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Journal of Bacteriology, August 1999, p. 4673-4675, Vol. 181, No. 15
E. C. Slater Institute/BioCentrum
Amsterdam, The University of Amsterdam, 1018 TV Amsterdam, The
Netherlands
Received 16 February 1999/Accepted 20 May 1999
A set of Saccharomyces cerevisiae strains with variable
expression of only the high-affinity Hxt7 glucose transporter was constructed by partial deletion of the HXT7 promoter in
vitro and integration of the gene at various copy numbers into the
genome of an hxt1-7 gal2 deletion strain. The glucose
transport capacity increased in strains with higher levels of
HXT7 expression. The consequences for various physiological
properties of varying the glucose transport capacity were examined. The
control coefficient of glucose transport with respect to growth rate
was 0.54. At high extracellular glucose concentrations, both invertase
activity and the rate of oxidative glucose metabolism increased
manyfold with decreasing glucose transport capacity, which is
indicative of release from glucose repression. These results suggest
that the intracellular glucose concentration produces the signal for glucose repression.
Metabolism of glucose in
Saccharomyces cerevisiae proceeds via sugar transport across
the plasma membrane and oxidation to pyruvate via the common glycolytic
pathway. The flux through these steps determines the rates of
fermentation and respiration. The contribution of the individual
enzymatic steps of glycolysis to flux through the pathway has been
examined in S. cerevisiae, with the surprising conclusion
that the enzymes can be overexpressed up to 10-fold without significant
effects on growth or ethanol production (26, 27). These
observations lend support to the proposal (11) that glucose
transport limits the rate of glycolysis. Control of flux by the
transport step may be more pronounced in cells growing at low glucose
concentrations and expressing high-affinity glucose transporters
(28).
Evaluation of the degree to which glycolysis in S. cerevisiae is limited by glucose transport is complicated by the
large number of transporters expressed by that yeast (16).
Metabolic control analysis offers both a theoretical basis and a set of experimental approaches for that evaluation. Metabolic control analysis
describes the control of flux through a metabolic pathway in terms of
the control coefficient of each step. In principle, every step in a
pathway shares the control of that pathway; the sum of the control
coefficients in a pathway is 1 (7, 15). In order to estimate
the control coefficient of an individual step under defined conditions,
its activity should be varied by small amounts and the magnitude of the
effect of each variation on flux should be measured.
Glucose plays a regulatory role in yeast in addition to its importance
as a nutrient. When glucose is available, it is used preferentially to
other carbon sources. This is achieved, in part, by transcriptional
repression of genes that are required for respiratory metabolism and
utilization of other carbon sources (12). The molecular
mechanisms of this signal transduction pathway have been described in
considerable detail (2, 14). However, the nature of the
signal that the cell perceives from glucose in its environment is still unknown.
The promoter of the HXT7 gene in plasmid p21 (encoding
the high-affinity glucose transporter Hxt7; reference
25) was progressively deleted by exonuclease III
deletion (13). Selected deletion mutants were integrated at
the URA3 locus of S. cerevisiae KY73 (MAT Cells of four selected HXT7 integrant strains and isogenic
strains MC996A (wild type; MAT
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Growth and Glucose Repression Are Controlled by
Glucose Transport in Saccharomyces cerevisiae Cells
Containing Only One Glucose Transporter
ABSTRACT
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hxt1
::HIS3::hxt4
hxt5::LEU2 hxt2::HIS3
hxt3::LEU2::hxt6 hxt7::HIS3
gal2::DR ura3-52 MAL2 SUC2 MEL; reference
17). The endpoints of the deletions were determined
by DNA sequence analysis, and the locations and copy numbers of the
integrated HXT7 genes were determined by Southern blotting
with an HXT7-specific oligonucleotide probe (4)
and a DNA probe from the URA3 gene. Isolates with various
HXT7 promoter lengths and copy numbers were selected for
further study based on their growth characteristics on solid glucose
medium (unpublished data).
ura3-52 his3-11,15
leu2-3,112 MAL2 SUC2 GAL MEL; reference
25) and RE607B (HXT7 only; MAT
hxt1::HIS3::hxt4 hxt5::LEU2
hxt2::HIS3 hxt3::LEU2::hxt6
ura3-52 MAL2 SUC2 GAL MEL; reference 25)
were grown in liquid medium containing 1% yeast extract, 2% peptone,
and 1% (approximately 55 mM) glucose. Growth was monitored by
measurement of the optical density at 600 nm (OD600) at
various time points. The residual glucose in the medium at each time
point was determined enzymatically (1). Cells were harvested
at a residual glucose concentration of approximately 40 mM for the
following analyses. Transport of glucose was measured with the 5-s
[14C]glucose uptake assay described by Walsh et al.
(30). For strains containing only HXT7, the
glucose concentrations in the assay were 1 and 10 mM; for wild-type
strain MC996A, transport was assayed at 10 glucose concentrations
ranging from 0.25 to 250 mM. Invertase activity was measured as
described by Walsh et al. (29). The rate of oxygen
consumption by the cultures was measured in an Oxygraph equipped with a
Clark oxygen electrode. Hxt7 protein abundance was estimated by Western
blotting of 10-µg samples of whole-cell extracts with anti-Hxt7
antibody (kind gift of E. Boles) as previously described
(17) and densitometric scanning of the resulting
chemiluminograms. Total cell protein was estimated by the method of
Lowry et al. (18) using bovine serum albumin as the
standard. The results of these analyses are shown in Table 1.
TABLE 1.
Effect of glucose transport on growth rate and glucose
repression of wild-type and Hxt7-only S. cerevisiae strainsa
Glucose transport exerts a high level of control over growth. Wild-type strain MC996A grew faster than all other strains. At this stage of growth, Hxt7 protein was not detected in the wild-type strain; HXT7 expression is low in wild-type S. cerevisiae at glucose concentrations of >20 mM (data not shown; see also reference 4). For the HXT7-only strains, the growth rate correlated well with the level of Hxt7 protein expressed.
The glucose transport capacity of these strains was also correlated with the growth rate. When the logarithm of the growth rate is plotted against the logarithm of the Vmax for glucose transport (Fig. 1), the data fall on a straight line with a slope of 0.54 ± 0.04 (R2 = 0.96). According to metabolic control analysis theory, the slope of the line produced by plotting a flux versus a catalytic activity in double-logarithmic space is equal to the control coefficient of that activity over that flux (8).
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Glucose transport affects glucose repression.
The status of
glucose repression in these cultures was determined by measuring their
invertase activity and oxygen consumption rate. The strains with
reduced glucose transport capacity expressed higher levels of invertase
activity (Table 1). Similarly, the specific oxygen consumption rate was
inversely correlated with transport capacity. The invertase assay used
here measures the total cellular activity of this enzyme. Using
standard culture conditions for repression and derepression of secreted
invertase (23), we found that the repressed level of total
invertase in the wild-type MC996A strain was 361 nmol · min
1 · mg of protein1, and the
derepressed level is 3,897 nmol · min1 · mg
of protein1. By comparison with Table 1, these values
demonstrate that invertase was fully repressed at the highest glucose
uptake capacities and was significantly derepressed at the lowest
uptake capacity.
-galactosidase activity was fully repressed during
growth on glucose. Null mutations of either gene resulted in partial
derepression of -galactosidase, and in a double null mutant strain,
the activity was completely derepressed (31). In S. cerevisiae, the dominant mutations HTR1-23 and
DGT1-1 resulted in decreased levels of HXT gene
expression and glucose transport activity. Both mutations alleviated
glucose repression of enzymes such as invertase, maltase, malate
dehydrogenase, glutamate dehydrogenase, and cytochrome c
oxidase (10, 22). However, it was not resolved whether the
reduced repression levels were consequences of the mutations or of the
reduced glucose transport activities. In an S. cerevisiae
strain with null mutations in HXT1 to HXT7,
glucose repression of maltase was completely relieved. In related
strains with single HXT genes, the extent of glucose
repression was strongly correlated with the glucose consumption rate of
the strain. In particular, increasing the copy number of
HXT1 stepwise from 1 to 3 in this hxt null strain
increased the glucose consumption rate and decreased the maltase
activity (24).
In contrast to these results that suggest that the flux of glucose into
the cell determines the degree of glucose repression, Meijer et al.
(20) found that repression of the SUC2 gene was dependent on the external glucose concentration and was fully derepressed at glucose concentrations of <14 mM. In contrast, the
level of SUC2 expression was independent of the glucose flux.
Mutations of HXK2, encoding hexokinase II, also lead to
relief of glucose repression (6, 19, 21). It has been
pointed out that intracellular glucose is the metabolite that links
glucose transport and hexokinase and that the intracellular glucose
concentration is a likely signal for the glucose repression pathway
(28).
We observed that at lower rates of transport, a higher fraction of
glucose was oxidized via the respiratory pathway (Table 1). Therefore,
the effect on the growth rate of the decrease in glucose uptake was
partly compensated for by a difference in glucose metabolism, with
relatively more glucose being metabolized by oxidative phosphorylation
(which generates more ATP per mole of glucose) at low rates of glucose
uptake. These results demonstrate that glucose transport plays
important roles in determining the relative activities of the
fermentative and respiratory pathways of glucose metabolism, both by
delivering glucose across the plasma membrane to the glycolytic pathway
and by influencing the glucose repression status of various metabolic activities.
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ACKNOWLEDGMENTS |
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This work was financially supported by the Association of Biotechnology Centers in the Netherlands (ABON).
We are grateful to Marco de Groot for constructing plasmid pBCY7.
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FOOTNOTES |
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* Corresponding author. Mailing address: E. C. Slater Institute/BioCentrum Amsterdam, The University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands. Phone: 31 20 525 5125. Fax: 31 20 525 5124. E-mail: k.van.dam{at}chem.uva.nl.
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REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. | Bergmeyer, H. U. 1974. Methods of enzymatic analysis. Verlag Chemie GmbH, Weinheim, Germany. |
| 2. | Carlson, M. 1998. Regulation of glucose utilization in yeast. Curr. Opin. Genet. Dev. 8:560-564[Medline]. |
| 3. | Davies, S. E., and K. M. Brindle. 1992. Effects of overexpression of phosphofructokinase on glycolysis in the yeast Saccharomyces cerevisiae. Biochemistry 31:4729-4735[Medline]. |
| 4. |
Diderich, J. A.,
M. Schepper,
P. van Hoek,
M. A. H. Luttik,
J. P. van Dijken,
J. T. Pronk,
P. Klaassen,
H. F. M. Boelens,
M. J. Teixeira de Mattos,
K. van Dam, and A. L. Kruckeberg.
1999.
Glucose uptake kinetics and transcription of HXT genes in chemostat cultures of Saccharomyces cerevisiae.
J. Biol. Chem.
274:15350-15359 |
| 5. | Diderich, J. A., B. Teusink, J. Valkier, J. Anjos, I. Spencer-Martins, K. van Dam, and M. C. Walsh. Inhibition characteristics of glucose transport and strategies to determine the control of glucose transport on glycolysis in yeast. Submitted for publication. |
| 6. | Entian, K. D. 1980. Genetic and biochemical evidence for hexokinase PII as a key enzyme involved in carbon catabolite repression in yeast. Mol. Gen. Genet. 178:633-637[Medline]. |
| 7. | Fell, D. A. 1992. Metabolic control analysis: a survey of its theoretical and experimental development. Biochem. J. 286:313-330. |
| 8. | Fell, D. A. 1997. Understanding the control of metabolism, 1st ed. Portland Press, London, England. |
| 9. | Galazzo, J. L., and J. E. Bailey. 1990. Fermentation pathway kinetics and metabolic flux control in suspended and immobilized Saccharomyces cerevisiae. Enzyme Microb. Technol. 12:162-172. |
| 10. |
Gamo, F. J.,
M. J. Lafuente, and C. Gancedo.
1994.
The mutation DGT1-1 decreases glucose transport and alleviates carbon catabolite repression in Saccharomyces cerevisiae.
J. Bacteriol.
176:7423-7429 |
| 11. | Gancedo, C., and R. Serrano. 1989. Energy-yielding metabolism, p. 205-259. In A. H. Rose, and J. S. Harrison (ed.), The yeasts, 2nd ed., vol. 3. Academic Press, London, England. |
| 12. |
Gancedo, J. M.
1998.
Yeast carbon catabolite repression.
Microbiol. Mol. Biol. Rev.
62:334-361 |
| 13. | Henikoff, S. 1984. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351-359[Medline]. |
| 14. | Johnston, M. 1999. Feasting, fasting and fermenting: glucose sensing in yeast and other cells. Trends Genet. 15:29-33[Medline]. |
| 15. |
Kacser, H., and J. A. Burns.
1981.
The molecular basis of dominance.
Genetics
97:639-666 |
| 16. | Kruckeberg, A. L. 1996. The hexose transporter family of Saccharomyces cerevisiae. Arch. Microbiol. 166:283-292[Medline]. |
| 17. | Kruckeberg, A. L., L. Ye, J. A. Berden, and K. van Dam. 1999. Functional expression, quantitation and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein. Biochem. J. 339:299-307. |
| 18. |
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr, and R. J. Randall.
1951.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:265-275 |
| 19. |
Ma, H.,
L. M. Bloom,
C. T. Walsh, and D. Botstein.
1989.
The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae.
Mol. Cell. Biol.
9:5643-5649 |
| 20. |
Meijer, M. M.,
J. Boonstra,
A. J. Verkleij, and C. T. Verrips.
1998.
Glucose repression in Saccharomyces cerevisiae is related to the glucose concentration rather than the glucose flux.
J. Biol. Chem.
273:24102-24107 |
| 21. |
Michels, C. A.,
K. M. Hahnenberger, and Y. Sylvestre.
1983.
Pleiotropic mutations regulating resistance to glucose repression in Saccharomyces carlsbergensis are allelic to the structural gene for hexokinase B.
J. Bacteriol.
153:574-578 |
| 22. |
Özcan, S.,
K. Freidel,
A. Leuker, and M. Ciriacy.
1993.
Glucose uptake and catabolite repression in dominant HTR1 mutants of Saccharomyces cerevisiae.
J. Bacteriol.
175:5520-5528 |
| 23. | Özcan, S., L. G. Vallier, J. S. Flick, M. Carlson, and M. Johnston. 1997. Expression of the SUC2 gene of Saccharomyces cerevisiae is induced by low levels of glucose. Yeast 13:127-137[Medline]. |
| 24. | Reifenberger, E., E. Boles, and M. Ciriacy. 1997. Kinetic characterization of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression. Eur. J. Biochem. 245:324-333[Medline]. |
| 25. | Reifenberger, E., K. Freidel, and M. Ciriacy. 1995. Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on glycolytic flux. Mol. Microbiol. 16:157-167[Medline]. |
| 26. | Rosenzweig, R. F. 1992. Regulation of fitness in yeast overexpressing glycolytic enzymes: parameters of growth and viability. Genet. Res. 59:35-48[Medline]. |
| 27. | Schaaff, I., J. Heinisch, and F. K. Zimmermann. 1989. Overproduction of glycolytic enzymes in yeast. Yeast 5:285-290[Medline]. |
| 28. |
Teusink, B.,
J. A. Diderich,
H. V. Westerhoff,
K. van Dam, and M. C. Walsh.
1998.
Intracellular glucose concentration in derepressed yeast cells consuming glucose is high enough to reduce the glucose transport rate by 50%.
J. Bacteriol.
180:556-562 |
| 29. |
Walsh, M. C.,
M. Scholte,
J. Valkier,
H. P. Smits, and K. van Dam.
1996.
Glucose sensing and signalling properties in Saccharomyces cerevisiae require the presence of at least two members of the glucose transporter family.
J. Bacteriol.
178:2593-2597 |
| 30. |
Walsh, M. C.,
H. P. Smits,
M. Scholte, and K. van Dam.
1994.
Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose.
J. Bacteriol.
176:953-958 |
| 31. | Weirich, J., P. Goffrini, P. Kuger, I. Ferrero, and K. D. Breunig. 1997. Influence of mutations in hexose-transporter genes on glucose repression in Kluyveromyces lactis. Eur. J. Biochem. 249:248-257[Medline]. |
Journal of Bacteriology, August 1999, p. 4673-4675, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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