MICROBIOLOGY LETTERS

ELSEVIER FEMS Microbiology Letters 166 (1998) 267-273

Glucose transport in a methylotrophic yeast Hansenula polymorpha

Helen Karp, Tiina Alamge *

Institute of Mokcular and Cell Biology, University of Tam, Riia 23, EE-2400 Tam, Estonia

Received 28 May 1998; revised 17 July 1998; accepted 23 July 1998

Abstract

Glucose transport was studied in a methylotrophic yeast Hansenula polymorpha. Two kinetically different glucose transport systems were revealed in cells grown under different growth conditions. Glucose-repressed cells exhibited a low-affinity transport system (K, for glucose 1.75 mM) while glucose-derepressed and ethanol-grown cells had a high-affinity transport

system (K, for glucose 0.054.06 mM). The high- and low-affinity transport systems differed in substrate specificity, sensitivity to pH, dinitrophenol and protonophore carbonyl cyanide-m-chlorophenyl-hydrazone. The kinetic rearrangement of the

glucose transport system in response to altered growth conditions was dependent on de novo protein synthesis. 0 1998

Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Glucose transport; Methylotrophic yeast; Hansenula polymorpha

1. Introduction

Glucose is an effector of several regulatory re-

sponses in yeasts: expression of a large number of

genes is repressed by glucose, and expression of

others is induced [l]. The precise mechanism of glu-

cose signalling in yeasts is not clear yet but it has

been assumed that it could be similar to pancreatic p-cells of humans, i.e. glucose sensing needs both a glucose transporter and a glucose phosphorylating enzyme [2]. Both hexose kinases and glucose trans-

port in yeasts have been most thoroughly studied in

* Corresponding author. Tel.: +372 (7) 465 013; Fax: +372 (7) 420 286; E-mail: talamae@ebc.ee

Abbreviations: CCCP, carbonyl cyanide-m-chlorophenyl- hydrazone; DNP, 2,4-dinitrophenol

Saccharomyces cerevisiae as a model system. In this yeast the genes of 20 different hexose transporter-

related proteins have been identified [3]. Two of these proteins, Rgt2p and SnfJp, probably act as glucose sensors that mediate generation of an intra- cellular glucose signal [4]. The kinetics of several

distinct hexose transporter proteins (Hxtl-7p) has been evaluated [5] and it has been generally accepted that apparent kinetics of glucose transport in S. cer-

evisiae grown under different conditions is deter- mined by numerous factors, including regulation of expression of different sets of transporters with sig- nificantly different affinities to the sugar, and the removal and inactivation of transporter proteins [3]. However, according to Walsh et al. affinity of glucose transport for the sugar during the transition of S. cerevisiae from glucose-repressed to -dere-

0378-1097/98 /$19.00 0 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

PII: SO378-1097(98)00342-S

pressed conditions can be increased through binding

of sugar kinases to the glucose carriers constitutively present in the cell [6]. This interaction would serve to

alter the kinetics of transport rather than expression of a different transporter protein. The dependence of the high-affinity glucose transport in S. wrcvi.riuc on

glucose phosphorylating enzymes has been earlier

reported also by Bisson and Fraenkel [7]. Glucose transport has also been studied in some

other yeasts, e.g. Khry~rorn~~s murxiunus [8], Pi-

chia ohmeri [9], and in Cundidu utilis [lo]. In all of these yeasts a proton symport exhibiting a high af- finity for glucose is present in the cells when there is

no or a very low concentration of glucose in the medium, and a facilitated diffusion system with a

low affinity for glucose is present in cells grown on a high concentration of glucose. This is in contrast to S. cwwisiuc~ where both low- and high-affinity trans-

port is mediated by facilitated diffusion. Genetics of glucose transport among non-Succhrl-

rornyces yeasts has been most extensively studied in

Kluyveromyces Iactis. This yeast has one major low-

affinity glucose transporter, encoded by the RAG1

gene, and one high-affinity glucose transporter, en- coded by the HGTI gene, and expression of both genes is positively affected by the RAG5 gene encod-

ing hexokinase, the only glucose phosphorylating en- zyme of this yeast [ 11,121. However, the precise role

of hexokinase in the regulation of glucose transport in K. lactic has still to be elucidated.

Methylotrophic yeasts, Hunsrnuku poly~7or;nl7u in

particular, have become popular tools for the expres- sion of foreign proteins mainly under the control of powerful methanol-induced promoter of alcohol ox-

idase [l3]. Besides induction by methanol, transcrip- tion from the alcohol oxidase promoter is regulated by glucose repression and derepression [14]. There- fore, study of mechanisms of glucose repression in methylotrophic yeasts should be an obligatory pre- requisite for the controlled production of foreign proteins in these yeasts. Moreover, due to the pres- ence of numerous glucose-regulated functions. e.g. synthesis of methanol-specific enzymes and organ- elles, synthesis of enzymes not related to methanol metabolism, such as maltase, and inactivation of methanol-specific enzymes. methylotrophic yeasts should also be considered attractive objects for the study of glucose-sensing mechanisms in non-Scrcrlra-

~~77~~s yeasts. In our earlier studies on a methylo- trophic yeast. Pichiu pinus. we have shown that the

glucose transport system can participate in glucose repression in methylotrophic yeasts [15]. In this pa-

per we present for the first time data on glucose transport and its regulation in H. pol_ymorphu.

2. Materials and methods

Uracil-auxotrophic strain LR9, derived from Hun-

.senuh polymorpha strain ATCC 34438 kindly do-

nated by Dr. R. Roggenkamp (Dtisseldorf), was used throughout the study.

To obtain cells of different states of glucose repres-

sion, yeasts were pregrown in YEP (1% yeast extract. 2% peptone) medium supplemented with 2% glucose to mid-exponential growth phase. Thereafter cells

were harvested by centrifugation at 2000 X g at 4°C

for 5 min, and incubated during 3 h in YEP medium

supplemented with either 2% glucose (glucose-re- pressed cells) or 0.05% glucose (glucose-derepressed cells). In order to study the rearrangement of the

glucose transport system, cells grown on YEP me- dium containing either 5% glucose or 0.7% ethanol

were divided into two batches and transferred to YEP medium with either 0.05% or 2% glucose, re-

spectively. Geneticin at final concentration of 1 mg nil- ’ was added to one parallel culture to inhibit protein synthesis. Culture samples were withdrawn at 0, 30, 90 and I80 min after the transfer, harvested

by centrifugation, washed and used for the measure- ment of glucose transport. For the study of the effect of carbon source on glucose transport, cells were grown to mid-exponential growth phase on YEP me-

dium supplemented with 2% glucose, 2% fructose, 0.1% glucose, 0.5% methanol, 0.7% ethanol or 2% glycerol, washed and used for the measurement of glucose transport as described above.

2.2. Preparation oj’suspensions fix transport measurements und determination of dry weight

Washed cells were suspended in 100 mM K-phos- phate buffer (pH 6.5) and kept in an ice-bath under stirring during the experiment. Dry weight content of

H. Karp, T Alamdel FEMS Microbiology Letters 166 (1998) 267-273 269

suspensions was determined by filtration of a fixed

amount of suspension onto a nitrocellulose filter. Filters were dried up to constant weight in the mi- crowave oven before and after the filtration, and weighed. The dry weight was calculated from the

difference.

2.3. Concentration of the unlabeled competing sub-

strate was 100 times higher than that of the labeled

glucose.

2.5. Inhibition studies

2.3. Transport assay

The method used was a combination of methods described by Bisson and Fraenkel [7] and Walsh

et al. [6]. 50 ul of labeled glucose (o-[14C]glucose, 0.24 mCi mM_‘) prewarmed to 37°C was added

at different concentrations to 50 ul of cells preincu- bated in a conical test tube at 37°C for 2 min, and

exactly after 10 s, the reaction was terminated by the addition of 10 ml of ice-cold 100 mM K-phosphate buffer (pH 6.5) containing 400 mM unlabelled glu-

cose. The cells were immediately filtered onto a glass- fiber filter under reduced pressure, and washed on the filter with 10 ml of ice-cold distilled water. The

filters were then transferred into scintillation vials containing 5 ml of scintillant, and radioactivity was

measured with a Beckman liquid scintillation coun- ter. Analogous measurements, but performed at 0°C

were used as control blanks that were subtracted

from readings.

The inhibitors of glucose transport carbonyl cya-

nide-m-chlorophenyl-hydrazone (CCCP) and 2,4-di- nitrophenol (DNP) were dissolved in ethanol (final concentration of ethanol in the incubation mixture 9

ul in 100 ul), and added to the cells 1 min before the

addition of labeled glucose. CCCP was used at two final concentrations, 0.05 mM and 1 mM, and DNP

at 5 mM. Ethanol was also added to the unpoisoned control cells, because it reduced the velocity of glu- cose transport.

2.6. Calculation of kinetic properties of transport

systems

Eadie-Hofstee plots were used to calculate K, and V max values for glucose transport systems.

3. Results and discussion

3.1. Kinetic characteristics of glucose transport

systems 2.4. Competition studies

To characterize the substrate specificity of glucose

transport systems, inhibition of uptake of labeled glucose by following unlabeled substrates was

studied : glucose, fructose, mannose, xylose, xylitol, 2-deoxy-n-glucose (2DG), maltose, sucrose and sor- bitol. Unlabeled competing substrates were added to

the cells 1 min before the addition of labeled glucose,

and the cells were processed as described in Section

Eadie-Hofstee (V vs. V/S) curves were used to evaluate presence of kinetically different transport

systems in cells grown under different conditions. The curves obtained for glucose-repressed, glucose- derepressed and ethanol-grown cells were all mono-

phasic and revealed the presence of a low-affinity system in glucose-repressed cells, and a high-affinity

system in either glucose-derepressed or ethanol-

grown cells. Monophasic curves typical for glucose-

Table 1

Kinetic characteristics of glucose transport systems in H. polymorpha grown under different conditions

Characteristic Cells

Glucose-repressed Glucose-derepressed Ethanol-grown

Km MM) 1.75 k 0.25 0.06 k 0.01 0.05 + 0.01

V,,,, (nmol min-’ (mg dry weight)) 145+15 61 +9 40+2

Glucose-repressed and -derepressed cells were obtained by transferring glucose-grown cells for 3 h to 2% glucose and O.OS% glucose,

respectively. Ethanol-grown cells used were from the exponential growth phase. Mean va1uesfS.D. of 2-4 independent experiments are

presented.

0 100 200 300 400 500 600 700 800 900 1000

v/s

160 , I

0 100 200 300 400 500 600 700 BOO 900 1000 VIS

160 , I

glucosederepressed for 180 min 4; 1 0 100 200 300 400 500 600 700 800 900 ,000

“IS

Fig. 1. Eadie-Hofstee plot of kinetic rrarrangement of glucose

transport after the transfer of H. po!,~wwphu grown on 2% glu-

cose (glucose-repressed cells) to the medium with 0.05% glucose.

V. glucose uptake rate, nmol min-’ (mg dry weight). S. glucose

concentration in the uptake mixture. mM

repressed and glucose-derepressed cells can be seen in Fig. lA,D. The Eadie-Hofstee curve of glucose

transport for ethanol-grown cells was very similar to that present in Fig. 1D (not shown). K,,, values for glucose, calculated from Eadie-Hofstee plots for the low- and high-affinity systems were 1.75 mM and

0.05-0.06 mM, respectively. The I’,,,;,, value of the low-affinity system was about 2-3 times higher than that of the high-affinity system (Table 1). The K,,,

values of the high- and low-affinity glucose transport systems in H. po/_vrnorphu were in good agreement

with literature data on glucose transport systems in some other yeasts. For Pi&u ohmeri the respective

K,,, values were 0.05-o. 15 mM and l-5 mM [9], for Cundidu intermediu 0.16 mM and 2.0 mM [16], and

for Pi&u pinus 0.1 mM and 4.6 mM [15].

3.2. Kinetic rrurrungement of’f’glucosr trunsport during

If glucose-repressed cells were transferred to me-

dium containing 0.05% glucose, and glucose trans- port kinetics was studied in 0, 30, 90 and 180 min after the transfer, smooth transition of a low-affinity

system to a high-affinity system was observed (Fig. IA-D). While a high-affinity transport system for

glucose was only emerging in cells after 30 min of glucose derepression (Fig. lB), it became a dominat- ing system by 90 min of derepression (Fig. lC), and

was the only glucose transport system found after 180 min of derepression (Fig. 1 D).

3.3. E.upression of’ the high-ufinity glucose transport

s,vstem during growth qf’ H. polymorphu on

deferent curhon sources

Carbon source-dependent expression of the high-

affinity glucose transport system was evaluated by

measurement of uptake of 0.05 mM glucose. At this concentration uptake of glucose through the low-affinity system was negligible and could there-

fore be ignored. Analogous approach has earlier been used for the study of expression of high-affinity glucose uptake in S. cerevisiue [17] and in Cundidu

utilis [lo]. According to Table 2, the high-affinity system was highly expressed in H. polymorphu cells

-fable 2

Expression of high-aJ?inity glucose transport system in H. &I‘-

~wrpho grown on different carbon sources evaluated ah velocity

of uptake of 0.05 mM glucose ._

Carbon source Uptake of 0.05 mM glucose

(nmol min-’ (mg dry weight)) ~~~ ~

Glucose 2% 3.8 f 0. I ‘0. I’!4 Glucose 24.9 + 2.2

Fructose 2’i’ 0 4.6 f 0.2

Ethanol 0.7% 19.8 i 0.3

Methanol II.~‘%~ 19.0* I.8 _____

Mean values? S.D. of two experiments are presented.

H. Karp. T. AlamdeIFEMS Microbiology Letters 166 (1998) 267-273 211

grown on low-glucose medium (O.l%), on ethanol and methanol, and was strongly reduced if the cells were grown on either 2% glucose or 2% fructose. Thus, the expression pattern of high-affinity glucose

transport system in H. polymorpha was quite similar

to C. utilis [lo] and S. cerevisiue [17].

3.4. The nature of kinetic rearrangements of glucose transport in response to altered growth conditions

It has been proposed that the glucose transport

system in yeasts can be adapted to changing growth conditions either through the synthesis of new trans- porters [3], or through modulation of the affinity of

preexisting transporter(s) due to the assembly with other existing components [6]. These two models could be distinguished by evaluating the requirement

for new protein synthesis during the kinetic rear- rangement. To assess the role of protein synthesis

in kinetic rearrangement of glucose transport in

H. polymorpha, an inhibitor of protein synthesis, ge- neticin, was used. Cycloheximide, which is tradition- ally used to inhibit protein synthesis in S. cerevisiae,

had no effect on H. polymorpha (not shown). Expres- sion of high-affinity transport was again evaluated by the uptake of 0.05 mM glucose. Fig. 2A shows

a gradual increase in the high-affinity glucose uptake during glucose derepression. Since geneticin pre-

vented formation of the high-affinity system, synthe-

B LR9 bmsfmed from elhanol lo 2% #mse

OtTIll 3Ordn 9Omn

Adaptabon bn?e

180 mn

Fig. 2. Rearrangement of glucose transport in H. polymorpha in

the presence (dark bars) and in the absence (white bars) of 1 mg

ml-’ geneticin in the transfer medium. A: Cells grown on 5%

glucose were transferred to medium with 0.05% glucose and incu-

bated during 180 min. 100% value corresponds to the glucose up-

take velocity 9.4 nmol mint (mg dry weight). B: Ethanol-grown

cells were transferred to medium with 2% glucose and incubated

during 180 min. 100% value corresponds to the glucose uptake

velocity 35.3 nmol min-’ (mg dry weight).

Table 3

Inhibition of uptake of labeled glucose by different unlabeled substrates

Competitor Velocity of glucose uptake (% of control)

No competitor (control) 100”

Glucose 6.3 If: 0.6

2-Deoxy-o-glucose 8.3 t 0.7

Fructose 28.652.0

Mannose 27.9 + 0.7

Maltose 25.6 f 2.5

Sorbitol 97.7 * 3.4

Sucrose 101.2f2.4

Xylitol 91.7kO.4

Xylose 53.7 k4.6

Low-affinity system High-affinity system

100s

2.6kO.l

8.9? 0.3

102.0* 1.2

70.4 f 1.2

92.2 + 2.4

101.4+3.0

101.1 f 1.2

107.6 f 1.5

87.9 ? 5.4

To characterize the low- and high-affinity glucose transport systems, glucose-repressed and glucose-derepressed cells were used, and uptake of

1 mM and 0.05 mM labeled glucose was measured, respectively. Mean values + SD. of two experiments are presented.

“100% value corresponded to a glucose uptake rate of 43.3 nmol mint (mg dry weight).

blOO% corresponded to a glucose uptake rate of 22.4 nmol mint (mg dry weight).

212 H. Burp. lY Alum&/ FEMS Microbiology Letters 166 119981 267 273

Table 4

Inhibition of glucose transport by CCCP and DNP

Inhibitor Velocity of glucose transport (% of control)

High-affinity system” Low-affinity system”

No inhibitor added (control) 100 100

CCCP I mM 0.9 ?r 0.2 45.0+ 5.0

CCCP 0.05 mM Il-_+O.4 86.4 + 1.2

DNP 5 mM 0.7 + 0.2 74.2 2 I .8 ..___-

To characterize the low-affinity and the high-aflinity glucose transport systems, glucose-repressed and glucose-derepressed cells were used, and

transport of 2 mM and 0.05 mM labeled glucose was measured respectively. Mean values + SD. of two experiments are presented.

“100% value corresponded to 3.6 nmol min-’ (mg dry weight).

“100% value corresponded to 26.1 nmol min-’ (mg dry weight).

sis of new carrier proteins was probably needed for

this kinetic rearrangement. If ethanol-grown cells were transferred to the me-

dium with 2% glucose, and uptake of 2 mM glucose

(the concentration close to the K,,, value of a low- affinity system) was measured 30, 90 and 180 min

after the transfer (Fig. 2B), the capacity of glucose

transport initially decreased, and then began to in- crease. The initial decrease of uptake can probably be described as glucose-induced inactivation of the high-affinity glucose transport system initially

present in ethanol-grown cells. The following in- crease in uptake rate can be attributed to the syn-

thesis of a new glucose transport system since it was

prevented by geneticin. Because fully glucose-re- pressed cells had only a low-affinity glucose trans- port system we consider that the system emerging

during the transfer of ethanol-grown cells to high- glucose medium is a low-affinity glucose transport

system.

3.5. Substrute specl$city qf glucose trunsport systems

Competition experiments (Table 3) showed that

the two glucose transport systems in H. polymorphu

exhibited different substrate specificities. The high-

affinity system was specific for glucose, and only 2DG could efficiently compete with glucose for the entry. At the same time, several sugars such as fruc- tose, maltose, mannose and xylose acted as glucose competitors in the case of the low-affinity system. Glucose, mannose and fructose share a common fa- cilitator also in S. cerevisiue [7]. Xylose in yeasts has also been shown to share a common carrier with glucose [18]. According to our data, sucrose, xylitol

and sorbitol did not compete with glucose for the

entry in the case of either a low- or high-affinity glucose transport system in H. polymorpha. Inhibi-

tion of low-affinity glucose transport by maltose was quite unexpected since the other disaccharide used,

sucrose, showed no inhibition. For S. cerevisiae a high-affinity proton symport specific for maltose

has been described in maltose-grown cells [19]. Su- crose is hydrolyzed in S. cerevisiae outside the cell to glucose and fructose by invertase, and the resulting

monosaccharides enter the cell via glucose facilita- tors [3]. According to our unpublished data, maltose

and sucrose in H. polymorpha are both hydrolyzed

inside the cell by maltase, and probably both of these disaccharides have to be transported into the cell prior to their intracellular splitting. However, inhib-

ition of glucose transport by maltose shown by us, does not rule out the possibility that maltose might have a transporter of its own induced during the

growth on maltose.

3.6. Efftict of’ metabolic inhibitors und pH on glucose

trunsport systems

To obtain information on the nature of the two different glucose transport systems, sensitivity of glu-

cose transport to the protonophore CCCP and the inhibitor of ATP formation 2,4-dinitrophenol (DNP) was studied. The high-affinity system was highly sen- sitive to the presence of both CCCP and DNP (Table 4). Even 0.05 mM CCCP caused almost complete inhibition of glucose transport. The low-affinity sys- tem, at the same time, was much less sensitive to these inhibitors. Moreover, the dependence of glu- cose transport velocity on pH was also different for

H. Karp, T Alam&/ FEMS Microbiology Letters 166 (1998) 267-273 213

these two transporters: the high-affinity system was

clearly more sensitive to pH than the low-affinity one (not shown). Both glucose-repressed and glucose- derepressed cells concentrated labeled the nonmeta-

bolizable glucose analogue 2DG inside the cell,

although the concentrating ability of the high-affinity system was about five times higher. Therefore, it

could be supposed that the high-affinity glucose transport in H. polymorpha is a proton symport. At the same time, the nature of the low-affinity glu-

cose transport is not that clear. It might be facili-

tated diffusion, and sensitivity of glucose transport to pH and inhibitors in glucose-repressed cells might

be due to the presence of a small amount of high-

affinity transport component in these cells that was not detected by kinetic analysis. However, alternative

explanations can be considered. For example, low

affinity of glucose transport can be due to two types of independent transporters, one that is an active transport system, and one that is facilitated diffu-

sion. In this case the high-affinity system might be expressed at high sugar concentrations but should have different kinetics. Hopefully, isolation and

study of glucose transport in H. polymorpha mutants lacking the high-affinity glucose transport system would clarify the situation.

Acknowledgments

This work was supported by the Estonian Science Foundation. The authors wish to thank Dr. R. Rog-

genkamp for providing the yeast strain and Dr. A. Kahru for revising the manuscript.

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