1023. Fuhrmann, G.F. and V61ker, B. (1992) Regulation of glucose transport in Saccharomyces ceret~isiae. J. Biotechnol. 27, 1-15. Kreutzfeldt, Ch. and Fuhrmann, G.F. (1984) Sugar transport in Saccharomyces cere~'isiae H 1022. Swiss Biotechnol. 2, 24-27. Kruckeberg, A.L. and Bisson, L.F. (1990) The HXT2 gene of Saccharornyces ceret,isiae is required for high-affinity glucose transport. Mol. Cell. Biol. 10, 5903-5913. Motulsky, H.J. (1985-1989) GraphPAD software, San Diego CA, USA. Neigeborn, L. and Carlson, M. (1984) Genes affecting the regulation of SUC2 gene expression in Saccharomyces ceret,isiae. Genetics 108, 845-858. Neigeborn, L., Schwartzberg, P., Reid, R. and Carlson, M. (1986) Null mutations in the SNF3 gene of Saccharomyces cerevisiae cause different phenotype than do in previously isolated missense mutations. Mol. Cell. Biol. 6, 3569-3574. Wilbrandt, W. and Rosenberg, T. (1961) The concept of carrier transport and its corollaries in pharmacology. Pharmacol. Rev. 13, 109-183. Zimmermann, F.K. (1992) Glycolytic enzymes as regulatory factors. J. Biotechnol. 27, 17-26.
47
BIOTEC 00844
Kinetic analysis of glucose transport in wild-type and transporter-deficient Saccharomyces cerevisiae strains under glucose repression and derepression C. W r e d e a B. V61ker a, H. Kiintzel b and G.F. F u h r m a n n a a Department of Pharmacology and Toxicology, Philipps-Universitiit Marburg, Germany, and b Max-Planck-lnstitut fi~r Experimentelle Medizin, G6ttingen, Germany (Received 26 September 1991; accepted 15 December 1991)
Summary Glucose transport in Saccharomyces cerevisiae is under regulatory control by glucose. After transition from glucose repression to derepression, the affinity of facilitated glucose transport into the cells increases from K m values around 7 mM to 1-2 mM. This change is accompanied by a significant increase of Vm under these conditions. Published glucose-transport data obtained from the transporter-deficient single mutants snf3, hxt2 and the double mutant snf3,hxt2 (Kxuckeberg and Bisson, 1990) were evaluated by computer-assisted nonlinear regression analysis, which indicates that additional glucose transporters are encoded by other genes different from SNF3 and HXT2. Regulation of glucose transport under glucose repression and derepression requires the presence of the SNF3 gene.
Saccharomyces cerevisiae; Glucose transport; Mutants snf3, t~t2; Repression; Derepression
Introduction Glucose repression and derepression in Saccharomyces cerevisiae cells is a rather complex regulatory mechanism affecting the expression of many genes and cellular systems (reviewed by Fiechter et al., 1981, and Zimmermann, 1992, this issue). Correspondence to: G.F. Fuhrmann, Department of Pharmacology and Toxicology, Philipps-Universit~it Marburg, Karl-von-Frisch-Strasse, 3550 Marburg, Germany.
48 Glucose repression and derepression effects have been reported in glucose transport experiments in Saccharornyces ceret;isiae cells (Bisson and Fraenkel, 1984; Bisson et al., 1987; Bisson, 1988; Fuhrmann et al., 1989; Does and Bisson, 1989; Kruckeberg and Bisson, 1990; Fuhrmann and V61ker, 1992, this issue). However, there are main differences in evaluation and interpretation of the kinetics of glucose transport in Saccharornyces cerel.,isiae between the laboratory of Bisson and that of Fuhrmann. There are different working hypotheses concerning kinetics after repression and derepression of the glucose carrier. In many experiments on initial glucose uptake, analysed graphically by EadieHofstee plots, two pseudolinear slopes are obtained. In the laboratory of Bisson these two slopes are representative for two glucose-carrier systems, namely a 'high-affinity' system with a K m of 1 to 2 mM and a constitutive 'low-affinity' system with a K m of about 20 mM. In the laboratory of Fuhrmann evaluation of initial glucose uptake kinetics was done by computer-assisted nonlinear regression analysis (Fuhrmann and V61ker, 1992). By this analysis there are two transport forms apparent; one demonstrates Michaelis-Menten kinetics and the second can be described by a first-order term, which we interpret as simple diffusion. Our mathematical analysis demonstrates, that the graphical analysis used in nonlinear Eadie-Hofstee plots causes significant deviations from the true kinetics especially for the so-called 'low-affinity' system ( K m around 20 mM), which in our interpretation rather represents a diffusion. Expression of the SUC2 (invertase) gene is regulated only by glucose repression and is modulated over a greater than 200-fold range (Neigeborn and Carlson, 1984). The SUC2 gene encodes both intracellular and secreted forms of invertase; the latter enzyme is responsible for the extracellular hydrolysis of sucrose and the trisaccharide raffinose. Six mutants have been studied (snfl through snf6); the name snf (sucrose non fermenting) is used only loosely, since all these mutants exhibited decreased levels of secreted invertase activity, and many of them were able to grow on sucrose. Thus, the six genes (SNF1 through SNF6) are essential for regulation of SUC2 expression. A special interesting feature of the snf3 mutants was, that all these mutants were more defective in growth on sucrose and raffinose than would have been predicted from the presence of invertase activity. This fact led to the suggestion, that the snf3 mutants might be defective in uptake of glucose and fructose, which are released by the extracellular hydrolysis of sucrose and raffinose (Neigeborn and Carlson, 1984). Neigeborn et al. (1986) have cloned the SNF3 gene by complementation and demonstrated linkage of the cloned DNA to the chromosomal SNF3 locus. The gene encodes a 3-kb poly(A)-containing RNA, which was five-fold more abundant in cells deprived of glucose. The SNF3 gene was disrupted to create null mutations (snf3). Disruption resulted in growth phenotypes consistent with a defect in glucose uptake. Surprisingly, gene disruption did not cause aberrant regulation of SUC2 expression as was observed in the above missense mutations. Here we demonstrate characteristic changes in affinity and Vm under glucose repression and derepression. This regulation is impaired by eliminating the SNF3 function. Furthermore, we showed that after elimination of the SNF3 function
49 (our experiments and our mathematical analysis of data of the following authors) there is still glucose transport with K m values around 2 to 4 mM. This result is superficially in accordance with the presence of the so-called 'high-affinity' slope in Eadie-Hofstee plots for some snf3 mutants (strain with a genetic background DFY1 (Bisson, 1988) and strains LBY411 and LBY416 (Kruckeberg and Bisson, 1990)), but not for the snf3-72 mutant strain MCY657 (Bisson et al., 1987).
Materials and Methods
Strains and growth conditions.
The strains of Saccharomyces cerevisiae used in this study were: H 1022 (wild strain, obtained from E T H Zfirich) H K 2 MA Ta ura3 his3 H K 3 MATa / M A T a ura3 /ura3 his3~ + leu2 / + H K 60 MA Ta snf3 :: HIS3 ura3 his3 The wild strain H K 2 was used for disruption of the SNF3 gene. The cells were grown in batch cultures under aeration at 30°C. The composition of the growth medium was 2% peptone, 1% yeast extract and 2% carbohydrate. Growth phases were determined by counting the cells in a Neubauer-counting chamber.
Vesicle preparation. For vesicle preparation cells were harvested by centrifugation at the middle of the exponential growth phase. Plasma membrane vesicles were prepared as described by Kreutzfeldt and Fuhrmann (1984). Countertransport experiments. Countertransport experiments with 2% plasma membrane vesicles were carried out under ice bath conditions in 0.4 M KC1 solution adjusted to p H 4.5. As shown by Fuhrmann et al. (1976) the plasma membrane vesicles are only sealed between pH 4 and 5 and diffusion of glucose is negligibly small in countertransport experiments. The amount of glucose in vesicles was analysed by 14C labeled glucose. Separation of the vesicles was done by the Millipore filter technique (Fuhrmann et al., 1976 and Kreutzfeldt and Fuhrmann, 1984). The techniques and mathematics of countertransport are described in detail by Fuhrmann et al. (1989). The K m value was calculated according to Wilbrandt and Rosenberg (1961) at the tmax of countertransport by: K m = [S i -- ( R i / R o ) S o ] / [ ( R i / R o ) - 1] S i, S o and Ri, Ro are the concentrations of unlabeled and labeled glucose inside (i) and outside (o) the vesicles.
Initial glucose uptake experiments. Before measuring glucose uptake the cells were washed three times in ice-cold distilled water and three times in 100 mM sodium phosphate buffer, pH 6.5. The cytocrit was adjusted to 10% cells, Kinetic studies
50 for glucose transport were conducted in the concentration range of 0.5-200 mM (0.075 to 6/xCi /xmo1-1 D-[U-14C]glucose) and a cell density of 50 mg wet weight per ml. Glucose uptake was started by addition of 0.1 ml radioactively labeled glucose solution to 0.1 ml of 10% cells at 25°C. After exactly 5 s uptake was stopped by addition of 10 ml ice-cold 200 mM sorbitol solution, cells were filtered on a glass fiber filter (GF 92, Schleicher and Schuell, Dassel, F R G ) and rinsed with two 10 ml portions of ice-cold distilled water. The filters with the cells were immersed into 5 ml scintillation cocktail (Rotiscint eco plus, Roth, Karlsruhe, F R G ) and counted in a Beckman LS 6000IC scintillation counter.
Analysis of transport kinetics. Experimental data were analysed by computer assisted nonlinear regression analysis using GraphPAD software (Motulsky, 19851989). Materials. Peptone from casein and yeast extract were purchased from E. Merck, Darmstadt, FRG. D-[U-14C]Glucose, specific activity 300 mCi mmo1-1, was obtained from Amersham Buchler, Braunschweig, FRG. All other chemicals were of analytical grade quality.
Results
Glucose countertransport and efflux in plasma membrane vesicles prepared from repressed and derepressed cells The galactose transport system in Saccharomyces cerevisiae is very inefficient and, consequently, growth on galactose prevents glucose repression (Fiechter et al., 1981; Fuhrmann et al., 1989; Fuhrmann and V61ker, 1992). Therefore we used this sugar in our studies to derepress cells. Repressed cells were grown on high glucose concentrations and derepressed cells on galactose up to the middle of the exponential growth phase. The countertransport experiments in vesicles prepared from repressed and derepressed H K 2 cells are depicted in Fig. l a and b. As previously shown (Fuhrmann et al., 1989) in vesicles prepared from H 1022 cells, the vesicles prepared from repressed H K 2 cells also demonstrate a low countertransport maximum (tmax) of 3.1 _+ 0.08 SE ( R i / R o, Fig. la). In vesicles prepared from galactose derepressed cells tma x is significantly increased to a value of 5.5 + 0.49 SE (Ri/Ro, Fig. lb). The tm~ values are related to the affinity of the transport system. A high /max is proportional to high affinity and a low tmax to low affinity. A similar result is further obtained in countertransport experiments with vesicles prepared from another wild strain H K 3 (countertransport experiments not shown). Glucose countertransport and efflux in plasma membrane vesicles prepared from a snf3 mutant grown on high glucose or galactose The countertransport experiment with vesicles prepared from the snf3 mutant (strain H K 60), which was grown under repressed (glucose) and derepressed (galactose) conditions up to the middle of the exponential growth phase, is depicted in Fig. 2a and b. In contrast to the countertransport experiments in
51 I0
(a)
i I e o I
Ln" x
o-
II
0
. . . .
'
5
. . . . . . . . .
~0
'
.
.
.
.
.
.
15 20 minutes
I0
.
.
.
.
.
.
25
.
.
30
(b)
m i m
x
5
u
0
5
I0
~5 20 minutes
25
30
Fig. 1. Glucose countertransport and effiux in plasma membrane vesicles prepared from HK 2 cells. (a) The cells were grown to the middle of the exponential growth phase on glucose (2% in the medium) and in (b) on galactose (2% in the medium). R i / R o is the ratio of 14C-labeled glucose concentration inside to outside; S i is the inside concentration of unlabeled glucose. Mean values of 5 (a) and 4 (b) experiments _+SE.
vesicles from the strain H K 2 (Fig. la and b) there is no significant difference in tmax noticeable when the ceils were grown repressed and derepressed. In both cases a relatively high affinity can be assumed from the high tmax of 5.6 ± 0.18 SE and 6.38 + 0.40 SE (Fig. 2a and b). In Fig. 3 the K m values have been calculated from the corresponding tmax values and unlabeled glucose inside and outside concentrations (Si, S o) at the tmax. In vesicles p r e p a r e d from cells of H 1022, H K 3 and H K 2 the effects of glucose repression are clearly visible by the large changes in K m values from 8.8 ___0.8 SE, 7.5 + 0.2 SE and 6.9 _+ 1.6 SE m M to galactose derepressed K m values of 2.8 _+ 0.2 SE, 2.7 _+ 0.4 SE and 4.4 _+ 0.6 SE mM. In contrast to these results a high affinity is present under repressed and derepressed conditions in the snf3 mutant and the Krn values are not significantly different with 3.7 _+ 0.2 SE m M to 3.5 _+ 0.8 SE mM.
The effect of glucose repression and derepression in initial uptake experiments in H 1022 cells Since glucose transport, as shown by the countertransport experiments, occurs by facilitated diffusion (net flux = influx-effiux) it is necessary to keep the uptake
52 10
(a)
m i i o I
x
o
~
5
o
II
0
5
10
20
t5
25
30
minutes 10
(b)
i I m o I
unx
o
qm II
5
10
15 20 minutes
25
30
Fig. 2. (a and b) Glucose countertransport and effiux in plasma membrane vesicles prepared from a snf3 mutant. Conditions are given in Fig. la and b. Mean values of 4 experiments_+ SE.
]
repressed
10
sed
0 H t022
HK 3
HK 2
HK 60 (snf3-)
Fig. 3. Bar graph of K m values calculated from the countertransport maximum values (plasma membrane vesicles prepared from H 1022, HK 3, HK 2 and snf3 cells) according to Wilbrandt and Rosenberg (1961). Mean values _+SE of 4 to 5 experiments.
period short enough (5 s) in order to create no significant effiux (initial uptake experiments, Fuhrmann and V61ker, 1992). As already recalculated for the DFY1 strain (Fuhrmann and V61ker, 1992) in two independent experiments in 2%
53 TABLE 1 Nonlinear regression analysis of initial glucose uptake in H 1022 cells from early exponential, late exponential, early stationary and late stationary growth phases Strain H 1022
Condition early exp. late exp. early stat. late stat.
Km
Vm
4.03 + 0.53 1.91 +0.41 1.60± 0.26 2.49 + 0.33
5.85 + 0.51 17.69+ 1.91 27.83 _+2.22 18.41 _+1.44
Kd 0.027 + 0.004 0.031 +0.016 0.043 + 0.020 0.012 ± 0.011
in mM, Vm in nmol glucose mg I min-1 cells and Ka in/zl mg-1 rain i cells. Standard error (SE) is estimated by GraphPAD (Motulsky, 1985-1989).
Km
glucose grown cells the K m values decreased from 6.3 and 5.2 mM in the early exponential phase to high affinity values when glucose was used up to K m values of 2.4 and 1.2 mM. A similar result is obtained with the strain H 1022 (Table 1). The K m value decreased from 4 mM in the early exponential phase to 1.9 mM in the late exponential, to 1.6 mM in the early stationary and 2.5 mM in the late stationary phase. The increase in Vm during the first three phases and the decrease in the late stationary phase is typical for the regulatory behaviour of glucose in glucose transport of Saccharomyces cerevisiae (Fuhrmann and V61ker, 1992). In addition to Michaelis-Menten transport the kinetic analysis demonstrates a firstorder term, which we have assumed to be diffusion ( K d = diffusion constant).
The effect of glucose repression and derepression in initial uptake experiments in wild-type, hxt2 and snf3 strain cells Instead of graphical analysis of initial uptake experiments by Eadie-Hofstee plots, the experimental data of Kruckeberg and Bisson (1990) were analysed by computer-assisted nonlinear regression analysis. For this purpose their experimental data points were digitized with the help of a Hewlett-Packard-Plotter and the G r a p h P A D computer program (Motulsky, 1985-1989). Then V/S-values (velocity of transport/glucose concentration) in the Eadie-Hofstee plots were transformed to S values. By choosing adequate equations (Michaelis-Menten and first-order term) given in the computer program the V versus S values were fitted by nonlinear regression and a new curve was calculated and fitted to the data points. In Table 2 our recalculated values for Kin, Vm and K d of the experimental data of Kruckeberg and Bisson (1990) are given. The HXT2 ( h e x o s e transporter) gene of Saccharomyces cereuisiae was identified by Kruckeberg and Bisson (1990) on the basis of its ability to complement the defect in glucose transport of a snf3 mutant. In order to achieve derepression, the cells were shifted to low glucose concentrations of 0.05% for 90 min and for repression to 2% glucose for 150 min. The strain with the intact HXT2 and SNF3 genes behaved similarly in K m and Vm changes by derepression and repression as did DFY1, H 1022, HK 3 and HK 2 strains. The hxt2 mutant showed about normal changes in K m o n derepression and repression, but no significant increase in Vm on derepression, and the snf3 mutant had a decreased Vr, under derepression. The double mutant hxt2,snf3
54 TABLE 2 Nonlinear regression analysis of initial glucose uptake in strain LBY410 (HXT2 SNF3), LBY413 (hxt2 SNF3), LBY411 (HXT2 snf3) and LBY416 (hxt2 snf3) grown under low glucose (0.05%) and high glucose (2%). Strain
Condition
HXT2 SNF3 HXT2 SNF3
low glucose high glucose
hxt2 SNF3 hxt2 SNF3
Km
Vm
Kd
2.85 _+0.78 7.25 + 1.72
11.49 + 2.13 5.82 _+1.06
0.106 + 0.037 0.029 + 0.009
low glucose high glucose
3.34 +_0.22 13.34 + 2.30
6.74 + 0.29 7.93 + 1.16
0.008 _+0.002 0.013 + 0.007
HXT2 snf3 HXT2 snf3
low glucose high glucose
2.81 + 0.42 6.68 + 1.35
4.39 + 0.45 7.71 _+1.13
0.013 + 0.006 0.005 + 0.006
hxt2 snf3 hxt2 snf3
low glucose high glucose
1.88 + 0.49 2.37 _+0.36
2.48 _+0.43 3.87 + 0.43
0.024 + 0.010 0.065 + 0.010
The experimental data points were taken from Fig. 8A-D of Kruckeberg and Bisson (1990). Parameters as in Table 1. s h o w e d d e r e p r e s s e d a n d r e p r e s s e d a high affinity with K m values o f 1.9 and 2.4 m M a n d very low Vm values of 2.5 a n d 3.9 n m o l m g - ~ m i n - ~ cells. Thus, this d o u b l e m u t a n t still h a d glucose t r a n s p o r t activity, b u t was i m p a i r e d in glucose regulation.
Discussion T h e S N F 3 g e n e o f Saccharomyces cerevisiae has b e e n s e q u e n c e d by C e l e n z a et al. (1988). It c o d e s for a p r o t e i n of 97 k D a which is 28% h o m o l o g o u s to m a m m a l i a n glucose t r a n s p o r t e r s a n d to Escherichia coli a r a b i n o s e - H + a n d xyloseH + t r a n s p o r t e r s . T h e h y d r o p h o b i c i t y profile i n d i c a t e s twelve p u t a t i v e m e m b r a n e s p a n n i n g r e g i o n s similar to o t h e r sugar t r a n s p o r t e r s , a n d the g a l a c t o s i d a s e - a s s a y with SNF3-1acZ fusion p r o t e i n s u g g e s t e d a cell surface p r o t e i n , which is u n d e r glucose r e p r e s s i o n c o n t r o l (Bisson, 1988; C e l e n z a et al., 1988). A n o t h e r g e n e H X T 2 of Saccharomyces cerevisiae was i d e n t i f i e d by the ability of c o m p l e m e n t i n g t h e s o - c a l l e d ' h i g h - a f f i n i t y ' glucose t r a n s p o r t of snf3 m u t a n t s ( K r u c k e b e r g a n d Bisson, 1990). T h e h o m o l o g y o f the p r e d i c t e d a m i n o acid seq u e n c e to S N F 3 is 31% a n d to t h e g a l a c t o s e c a r r i e r gene, G A L 2 , 65%. T h e H X T 2 g e n e c o d e s for a p r o t e i n of 59.8 k D a with also twelve m e m b r a n e s p a n n i n g domains. T h e p o s s i b l e functions o f the S N F 3 a n d t h e H X T 2 gene p r o d u c t s in glucose t r a n s p o r t w e r e m a i n l y d e r i v e d f r o m e v a l u a t i o n o f kinetics by g r a p h i c a l analysis of n o n l i n e a r E a d i e - H o f s t e e plots (Bisson et al., 1987; Bisson, 1988; K r u c k e b e r g a n d Bisson, 1990). O n e m a i n i n d i c a t i o n for the c a r r i e r functions of t h e s e g e n e p r o d u c t s was t h e study of initial glucose u p t a k e in cells which w e r e grown u n d e r glucose r e p r e s s i o n ( 2 % glucose) a n d u n d e r glucose d e r e p r e s s i o n (0.05% glucose). T h e
55 results of Kruckeberg and Bisson (1990) are interpreted by the authors in the following way: " W h e n LBY410 (HXT2 SNF3; Fig. 8A) was grown on high glucose, only low-affinity transport (Km around 20 mM) was apparent, whereas a significant high-affinity transport system ( K m 1-2 mM) was evident after the cells were shifted to low glucose for 90 min, as expected. Transport by LBY413 (hxt2 SNF3; Fig. 8B) was not significantly different from that by the wild-type grown on high glucose. However, this strain lacked a major component of the high-affinity system after being shifted to low glucose. LBY411 (HXT2 snf3; Fig. 8C) was also deficient in a component of the high-affinity system when shifted to low glucose, as previously reported (Bisson et al., 1987). Furthermore, the residual high-affinity system of LBY411 was not significantly repressed in cells from high-glucose medium. In the double null strain LBY416 (hxt2 snf3; Fig. 8D), high-affinity transport was severely diminished. The presence of residual high-affinity transport in cells from high glucose was also seen in this strain. Patterns of transport kinetics identical to the pattern for LBY413 were observed for LBY406 (the original hxt2::LEU2 disruptant) and for LBY414, which is isogenic to LBY413. Similarly, other wild-type and snf3 strains gave results identical to those for the strains of the genotypes depicted in Fig. 8." We have also analysed, but by computer-assisted nonlinear regression analysis, the experimental data of Kruckeberg and Bisson (1990). As can be seen from our analysis (Table 2), there is no constitutive 'low-affinity' system with a K m value around 20 mM detectable in the strain LBY410 (HXT2 SNF3) as well as in the other mutants. Instead of this our mathematical analysis is consistent with a diffusion with the K d values given in the Table. The LBY410 (HXT2 SNF3) strain grown under glucose repression and derepression conditions showed K m values of 7.2 mM and 2.8 mM. This change in K m values is characteristic for glucose repression and derepression (Fuhrmann et al., 1989; Fuhrmann and V61ker, 1992; Fig. 1, Fig. 3 and Table 1). Also the increase in the Vm value by a significant factor of about 2 under derepression in comparison to repression is typical for many wild-type strains (example H 1022 Table 1, and reviewed by Fuhrmann and V61ker, 1992). This analysis clearly shows, that our method allows a precise description of the experimental data by the parameters given. As can be followed from the authors' description of their results in LBY410 (HXT2 SNF3) and other wild strains (Kruckeberg and Bisson, 1990) under glucose repression and derepression, there is no agreement with our analysis. The only qualitative description which might come close is that for the 'high-affinity' system ( K m 1-2 mM), but for example the K m changes under repression and derepression conditions were not calculated. Further the qualitative descriptions given by Kruckeberg and Bisson (1990) for the mutants hxt2 SNF3 (LBY413), HXT2 snf3 (LBY411) and the double mutant hxt2,snf3 (LBY416) in respect to the 'low-' and 'high-affinity' system are not reflected for the first and only superficially for the second system in our quantitative analysis (Table 2). The important facts described here are: In strain hxt2 SNF3 no significant increase in the Vm value under derepression occurs and for the
56 mutant HXT2 snf3 there is even a slight decrease in V,n noticeable under derepression. The double mutant hxt2 snf3 behaves quite unexpected by fact that the Vm value of 2.5 nmol mg 1 m i n - 1 is very low under derepression and does not further decrease under repression condition. As in the mutant HXT2 snf3 the reverse takes place: in both strains the Vm values increase on repression (see Table 2). The affinity, which remains a t K m values around 2 m M is further very remarkable in the mutant hxt2,snf3 and points to the presence of other high affinity transporters. The absence of typical changes in K m values are also documented in the snf3 strain investigated in Figs. 2 and 3. These results were obtained in countertransport experiments with plasma m e m b r a n e vesicles. Plasma m e m b r a n e vesicles from cells grown glucose repressed or galactose derepressed exhibited a relatively high affinity with K m values not significantly different, namely 3.7 and 3.5 mM. F u h r m a n n and V61ker (1992) also analysed data of another snf3-72 mutant strain MCY657 (Bisson et al., 1987, initial uptake experiments) under glucose repression and derepression and obtained no significant changes in Vm values, but K m changes of 7.3 to 4.0 mM. The wild-type SNF3 strain MCY638 (Bisson, 1988) demonstrated under glucose repression and derepression a significant 3-fold increase in Vm and the K m showed a slight decrease from 4.2 to 3.3 mM. Thus, from the glucose transport studies in snf3 mutants defects in regulation of glucose transport are obvious. Although the SNF3 and HXT2 gene products due to their primary structure (Celenza et al., 1988; Kruckeberg and Bisson, 1990) are probably involved in glucose transport, they remain to be identified kinetically as glucose transporters. The data of Bisson (1988) and Kruckeberg and Bisson (1990), as well as our mathematical analysis of them and our transport experiments suggest the presence of glucose transporters with K m values around 2 m M in strains lacking functional SNF3 a n d / o r HXT2 genes. On the other hand we could demonstrate that regulation of glucose transport capacity requires an intact SNF3 gene. The most important observation is that the typical increase in Vm on derepression is abolished in snf3 strains and that the characteristic affinity change under glucose repression and derepression can be absent.
Acknowledgments Supported by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie. It is a great pleasure to thank Professor K.J. Netter for his most valuable comments and suggestions.
References Bisson, L.F. and Fraenkel, D.G. (1984) Expression of kinase-dependent glucose uptake in Saccharomyces cerecisiae. J. Baeteriol. 159, 1013-1017.
57 Bisson, L.F., Neigeborn, L., Carlson, M. and Fraenkel, D.G. (1987) The SNF3 gene is required for high-affinity glucose transport in Saccharomyces cerevisiae. J. Bacteriol. 169, 1656-1662. Bisson, LF. (1988) High-affinity glucose transport in Saccharomyces cerevisiae is under general glucose repression control. J. Bacteriol. 170, 4838-4845. Celenza, J.L., Marshall-Carlson, L. and Carlson, M. (1988) The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein. Proc. Natl. Acad. Sci. USA 85, 2130-2134. Does, A.L. and Bisson, L.F. (1989) Comparison of glucose uptake kinetics in different yeasts. J. Bacteriol. 171, 1303-1308. Fiechter, A., Fuhrmann, G.F. and Kiippeli, O. (1981) Regulation of glucose metabolism in growing yeast cells. Adv. Microbiol. Physiol. 22, 123-183. Fuhrmann, G.F., Boehm, C. and Theuvenet, A,P.R. (1976) Sugar transport and potassium permeability in yeast plasma membrane vesicles. Biochim. Biophys. Acta 433, 583-596. Fuhrmann, G.F., V61ker, B., Sander, S. and Potthast, M. (1989) Kinetic analysis and simulation of glucose transport in plasma membrane vesicles of glucose-repressed and derepressed Saccharornyces ceret.,isiae cells. Experientia 45, 1018-1023. Fuhrmann, G.F. and V61ker, B. (1992) Regulation of glucose transport in Saccharomyces ceret~isiae. J. Biotechnol. 27, 1-15. Kreutzfeldt, Ch. and Fuhrmann, G.F. (1984) Sugar transport in Saccharomyces cere~'isiae H 1022. Swiss Biotechnol. 2, 24-27. Kruckeberg, A.L. and Bisson, L.F. (1990) The HXT2 gene of Saccharornyces ceret,isiae is required for high-affinity glucose transport. Mol. Cell. Biol. 10, 5903-5913. Motulsky, H.J. (1985-1989) GraphPAD software, San Diego CA, USA. Neigeborn, L. and Carlson, M. (1984) Genes affecting the regulation of SUC2 gene expression in Saccharomyces ceret,isiae. Genetics 108, 845-858. Neigeborn, L., Schwartzberg, P., Reid, R. and Carlson, M. (1986) Null mutations in the SNF3 gene of Saccharomyces cerevisiae cause different phenotype than do in previously isolated missense mutations. Mol. Cell. Biol. 6, 3569-3574. Wilbrandt, W. and Rosenberg, T. (1961) The concept of carrier transport and its corollaries in pharmacology. Pharmacol. Rev. 13, 109-183. Zimmermann, F.K. (1992) Glycolytic enzymes as regulatory factors. J. Biotechnol. 27, 17-26.