Journal of General Microbiology (1989, 131, 479-485. Printed in Great Britain
479
Sugar Transport and Hexose-ATP-Kinase Activity in a 2-Deoxy-D-glucose
Tolerant Mutant of the Yeast Rhodotovulu glutinis
By D I E T E R M A H L B E R G , M I L A N H O F E R * A N D A N D R E A S T A U B E R
Botanisches Institut der Universitat Bonn, Kirschallee I , 5300 Bonn I , FRG
(Received 17 May 1984; revised 4 September 1984)
Mutants of the obligatory aerobic yeast Rhodotorula glutinis were selected after treatment of
wild-type cells with N-methyl-N’-nitro-N-nitrosoguanidine
on glycerol/glutamic acid medium
containing 1 % 2-deoxy-~-glucose.Rates of D-glucose transport in the mutants were about onetenth that of the wild-type, whereas the transport of D-fructose and D-xylose was unaRected.
However, glucose transport in the mutants was inhibited by uncouplers (for example carbonyl
cyanide rn-chlorophenylhydrazone)and, therefore, remained an energy-dependent process. One
of the mutants, M8, was chosen for further characterization of the transport and metabolism of
hexoses. Biochemical analysis of the hexose-ATP-kinase activities revealed the absence of
glucokinase (EC 2.7.1 .2) activity in M8. Thus, the phosphorylation capacity was reduced to
one-tenth to one-fifteenth that of the wild-type, resulting in accumulation of D-glucose which, in
turn, slowed down net glucose uptake. With the help of the mutant, the stoicheiometry of the
H+/glucose symport in R. glutinis was determined to be one proton per molecule of D-glucose
transported.
INTRODUCTION
The uptake of monosaccharides in the yeast Rhodotorula glutinis seems to be mediated by a
common plasma membrane transport system (Kotyk & Hofer, 1965; Hofer & Kotyk, 1968;
Hofer & Dahle, 1972), being an electrogenic H+-symport (Hofer & Misra, 1978; Hauer & Hofer,
1978). Its substrate specificity is rather low since non-metabolizable ionic D-glucose analogues
such as glucosamineor glucuronate were found to share the monosaccharide carrier (Niemietz et
al., 1981 ; Niemietz & Hofer, 1984). The existence of a single constitutive carrier for all
monosaccharides in R . glutinis was questioned by Janda et al. (1976). On the basis of differing
relationships between transport velocity and pH with various substrates, and the lack of
competition for transport in certain cases, the authors postulated at least two distinct transport
systems for the uptake of monosaccharides. Yet another inducible membrane carrier of low
affinity, but specific for D-xylose, appears in R . glutinis after prolonged starvation (Alcorn &
Griffin, 1978).
Isolation of transport-defective mutants should enable the existence of common or more
specific transport systems in R . glutinis to be distinguished. Such mutants would be either pleiotropic, indicating a single common carrier, or cryptic for a group of monosaccharides. Since Dglucose plays a central role in monosaccharide transport (Kotyk & Hofer, 1965; Hofer & Dahle,
1972; Janda et al., 1976), we attempted to isolate glucose transport-defective mutants of R .
glutinis by selecting for resistance to 2-deoxy-D-glucose (2-DOG). Therefore, both transportdefective and metabolic mutants could have been selected. Consequently, the capacity to
phosphorylate hexoses was studied in addition to the transport properties of the mutants. A
preliminary communication of this work has been published (Mahlberg & Hofer, 1984).
METHODS
Organism and growth. The obligatory aerobic yeast Rhodotorula glutinis (synonym Rhodosporidium toruloides)
mating type a (ATCC 26194; CBS 6681) was grown in a medium containing the following (w/v): 0.1 % K2HP04,
Abbreciation: 2-DOG, 2-deoxy-~-glucose.
0001-1957
0 1985 SGM
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480
D. M A H L B E R G , M . H O F E R A N D A . T A U B E R
0.05% NaCl, 0.1 % MgSO, .7H20, 0.033 % CaClz.2H20, 0.005 % FeCI, .6H20, 0-5% L-glutamic acid, 2.5 %
glycerol and 0.033% yeast extract (Difco); the pH was adjusted to 3.1 with HCl before autoclaving. A preculture
(80 ml) was inoculated with 1-2 loops of wild-type cells and cultivated for 24-48 h on a gyratory shaker at 30 "C;
10 ml of this subculture were used to inoculate the main culture (160 ml). After a further 24 h, the cells were
harvested by centrifugation, washed three times with distilled water, and aerated for 4-12 h as a 5 % suspension
(wet wt/vol.).
Isolation ofmutants. A 0.1 % (wet wt/vol.) aqueous cell suspension of R. glutinis aerated overnight was exposed to
for 1 h in darkness at 30 "C.The cells were washed three times with
1.4 mM-N-methyl-N'-nitro-N-nitrosoguanidine
distilled water, and then selected on the glycerol/glutamic acid medium (see above) containing 1 % (w/v) 2-DOG.
After 10 d growth on a gyratory shaker at 30 "C the cells were plated on agar plates of the same composition. In two
independent treatments, 13 mutants were isolated altogether. All mutants were tested for energy-dependent
uptake of D-glucose, Dfructose and D-XylOSe. One of the mutants, M8, was chosen for further biochemical
characterization as well as for measuring H+/sugar stoicheiometry.
Growth ojmutants. The mutants were cultivated in two different media. Usually the culture medium was of the
above composition (glycerol/glutamic acid medium) : however, for measurements of H+-symport the cells were
grown in a medium (pH 5.4) in which glycerol and glutamic acid were replaced by 0.066% NH4N03 and 2.5% Dfructose (both w/v). In order to inhibit the growth of possible revertants, 0.3% (w/v) 2-DOG was present on a
routine basis (the frequency of reversion has not been followed). This concentration of 2-DOG completely
inhibited the growth of wild-type cells. The main culture was harvested after 36-48 h, washed three times and
aerated for 4-12 h.
Transport experiments. The uptake of D-xylose as well as D-glucose and mfructose was determined at transportsaturating concentrations of the individual monosaccharide, as described by Heller & Hofer (1 975) for D-xylose
and by Hofer & Dahle (1972) for D-glucose and Dfructose. H+-cotransport was monitored with a pH electrode
connected to the PHM 62 pH meter (both Radiometer, Copenhagen, Denmark) and the H+/sugar stoicheiometries
were calculated as described in detail by Hofer & Misra (1978) and Hauer & Hofer (1982). The intracellular glucose
level was measured enzymically (Bergmeyer et al., 1974) in hot water extracts of cells separated from the
incubation medium by membrane filtration (Heller & Hofer, 1975).
Separation and determination of hexose-ATP-kinase activity. Washed, but not aerated cells (about 15 g wet
weight) of both the wild-type strain and the mutant (M8) were resuspended~(50%,w/v) in 0.2 M-sodium phosphate
buffer, pH 7.2, containing 20 pM-phenylmethylsulphonyl fluoride and 7 mM-benzamidine to prevent proteolytic
degradation sfGellular enzymes. Cell disruption was carried out in a pre-cooled French press MP 025, at a pressure
of 8 MPa. The fraction of soluble enzymes, the crude extract, was obtained by centrifugation at 30000 g for 1 h at
0 "C.The enzymes of the crude extract were partially fractionated by ammonium sulphate precipitation (70%
saturation). After 1 h, the precipitate was collected by centrifugation at 48000g for 10 min. The pellet was
digested in 10 ml 20 mM-succinate/l mM-EDTA buffer, pH 6.5, and dialysed against 1 litre of the same buffer,
exchanged three times at 4 h intervals. The hexose-ATP-kinase activities were purified on a DE 52 anion
exchanger (Whatman), equilibrated in 20 mM-succinate/1 mM-EDTA buffer, pH 6.5 (modified procedure of
Rustum et al., 1971). About 100 mg of the dialysed protein mixture (10 ml) was applied to a 70 ml column (1.8 x
40 cm) and eluted with 300 ml of a linear sodium chloride gradient (9-0.4 M).Fractions (5 ml) were collected at a
flow rate of 20 ml h-l.
Protein concentration. During the chromatographic step, the protein content was monitored at A280. For
calculation of the specific activities of hexose-ATP-kinases, the protein concentration was determined by the
Lowry method.
Enzyme assay. The hexose-ATP-kinase activity was assayed by measuring the velocity of hexose 6-phosphate
formation in the presence of ATP at 25 "C. The reaction vessel contained 1 ml substrate buffer (0.1 Mtriethanolamine, 10 mM-MgSO,. 7Hz0, 0.2 M-hexose, pH 8.5) and 0.1 ml ATP (8.3 mM). A 20-50 pl sample of the
individual fraction, corresponding to approx. 30 pg protein, was used to start the reaction. The reaction was
stopped either by heat or by addition of perchloric acid to give a concentration of 0.5 M. The amount of hexose 6phosphate was determined enzymically as described by Lang & Michal(l974); the enzyme activity is expressed in
pmol hexose 6-phosphate formed min+l (U). D-Glucose, D-fructose or 2-DOG were utilized as substrates.
Chemicals. All enzymes and coenzymes were products of Boehringer. N-Methyl-N'-nitro-N-nitrosoguanidine,
carbonyl cyanide m-chlorophenylhydrazone and benzamidine were purchased from Sigma. Phenylmethylsulphonyl fluoride and 2-deoxy-~-glucosewere from Calbiochem. All other compounds were of Reagent grade
(Merck).
R E S U L T S A N D DISCUSSION
In accordance with our intention to isolate a glucose transport-defective mutant, the transport
properties of the mutants were analysed first. Fig. 1 (a) demonstrates that the maximal rate of
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48 1
2-DOG tolerant mutant of Rhodotorula gracilis
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Fig. 2
Fig. 1. Uptake of Dglucose (a) and Dfructose (b), measured as their consumption from the external
medium, and mxylose (c), determined in cell extracts, into wild-type (WS,0 )and M8 (a)cells. Cell
suspensions (about 4 mg dry wt ml-I) were incubated in 0.15 M-KH~PO,buffer, pH 4.5, at 30 "C.After
starting the experiment (1 mM-Dglucose, 2 mM-mfructose or 10 mM-Dxylose initial concentration),
samples of 0.8-1-0 ml were withdrawn at intervals and treated as described in Methods. Maximal
transport rates were [min-1 (mg dry wt)-']: V T , M 8 , 2 nmol D-glucose; VT,ws, 30 nmol D-glucose; V T , M ~ ,
16 nmol Dfructose; VT,ws, 10 nmol Dfructose; VT,M8, 7 nmol Dxylose; VT,ws, 8 nmol Dxylose.
Fig. 2. Variation of hexose-ATP-kinase activity with pH. Measurements were made in crude extracts
of the wild-type strain (WS,0 )and the mutant M8 (a).The pH optimum was at 8.5 in both cases.
uptake (VT) of D-glucose by cells of the mutant M8 was, at comparable affinities (data not
shown), significantly slower than that by wild-type cells (WS)[VT,MS,
2 nmol D-glUCOSe min-'
(mg dry wt)-l; VT,\rs, 30 nmol D-glucose min-l (mg dry wt)-l]. However, the uptake of Dfructose and D-xylose (Fig. 1b, c) proceeded with similar maximal velocities in both wild-type
and M8 cells [
16 nmol D-fructose min-l (mg dry wt)-l; VT,ws, 10 nmol D-fructose min-*
(mg dry wt)-' ; VT,M8,7 nmol D-xylose min-* (mg dry wt)-' ; VT,ws, 8 nmol D-xylose min-l
(mg dry wt)-*]. The strongly decreased uptake of D-ghCOSe into M8 cells was still inhibited by
uncouplers such as carbonyl cyanide m-chlorophenylhydrazone, indicating its tight coupling to
metabolic energy. The other 12 mutants selected were very similar in transporting D-glucose at
one-tenth to one-twentieth the velocity of the wild-type strain. All the mutants transported Dfructose and accumulated D-xylose at rates comparable with the wild-type cells. The transport
was energy-dependent and D-glUCOSe inhibited the uptake of other monosaccharides in all
mutant cultures, which was characteristic of wild-type cells (cf. Hofer & Dahle, 1972).
In the light of these experiments, a defect of the monosaccharide transport system in the
mutants appeared unlikely. Hence, we searched for a defect in the pathway of D-glucose
catabolism. Since the utilization of D-fructose showed no difference between the mutants and
the wild-type strain, a defect in an early step of glucose metabolism (e.g. glucose
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482
D. M A H L B E R G
M, . H O F E R
A N D A. T ~ U B E R
phosphorylation) seemed apparent. Based on these considerations, one of the mutants, M8, was
chosen and the hexose-ATP-kinase activity in the crude extracts of M8 cells and of the wild-type
cells was examined. Indeed, hexose-ATP-kinase activity of crude extracts from the mutant and
the wild-type strain differed by a factor of 15, being 16-19 mU (mg protein)-' and 230-270 mU
(mg protein)-', respectively. The values given were obtained with five different cell cultures
grown independently of each other. To prove that the observed difference was not caused by
different pH optima of the enzyme activity, pH dependency of hexose-ATP-kinase was
determined in both crude extracts. Fig. 2 shows that the two were almost identical with varying
PH.
In order to characterize the strongly lowered capacity of D-glucose phosphorylation in the
crude extracts from M8 cells, the hexose-ATP-kinase from both the mutant cells and the wildtype cells was purified by anion exchange chromatography. A DE 52 anion exchanger,
equilibrated in 20 mM-succinate/l mM-EDTA buffer, pH 6.5, bound all the kinase activity of the
dialysed crude extract. The bound protein was eluted by a linear sodium chloride gradient (00.4 M).The results of the column chromatography are summarized in Fig. 3, The elution profile
of the hexose-ATP-kinase from the wild-type strain, with D-glucose as substrate, revealed two
major activity peaks (fractions 23 and 41), called P, and P2. The enzyme activity in the shoulder
(fractions 18/19) was identical to that of P1 with respect to substrate specificity and kinetic
properties. The elution diagram of the mutant M8, however, displayed only one peak in the
fractions 36-40 (P2-M).The hexose-ATP-kinase activities in the three major peaks (PI and P2 of
the wild-type strain and P2-M of the mutant) were examined for their substrate specificity and
kinetic properties. Table 1 summarizes the kinetic data obtained for the two principal substrates
of glycolysis. The hexose-ATP-kinase of P1, kinase 1, exhibited a high affinity for D-ghCOSe and
a 1500-fold lower affinity for D-fructose. In uiuo, the enzyme phosphorylates D-glucose with
correspondingly high velocity. Despite comparable maximal phosphorylation rates with both
substrates, no D-fructose is phosphorylated under physiological concentrations. For this reason,
kinase 1 was assigned to glucokinase (EC 2.7.1 .2). The enzyme of P,, kinase 2, showed the
same affinity for D-glucose, but its affinity for D-frUCtOSe was more than 150 times higher than
that of the glucokinase. Since the maximal velocities for glucose are lower than for fructose,
kinase 2 is able to phosphorylate both hexoses with comparable rates under physiological
conditions. This behaviour is characteristic of hexokinase (EC 2.7.1 . 1). The M8 cells lack
glucokinase : their hexose-ATP-kinase activity, kinase 2-M, corresponded to hexokinase
(EC 2.7-1.l), being able to phosphorylate both D-glucose and D-fructose with comparable rates.
However, its capacity to phosphorylate any one of the two substrates was about one-quarter to
one-third that of the kinase 2 of wild-type cells.
2-DOG, the substrate used for selection of the mutant cells, was phosphorylated only by the
glucokinase of the wild-type cells at a measurable velocity, K , and V,,, being 41 mM and 0.02 U
(mg protein)-', respectively. The intracellular concentration of 2-DOG under the growth
conditions amounted to about 150-200 mM, which was high enough to saturate the enzyme in
the wild-type cells (data not shown). Thus, the growth of the wild-type and not the M8 cells was
inhibited in the presence of 2-DOG.
The glucose phosphorylation capacity of the M8 cells was lowered about 15-fold due to the
absence of glucokinase and the lower maximal velocity of hexokinase. In wild-type cells, the
translocation step in D-glucose catabolism seemed to be rate-limiting, as concluded from the
very low intracellular free sugar concentration (Hofer et al., 1970). The rate-limiting step of
glucose catabolism in M8 cells is obviously transferred from translocation to phosphorylation,
since M8 cells accumulated D-glucose (up to 20 mM intracellular concentration at 10 mM-Dglucose in the medium). The intracellular steady-state D-glucose concentration in wild-type cells
under comparable conditions was below 1 mM. Thus, the mutant M8 was not defective in
transport but in the metabolism of glucose: it is a glucokinase-negative mutant.
The mechanism by which the translocation step is slowed down in the mutant M8 has not
been elucidated. Both possibilities, a feed-back regulation of transport by an unknown
metabolic intermediate, or simply an enhanced efflux due to the considerably higher
intracellular D-glucose concentration in the mutant M8,may be effective.
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2-DOG tolerant mutant of' Rhodotorula gracilis
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Fig. 3. Elution profile of hexose-ATP-kinase from the wild-type strain with peaks P, and P2 (a) and
from the mutant M8 with peak P2-M(b). Chromatography column size, 1.8 x 40 cm; buffer, 20 mMsuccinate/l mM-EDTA, pH 6.5; elution, linear NaCl gradient (0-0-4 M);flow rate, 20 ml h-l ; volume
of the fractions, 5 ml. 0 ,Activity of hexose-ATP-kinase, determined with 0.2 M-D-glucose; 0,protein
concentration (Azs0).The columns give the protein content (mg ml-l) in the relevant fractions.
--
Table 1. Half-saturatwn constant (K,) and maximal velocity ( V,,,,,) of hexose-ATP-kinase
from the wild-type strain (WS) and the mutant (M8)
Data are means of four measurements from two or three preparations. The kinetic parameters were
estimated from double reciprocal plots.
D-Fructose
D-Glucose
Km
ws
M8
Kinase 1
Kinase 2
Kinase 2-M
Vmax
Km
Vmax
(mM)
[U (mg protein)-']
(mM)
[U (mg protein)-']
0.22
0.2 1
0.22
0.94
0.25
1.84
300
2.10
3.30
0.86
2.10
0.78
In spite of our failure to isolate a transport-defective mutant, the mutant M8 became
interesting also from the point of view of transport studies. In general, monosaccharides are
accumulated in R . glutinis by an electrogenic H+-symport (Hofer & Misra, 1978; Hauer & Hofer,
1978,1982).The stoicheiometry is one H+ per sugar molecule transported (Hofer & Misra, 1978;
Hauer & Hofer, 1982). However, H+/D-glucosecotransport could never be measured because of
an immediate strong acidification of the cell suspension by the onset of D-glucose uptake and
catabolism (see e.g. Conway & O'Maloney, 1946; Conway et al., 1954; Eddy, 1978; and Hofer &
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D.
484
MAHLBERG, M. HOFER A N D A. TAUBER
Table 2. Maximal velocities ( VT) of sugar uptake and H+-cotransport, and H+/sugar
stoicheiometry as a junction of external p H and temperature in the mutant
Mutant cells were grown and treated as described in Methods. A 5% (wet wt/vol.) cell suspension was
titrated with either 10 mM-Ca(OH)* or 10 mM-HCI to the desired pH. Maximal velocities of H+ and
sugar uptake were measured as described by Hofer & Misra (1978) and Hofer & Dahle (1972),
respectively.
VT
Conditions
pH 6.5
t 30°C
pH 6.0
t 30°C
pH 5.2
t 30°C
pH 4-5
t 30°C
pH 6.6
t 26°C
[nmol &glucose
min-' (mg dry wt)-'l
H+/D-glucose
7.38
7-25
0.98
6.63
7-03
1-06
5.99
6.16
1*03
5.69
5-01
0.88
6.16
6.40
1.04
VT
PH 5
t 28°C
PH 6
t 28°C
PH 5
t 25°C
VT
[nmol H+ min-'
(mg dry wO-'I
VT
[nmol D-xylose
min-' (mg dry wt)-'1
[nmol H+ min-'
(mg dry wt)-']
H+/D-xylose
21.1 1
23-87
1-13
17.69
17.36
0-98
13.13
14.21
1*08
Misra, 1978 for R . glutinis). Due to the slower catabolism of D-glucose in M8 cells, we succeeded
in completing the H+/D-glucose stoicheiometry measurements. Table 2 gives the fluxes of H+
and D-glucose under various physiological conditions (pH, temperature). The results
demonstrate that, as with other monosaccharides, one proton was cotransported with each Dglucose molecule. As a control, the H+/D-xylose stoicheiometry is also shown in Table 2. Since
the transport system in M8 cells did not appear to be affected by the mutation, the
stoicheiometry probably holds for the wild-type strain as well.
Although only the M8 mutant was further characterized as to its hexose phosphorylation
capacity, the other mutants isolated are probably of similar phenotype. This conclusion was
drawn on the basis of the similar transport properties of all the mutants isolated. Hence, other
mutants are needed to provide evidence for either a common, or more separate transport systems
for monosaccharides in R . glutinis.
This work was supported by the Deutsche Forschungsgemeinschaft (grant no. Ho 555).
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