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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.
E. C. Slater Institute/BioCentrum Amsterdam, The University of Amsterdam, 1018 TV Amsterdam, The Netherlands
Received 16 February 1999/Accepted 20 May 1999
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ABSTRACT |
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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.
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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
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).
Cells of four selected HXT7 integrant strains and isogenic
strains MC996A (wild type; MAT 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.
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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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