Transport of l -glucose by Rhodotorula glutinis

Biochimie 70 (1988) 183-185 © Soci6t6 de Chimie biologique/Elsevier, Paris

183

Research article

Transport of L-glucose by Rhodotorula glutinis Mark D. PINKERTON, Candace K. RITCHIE and Charles C. G R I F F I N *

Hughes Laboratories, Department of Chemistry, Oxford, 0 45056, U.S.A. (Received 10-8-1987, accepted 5-10-1987)

S u m m a r y - The kinetics of L-glucose transport by Rhodotorula glutinis were studied over a 720-fold range of sugar concentrations. Analysis of the saturation isotherm revealed the presence of a onecarrier system for L-glucose in the plasma membrane ofRhodotorula glutinis. This carrier exhibited a k,,, of 3.7 +_ 0.3 mM. D-Ribose was found to be a competitive inhibitor with a Ki of 19 + 1 mM. The results suggest that L-glucose is transported by the high-K,,, D-ribose carrier. L-Glucose was transported against a concentration gradient and the transport was inhibited by the proton conductor 2,4dinitrophenol.

L-glucose / active transport / Rhodotorula

Introduction The red yeast Rhodotorula glutinis is capable of transporting a wide variety of metabolizable and n o n - m e t a b o l i z a b l e m o n o s a c c h a r i d e s against considerable concentration gradients [1-3]. Earlier studies in our laboratory [3-5] have demonstrated the presence of two Dglucose carriers in the plasma membrane of this yeast. These carriers, which transport Dglucose with K,,'s of 0.020 4- 0.001 and 0.15 -40.01 raM, are also responsible for the transport of 2-deoxy-D- glucose [6] and D-xylose [4] and may transport other monosaccharides [5] such as D-mannose and D-allose which competitively inhibit D- glucose transport. D-Galactose is transported only by the low-K,,, D-glucose carrier [7]. A separate set of two carriers has been demonstrated to transport the pentose, D-ribose [2]. Recently, we have been examining the transport of D-glucose by plasma membrane vesicles prepared from Rhodotorula cells. LGlucose uptake has been used as a measure of vesicle 'leakiness' in other studies of D*Author Io whom correspondence shouhl b(, add;essed.

glucose transport (e.g., [8-11]). When our membrane vesicles were observed to accumulate significant quantities of L-glucose, it was necessary to ao,o.,,,.; . . . . . ~.o,r,. . . . . . . ;..,..~ were 'leaky' or whether the plasma membrane of Rhodotorula contains a transporter for this sugar. The results presented here demonstrate that L- glucose is transported by a single, saturable carrier in the Rhodotorula plasma membrane. This carrier appears to be the high-K.,, D-ribose carrier and is capable of catalyzing an active transport of L-glucose.

Materials and methods

Rhodotorula glutinis (Rhodospotidium toruloides, ATCC 26194) was grown at 30"C in liquid medium [4] and harvested at mid-log phase. The harvested cells were washed 3 times with 0.1 M KH2PO4 (pH 4.5), suspended in 0.1 M KH~PO4(10% wet wt/vol), vigorously aerated on a magnetic stirrer for 1.5 h at room temperature, then packed in ice and maintained with gentle stirring at 0°C. Sugar uptake was measured at 30"C with L-[I-~4C]glucose (2.15 GBq/ mmol, Amersham Corp.) by a filtration assay [2-41.

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M.D. Pinkerton et al.

Initial velocities were estimated from the differences between intracellular radioactivities of samples withdrawn after 0.5 and 3.5 min of incubation. The progress curves for uptake were reasonably linear over this time interval and uptake rates were directly proportional to cell concentrations. Initial velocity data, in the presence and absence of inhibitor, were analyzed by weighted (1/v21,b0, nonlinear regression techniques. Computer programs for fitting the transport models considered were written using the general approach described by Cleland [12] for the treatment of enzyme kinetic data. Weighted fits were used because in our experimental system (specific radioactivity decreased with increasing concentrations of L-glucose) the standard errors of the velocities generally increased with increasing velocity. Cell extracts for sugar analysis were prepared by extracting filtered cells with 60% (v/v) ethanol at room temperature for 3-4 h. The resulting suspensions were filtered and washed, and the filtrates were concentrated on a rotary evaporator (bath temperature ca. 30°C). Aliquots of these extracts were applied to thin layers of silica gel G (E. Merck) and the chromatograms were developed in one of the following solvent systems: I: ethyl acetate: acetic acid : methanol : water (11 : 3 : 3 : 3, v : v); II : acetone : water (9: 1, v : v). Radioactive spots were located on the chromatogram with a radiochromatogram scanner (Berthold, Model LB 276). lntracellular sugar concentrations were calculated from specific radioactivities and a value of 0.4 ml of intracellular water/g (wet wt) of yeast [13: 14].

Results In earlier studies [2-7] we r e p o r t e d s t i m u l a t i o n o f the initial velocity o f t r a n s p o r t by aeration o f freshly harvested g l u c o s e - g r o w n cells o f Rhodotorula glutinis. A similar effect o f aeration was o b s e r v e d with L-glucose transport. T h e initial velocity o f L-glucose transport was essentially zero in cells which had b e e n freshly h a r v e s t e d and m a i n t a i n e d at 0°C. D u r i n g aeration, however, the rate o f L-glucose t r a n s p o r t increased and b e c a m e c o n s t a n t after 60-120 min. All the data p r e s e n t e d in this report were o b t a i n e d with cells which had b e e n 'activated' by a 90 m i n aeration at r o o m t e m p e r a t u r e a n d t h e n m a i n t a i n e d at 0°C d u r i n g the experim e n t a l period (1-2 h). A double-reciprocal plot o f the data from the saturation i s o t h e r m for L-glucose transport by Rhodotorula cells is s h o w n as the lower line (A) in Fig. 1. T h e data set for that line contains 174 data points covering a 720-fold range o f Lglucose c o n c e n t r a t i o n s (0.0083_6.01mM). T h e s e

0.8 C

1IV 0.4

B

60 1/(L-GLUCOSE) mM-1

t

120

Fig. 1 Lincweaver-Burk plot of the uptake of L-glucose (A) and its inhibition by 10 mM (B) and 50 mM (C) I)-ribose. The lines are drawn for a non-linear fit of the data to a model for linear competitive inhibition of a single carrier. Velocities are in pmol/min/mg (wet wt).

data were t e s t e d for consistency with one-carrier (Model I) a n d two-carrier ( M o d e l II) m o d e l s for the t r a n s p o r t by w e i g h t e d , n o n - l i n e a r regressionsModel I" v = V ; . [S] K,,,, + [S] ModellI'v=

V"[S]

+ V.,.[5]

K,,,; + [SI

K,,,: + [SI

T h e c o n v e r g e d values o f the c o n s t a n t s and the variances for the overall fits are p r e s e n t e d in Table I. A l t h o u g h c o n v e r g e n c e was o b t a i n e d for both m o d e l s , the fit to the two-carrier m o d e l gave physically unrealistic values o f the p a r a m e t e r s for the s e c o n d carrier (Table I). In o r d e r to ascertain w h e t h e r L-glucose was Table I. L-Glucose uptake and its inhibition by Dribose; computed parameters for one- and twocarrier systems. Parameters V; Ve K,,,;

One-carrier 2.3 _ 0.1 3.7 +__0.3

K,,,_, K;;

K;.,

19

+__ 1

Two-carrier 2.3 ___0.2 0.00044 __+0.00030 3.8 + 0.3 - 0.25 + 0.20 2(~+ 1 2 x 1012 + x 1025

The parameters were obtained from weighted (I/v".,h,) non-linear regression Iits. Fis expressed in nmol/min/mg (wet wt) ; K,,,and K, in raM. The'parameters are reported + S.E. The data set contained 330 points.

L-glucose transport by Rhodotorula

being transported by one of the previously characterized [2-7] monosaccharide transporters, a n u m b e r of sugars were examined for their ability to competitively inhibit L-glucose transport. Although some inhibition of Lglucose uptake was observed with 2-deoxy-Dglucose, D-galactose and 3-O-methyl-Dglucose, significant inhibition required concentrations of these sugars greatly exceeding their K~'s for the inhibition of D-glucose transport [5]. D-Ribose, on the other hand, appeared to inhibit L-glucose uptake at concentrations in the range of the K,,,'s for its own transport [2]. Fig. 1 demonstrates that D-ribose is a competitive inhibitor of L-glucose transport and its K; (19 -!- 1 mM, Table I) is close to the K,,, (30 ___ 5) reported [2] for the low-affinity Dribose transporter. The transport of many monosaccharides by Rhodotorula glutinis results in accumulation of sugar against a concentration gradient. In order to determine whether Rhodotorula cells were capable of concentrative transport of Lglucose, we examined concentration ratios, [Lglucose]J[L-glucose]o,,, during 10.5 min incubations of the cells with radioactive sugar. Calculations based on total radioactivity taken up by the cells yielded concentration ratios varying from 3.7 to 6.2 at extracellular Lglucose concentrations of 2.0 and 0.021 mM, I U;~i3U;k, LI V ~ l y

.

['Of

L I I ~

t.,d.l k., UI li:l.L I U I I ~

LU

UII~

correct, it was necessary to demonstrate that the intracellular radioactivity did in fact correspond to the free sugar. In a separate experiment, radiolabeled L-glucose was incubated with Rhodotoru/a cells for 30 min. The cells were washed; the radioactive material was extracted and subjected to thin-layer chromatography. Only a single radioactive spot exhibiting an R I corresponding to that of authentic L- glucose was observed. Additional evidence for an energy-requiring, concentrative transport of L-glucose by Rhodotorula glutinis was the observation that the protonophore, 2,4dinitrophenol, inhibited the uptake of Lglucose by 87% at 5 ~M and 97% at 10l~M concentrations.

Discussion To date, there is not a single, simple monosaccharide which is known not to be transported by the yeast Rhodotorula glutinis. To the list of sugars known to be transported, must now be added the rare natural product L-glucose. The

185

data presented here convincingly demonstrate that Rhodotorula cells carry out an active transport of L-glucose catalyzed by a singl.e, saturable carrier exhibiting a Km of 3.7 mM and a V,,,~, of 2.3 n m o l / m i n / m g (wet wt). The transport of L-glucose is competitively inhibited by D-ribose with a K; of 19 mM. The close correspondence of this K, with the K,n for the lowaffinity D-ribose carrier [2] suggests that the latter is responsible for the transport of Lglucose.

Conclusions L-Glucose is transported by a single, saturable carrier in the plasma membrane of Rhodotorula glutinis. The transport is concentrative and energy-requiring, and appears to involve the high-K,,, D-ribose carrier.

Acknowledgments This work was supported in part by grants from the Faculty Research Committee of Miami University and from Hepar Industries, Inc., Franklin, Ohio. Taken in part from a M.S. Thesis submitted by M.D.P. to Miami University, Oxford, Ohio.

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