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Journal of Bacteriology, September 2001, p. 5223-5229, Vol. 183, No. 18
Institut für Genetik,
Martin-Luther-Universität Halle-Wittenberg, D-06099
Halle,1 and Institut für
Mikrobiologie, Heinrich-Heine-Universität Düsseldorf,
D-40225 Düsseldorf,2 Germany
Received 5 April 2001/Accepted 29 June 2001
In the respirofermentative yeast Kluyveromyces
lactis, only a single genetic locus encodes glucose
transporters that can support fermentative growth. This locus is
polymorphic in wild-type isolates carrying either KHT1
and KHT2, two tandemly arranged HXT-like genes, or RAG1, a low-affinity transporter gene that
arose by recombination between KHT1 and
KHT2. Here we show that KHT1 is a
glucose-induced gene encoding a low-affinity transporter very similar
to Rag1p. Kht2p has a lower Km (3.7 mM) and a more complex regulation. Transcription is high in the absence of glucose, further induced by low glucose concentrations, and repressed at higher glucose concentrations. The response of
KHT1 and KHT2 gene regulation to high but
not to low concentrations of glucose depends on glucose transport. The
function of either Kht1p or Kht2p is sufficient to mediate the
characteristic response to high glucose, which is impaired in a
kht1 kht2 deletion mutant. Thus, the KHT
genes are subject to mutual feedback regulation. Moreover, glucose
repression of the endogenous Most organisms have evolved
sophisticated regulatory strategies to adapt their metabolism to the
availability of nutrients. Substrate uptake is a first key function
that is regulated. Signals that control substrate uptake depend on the
nature and concentration of available nutrients and the nutritional
state of the cell. Since substrate uptake feeds back on the nutritional
state, a regulatory circuit exists, the components of which are only
beginning to be understood even in such intensely studied pathways as
glycolysis in Saccharomyces cerevisiae (see reference
20 for a recent review).
S. cerevisiae cells are apparently able to sense the
extracellular glucose concentration and transmit the signal over the membrane into the cytoplasm (20, 32). Two types of
receptors have been proposed: hexose transporter-like receptors, which
are involved in controlling hexose transporter (HXT) gene
expression (30), and a G protein-coupled receptor that is
required for the activation of the protein kinase A signaling pathway
by cyclic AMP (22, 32, 36).
Intracellularly, glucose-phosphorylating enzymes play an important role
in glucose regulation. Whether these enzymes exert their influence on
glucose regulation through their metabolic activity or whether they
function as intracellular signaling molecules is still a controversial
issue (13, 15, 19, 33).
A signaling function has clearly been established for the
galactose-phosphorylating enzyme of the yeast Kluyveromyces
lactis. This galactokinase (KlGal1p) is required to activate
transcription of lactose and galactose metabolic genes
(26). Upon binding of its substrates galactose and ATP,
KlGal1p can interact with the KlGal80 protein, an inhibitor of the
transcription activator KlGal4p (an ortholog of S. cerevisiae Gal4p) (45). KlGal1p-KlGal80p interaction
relieves KlGal4p inhibition and does not require galactokinase enzymatic activity. The KlGAL80 gene is also under control
of KlGal4p, and KlGAL80 induction counteracts KlGal4p
activation (44). Thus, the dynamics of induction depends
crucially on the dynamics of KlGal80p inactivation. This, in turn,
depends on the rate of lactose and galactose uptake, since the
signaling molecule is intracellular galactose.
The induction process can be impaired in the presence of glucose if the
concentration of KlGal4p is below a critical threshold (34,
43). Transcription of the LAC4-LAC12
genes, encoding By screening for reduced By complementation of these mutants, two new K. lactis
hexose transporter genes were isolated, KHT1 and
KHT2 (38). These genes are closely linked and
tandemly transcribed. Mutations in any of these genes slightly reduced
the repression by glucose of lactose induction, whereas in the
kht1 kht2 double mutant, the glucose effect was completely
abolished. Not only the inducible LAC/GAL regulon, but also
glucose repression of lactate dehydrogenase and malate dehydrogenase,
was affected in the kht1 kht2 mutant.
KHT1 and KHT2 map to the same chromosomal locus
as the low-affinity glucose transporter gene RAG1 described
earlier (11). The sequence of RAG1 is almost
identical to that of KHT1, except for the 3' end (encoding
K-A-M-L in RAG1 and K-R-F in KHT1), which is
identical to the 3' end of KHT2, indicating that
RAG1 arose by recombination between KHT1 and
KHT2 (38).
The presence of KHT1 and KHT2 correlated with a
higher sensitivity to glucose repression found in only a few K. lactis strains (7). A natural isolate that was
entirely insensitive to glucose repression was shown to carry a
defective rag1 allele (18). These findings
suggested that glucose repression in K. lactis depends on
particular glucose transporters.
The KHT1/RAG1-KHT2 gene cluster is the only genetic locus in
the respirofermentative yeast K. lactis that contains
glucose permeases relevant for fermentative metabolism.
Another hexose transporter gene, HGT1, encoding a
high-affinity transporter has a transport capacity too low to suppress
the so-called "Rag Strains and culture conditions.
The yeast strains used in
this study are listed in Table 1. Strains
JA6/CM57, JA6/CM58, DT12R/57, and DT12R/58 arose from ectopic
integration of BamHI-linearized plasmids pBM3157 and
pBM3158, respectively, into the genomes of strains JA6/DL4R
(lac4) and JA6/DT12R (kht1 kht2). JA6/CM57 and
JA6/CM58 carry single integrations, as shown by Southern blot
hybridization, whereas DT12R/57 and DT12R/58 carry multiple copies.
Cells were grown in batch culture at 30°C in synthetic minimal medium
containing (per liter) 6.7 g of yeast nitrogen base (YNB) without
amino acids (Difco) supplemented with required amino acids and bases.
Carbon sources were routinely added at 2% (wt/vol) for glucose and
galactose and 3% (wt/vol) for glycerol. Solid media were prepared by
adding Bacto agar (Difco) to a final concentration of 2% (wt/vol). The
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.18.5223-5229.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Feedback Regulation of Glucose Transporter Gene Transcription
in Kluyveromyces lactis by Glucose Uptake
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
-galactosidase (LAC4)
promoter and glucose induction of pyruvate decarboxylase were abolished
in the kht1 kht2 mutant. These phenotypes could be
partially restored by HXT gene family members from
Saccharomyces cerevisiae. The results indicate that the
specific responses to high but not to low glucose concentrations
require a high rate of glucose uptake.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
-galactosidase and lactose permease, respectively,
and controlled by KlGal4p from a large bidirectional promoter (9,
17, 35), is particularly sensitive to glucose.
-galactosidase expression in
glucose-lactose medium, mutations in glucose transporters were obtained that reduced the inhibitory effect of glucose (38).
" phenotype of
rag1 mutants (Rag+ = resistant against
antimycin A on glucose) (18). A rag1 hgt1 double mutant like the kht1 kht2 mutant is still able to
grow on glucose as a carbon source, indicating the presence of more still unidentified glucose transporters (5). Here we
extend the studies on glucose transport in K. lactis by
characterizing the Kht1p and Kht2p transport kinetics and by analyzing
regulation of the KHT1 and KHT2 genes. We show
that transporter gene regulation is influenced by transport activity
and that Kht1p and Kht2p mutually control each other, depending on
glucose availability. In the absence of both transporters, the high
glucose response is impaired, supporting the view that the rate of
sugar transport is a crucial parameter in intracellular glucose signaling.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
-galactosidase activity of K. lactis clones was checked
on solid medium containing X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside
[40 µg/ml]).
TABLE 1.
Yeast strains used in this study
Yeast transformation.
Competent cells of K. lactis were routinely prepared according to the method of Klebe et
al. (21) and stored at 
70°C (14). Transformation with linearized plasmid DNA for chromosomal integration was performed by the lithium acetate method (1).
DNA manipulation, preparation of yeast RNA, and Northern blot
analysis.
The plasmids used are listed in Table
2. pBM3157 and pBM3158 are derivatives of
plasmid YEp357R (28) carrying the promoter sequences of
KHT1 and KHT2, respectively, as
BamHI-EcoRI fragments fused to 'lacZ.
Both plasmids were kindly supplied by S. Özcan (Lexington, Ky.).
The isolation of total DNA from K. lactis was done as
described earlier (7). All DNA techniques were performed according to standard procedures (1, 25).
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70°C. Labeling of DNA probes was done by random-primed DNA
synthesis with [
-32P]dATP by using the Hexa
labeling kit (MBI Fermentas).
-Galactosidase assays.
-Galactosidase activity
(K. lactis Lac4p) was determined in crude extracts at 30°C
as described previously (43). The activity of LacZ fusion
proteins was measured in crude extracts at 37°C in LacZ buffer
(27). In JA6/DT12-derived strains containing lacZ fusion genes, LAC4-derived background
-galactosidase activity was subtracted from the total activity by
including parent strain DT12R in each experiment. The background never
exceeded 15% of the lacZ activity and was not influenced by
the glucose concentration. Protein concentrations were determined
according to the method of Lowry (24) with bovine serum
albumin as a standard.
Glucose determination. Glucose concentrations in the culture supernatants were determined by using the glucose oxidase/peroxidase assay (3).
Glucose transport measurements. Transport of [14C]glucose was determined in 5-s glucose-uptake assays as described previously (31).
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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KHT1 and KHT2 encode functional
glucose transporters with low and intermediate affinity,
respectively.
The fact that KHT1 and KHT2
are both able to complement the Rag phenotype
of a K. lactis rag1 mutant indicated that they encode functional glucose transporters (38). To further
characterize the gene products, we first compared the kinetics of
glucose uptake in a wild-type strain carrying both genes to that of a
strain containing only RAG1. Glucose transport kinetics was
determined in 5-s-uptake measurements (Fig.
1A). For strain JA6 (KHT1
KHT2), a biphasic curve with a high-affinity branch
(Km 
4.5 mM) was obtained. In strain
ST105 (RAG1), the high-affinity branch was missing. Exact
quantitation of Km was difficult in this
strain due to high background at the high substrate concentrations; a rough estimate of 80 mM may not be significantly different from the
Km of 20 to 50 mM described before for
RAG1 (39).
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Expression of KHT1 and KHT2 is
differentially regulated by glucose.
To study the regulation of
the KHT1 and KHT2 genes, KHT1 and
KHT2 promoter-driven lacZ reporter gene
expression was analyzed. The fusion genes were integrated into the
chromosome of K. lactis strain JA6/DL4R (KHT1 KHT2
lac4) mutated for the endogenous -galactosidase gene, and
reporter gene expression was measured in cells shifted from glycerol to
different concentrations of glucose (Fig.
2). For strain JA6/CM57 carrying the
KHT1-'lacZ fusion, -galactosidase activity was
low in glycerol and induced by glucose. Enzyme activity increased with
the external glucose concentration up to a concentration of about 2%
(100 mM) (Fig. 2) (data not shown) and proceeded slowly with about a
twofold increase in 4 h.
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, 0.1% glucose 
2% glucose 
, and 5% glucose 
). During
batch cultivation at 30°C, samples were taken at the indicated time
points for determination of lacZ activity (solid
symbols) and cell density (open symbols with dotted lines) OD600,
optical density at 600 nm.
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The kht1 kht2 double mutation eliminates the high glucose response of the KHT1 and KHT2 genes. To examine the dependence of gene regulation on the structural integrity of the KHT transporter genes, the kht1, kht2, and kht1 kht2 mutants described previously (38) were analyzed in parallel to the wild type (Fig. 3). Interpretation of the banding pattern in Northern blots is complicated by the fact that the kht2 mutant had been generated by introducing the RAG1 gene into the kht1 kht2 deletion instead of KHT1. Since RAG1 differs at the very 3' end from KHT1, it gives rise to an mRNA that is slightly longer than in the parent strain and migrates at the same position as the KHT2 transcript (Fig. 3A, lane 3). With a short 5' probe, the KHT1 and RAG1-specific signals were identified (Fig. 3B). KHT1 promoter activity could be monitored even in kht1 mutants, since a small kht1::URA3 fusion was formed. This fusion transcript migrated close to the HHT1 transcript used as a loading control (Fig. 3A); therefore, the two probes were applied sequentially to the same filter (Fig. 3B, top and middle panels).
The intensity of the KHT1- and kht1::URA3 band was stronger on glucose (2%) than on glycerol in all four strains examined (Fig. 3B, lanes 1, 3, 5, and 7). However, in the kht1 kht2 double mutant, the induced level was reduced compared to those of the wild type and single mutants, indicating that at least one of the KHT genes is required for full induction of KHT1 and that the two genes can substitute for one another. KHT2 transcription could not be analyzed in the same way, since no KHT2-specific transcript was formed in kht2 mutants. (Note that the signal in Fig. 3A, lane 3, is RAG1.) Therefore, the KHT-'lacZ fusions were integrated into the genome of the kht1 kht2 strain JA6/DT12R, and the influence of the mutations on KHT1 and KHT2 promoter activities was determined by reporter gene assays (Table 3). For the KHT1 promoter, no influence of the kht1 kht2 mutations was detectable in low glucose (0.1%). The wild type and mutant had roughly the same ratio of-galactosidase activity in glycerol versus glucose
(2.7- and 3.3-fold, respectively). However, in 2% glucose, induction
in the double mutant was weaker than in the wild type (3.3-fold versus 5.1-fold, respectively), in agreement with the results obtained by
Northern analysis (Fig. 3).
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Several transporter genes restore the high-glucose response in kht1 kht2 double mutants. To examine whether complementation of the glucose uptake deficiency was sufficient to restore the high-glucose response in the double mutant, transporter genes were introduced on centromeric plasmids into the kht1 kht2 strains carrying the integrated KHT1-lacZ and KHT2-lacZ fusions, respectively. (Table 3). In transformants with the KHT1-KHT2 tandem genes (plasmid pY10-23), the wild-type regulation of the KHT1 and KHT2 promoters was completely (KHT1-'lacZ) or almost completely (KHT2-'lacZ) restored. Partial induction of the KHT1 promoter and partial repression of the KHT2 promoter by glucose were observed with RAG1 (with plasmid pY10-7), and a weaker effect was seen with the S. cerevisiae HXT1 gene (on pVSH1), encoding a low-affinity hexose transporter.
We also assayed the ability of the S. cerevisiae GAL2 gene to complement the glucose uptake deficiency of the kht1 kht2 mutant strain. The galactose permease encoded by GAL2 had been shown to be able to mediate glucose uptake (23, 31), but the regulation of GAL2 (induction by galactose) suggested that it is not a component of a glucose signaling pathway. To overcome the requirement of galactose for the Gal4p-regulated GAL2 gene, the KlGAL80 gene was disrupted in the kht1 kht2 mutant background. The resulting triple mutant was Rag like the parent, but a centromeric
GAL2 plasmid clearly improved growth on 2% glucose plus
antimycin A, an effect not observed when the KlGAL80 gene
was functional (data not shown). Thus, Gal2p was able to function as a
glucose transporter in K. lactis.
The triple mutant was used to analyze regulation of the LAC4
promoter (Table 3). The KHT1 KHT2 Klgal80 strain showed
fivefold-lower LAC4-encoded -galactosidase activity on
glucose compared to glycerol (13,500 mU/mg of protein). This implies
that glucose not only inhibits galactose induction of the
LAC/GAL genes, but also represses the induced promoter to
some extent. This form of glucose repression was abolished in the
kht1 kht2 Klgal80 triple mutant. Again, repression could be
restored by transformation with the RAG1- as well as with
the GAL2-containing centromeric plasmid; ScHXT1
gave a weaker effect.
We also analyzed glucose induction of the glycolytic enzyme pyruvate
decarboxylase encoded by the unique glucose-inducible gene,
KlPDC1 (4, 12). In glycerol-grown cells,
pyruvate decarboxylase activities varied between 60 and 260 mU/mg of
protein. A fivefold induction by glucose observed in the
Klgal80 mutant was completely abolished in the kht1
kht2 Klgal80 mutant and could be fully or partially restored by
GAL2 and RAG1, respectively. No effect was observed with S. cerevisiae HXT1 in this case.
Thus, the kht1 kht2 mutation had a broad influence on
glucose-regulated gene expression in general and was not restricted to
the inducible LAC/GAL regulon (38). Formally,
the possibility exists that the KHT gene products have
regulatory activity in addition to their transport function, like the
S. cerevisiae Snf3p and Rgt2p proteins, which serve as
membrane-bound high- and low-affinity glucose receptors, respectively
(30).
However, there is no indication that Kht1p and Kht2p are such glucose
sensors. (i) Based on sequence similarity (65% identity), KHT1 and KHT2 clearly belong to the
HXT gene family, with much weaker resemblance to
SNF3 and RGT2 (25% identity), and the
cytoplasmic tail characteristic of the latter gene products is not
found in Kht1p and Kht2p. (ii) Both KHT1 and KHT2
encode functional glucose transporters, and the regulatory phenotype
displayed by the kht1 kht2 double mutant could be restored
by S. cerevisiae hexose transporter genes such as
HXT1 and GAL2. (iii) There is a correlation
between glucose uptake capacity and glucose signaling. The Hxt1p
transporter, which has a very low affinity for glucose and requires
high glucose concentrations for induction (29), had a
weaker effect on glucose repression and induction than RAG1
and GAL2. The correlation between glucose uptake and the
glucose response of gene expression is consistent with recent results
from S. cerevisiae showing a correlation between relief from
glucose repression and the decrease in glucose transport capacity. Even
under high-glucose conditions, an increase in SUC2
expression was detected when cells displayed reduced glucose uptake
(31, 42).
We conclude that Kht1p and Kht2p influence regulation through their
transport function and that glucose sensing occurs intracellularly.
An intracellular sensing mechanism for glucose does not exclude an
additional extracellular sensor. The RAG4 gene conferring a
Rag phenotype when mutated encodes an
Snf3/Rgt2-related protein with a long C-terminal cytoplasmic tail
containing the conserved sequence motif (3a). It is a
regulator of RAG1 and may mediate the low glucose response
of KHT1/RAG1, which we have shown here to be independent of
KHT1 and KHT2. A combination of extracellular and intracellular sensing mechanisms would ensure that the high-glucose response is only triggered if the cell is able to metabolize the available glucose.
We propose that in K. lactis glucose repression correlates
with fermentative metabolism. Both forms of regulation require the
transport capacity of the cell to exceed a threshold level. This level
cannot be reached in the kht1 kht2 or rag1
mutant. The presence of either KHT1/RAG1 or KHT2
alone is sufficient to overcome the threshold. By a self-sustaining
process caused by further induction of KHT1/RAG1 and other
fermentative genes (like the pyruvate decarboxylase gene
KlPDC1) and by favoring the utilization of glucose over
other carbon sources through glucose repression, the metabolism is
shifted towards fermentation. The presence of both genes
KHT1 and KHT2 favors this metabolic shift due to
the high basal expression of KHT2 resulting in a higher
uptake capacity in noninduced cells. Since KHT2 itself is
subject to glucose repression, the transporter with the higher
Km, Kht2p, is replaced by the one with the
lower Km if there is a high and constant
supply of glucose. The slow and concentration-dependent induction of
Kht1p/Rag1p enables the cells to monitor the availability of glucose
over time; its low-affinity ensures a concentration-dependent rate of
transport at subsaturating glucose levels.
In S. cerevisiae, the abundance of hexose transporter genes
and their specific regulation indicate that this yeast tries to sustain
the highest possible glucose transport activity, such that the
glycolytic flux is largely determined by the extracellular glucose
concentration. In contrast, the respirofermentative yeast K. lactis does not fully exploit its glucose uptake capacity
during oxidative growth. Sugar uptake may therefore be primarily
controlled by the availability of oxygen and by the cellular demand
rather than by the extracellular supply (8).
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ACKNOWLEDGMENTS |
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This work was supported by EU grant BIO4-CT96-0003 to K.D.B.
We are grateful to S. Özcan for providing plasmids and unpublished data, to M.Wésolowski-Louvel for communicating results prior to publication, to A. Kruckeberg for stimulating discussion, and to P. Kuger for excellent technical support.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Genetik, Martin-Luther-Universität Halle-Wittenberg, D-06099 Halle, Germany. Phone: 49-345-5526301. Fax: 49-345-5527151. E-mail: breunig{at}genetik.uni-halle.de.
Present address: Leibniz-Institut für Pflanzenbiochemie,
Abteilung Sekundärstoffwechsel, 06120 Halle (Saale), Germany.
Present address: Aventis Pasteur MSD Gmbh, 69181 Leimen, Germany.
§ Present address: Institut für Vegetative Physiologie, D-50931 Köln, Germany.
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Journal of Bacteriology, September 2001, p. 5223-5229, Vol. 183, No. 18
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.18.5223-5229.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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