Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK1
Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504 UR-INRA 792, Département de Génie Biochimique et Alimentaire, Institut National des Sciences Appliquées, 31077 Toulouse Cedex 04, France2
Department of Chemistry, Cardiff University, PO Box 912, Cardiff CF10 3TB, UK3
Cardiff School of Biosciences, Cardiff University, PO Box 915, Cardiff CF10 3TL, UK4
Author for correspondence: Alistair J. P. Brown. Tel: +44 1224 273183. Fax: +44 1224 273144. e-mail: al.brown{at}abdn.ac.uk
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ABSTRACT |
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Keywords: Glycolysis, metabolic flux, pyruvate kinase, phosphofructokinase, yeast physiology
Abbreviations: MCA, metabolic control analysis; Pf1k, 6-phosphofructo-1-kinase; Pyk1, pyruvate kinase
a Present address: MRC Radiation and Genome Stability Unit, Harwell, Didcot, Oxfordshire OX11 ORD, UK.
b Present address: Quest International, Menstrie, Clackmannanshire SK11 7ES, UK.
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INTRODUCTION |
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Once hexoses have been transported into the yeast cell and phosphorylated, they have several alternative metabolic routes. Hexose phosphates may be utilized for trehalose, glycogen or cell-wall biosynthesis, or catabolized via the glycolytic or pentose phosphate pathways. The first glycolytic-specific reaction involves the generation of fructose 1,6-bisphosphate. Most glycolytic enzymes catalyse reversible reactions that are also utilized for the opposing pathway, gluconeogenesis. Therefore, early studies on glycolytic regulation focused on those enzymes that catalyse essentially irreversible reactions in this pathway and hence are specific to glycolysis such as 6-phosphofructo-1-kinase (Pf1k) and pyruvate kinase (Pyk1). Both of these enzymes are subject to significant allosteric regulation (Hunsley & Suelter, 1969 ; Bañuelos et al., 1977
; Avigad, 1981
; Bartrons et al., 1982
; Fraenkel, 1982
; Arvantidis & Heinisch, 1994
; Heinisch et al., 1996
). Hence, Pf1k and Pyk1 were viewed as catalysing rate-limiting steps in glycolysis; this view still dominates conventional thinking. In addition, the genes encoding Pf1k and Pyk1 appear to be regulated more tightly than other glycolytic genes. Pyk1 is a homotetramer, the monomer being encoded by PYK1 (Burke et al., 1983
). Pf1k is a heterooctamer, the
and ß subunits being encoded by PFK1 and PFK2 (Heinisch, 1986
). The PYK1, PFK1 and PFK2 genes appear to be regulated at both the transcriptional and translational levels (Moore et al., 1990a
, b
, c
, 1991
). From a genetic viewpoint, it seemed attractive to suggest that these regulatory phenomena might contribute to the control of glycolysis.
Metabolic control analysis (MCA) suggests that all enzymes in a biochemical pathway contribute to some extent to metabolic flux through that pathway (Kacser & Burns, 1973 , 1979
; Fell, 1997
). Under some specific circumstances a particular enzyme might exert a high degree of control over the flux through a pathway and hence be rate limiting with respect to this flux. However, this is unlikely to be the case for enzymes that are subject to allosteric regulation, because according to MCA theory, a consequence of allosteric regulation is the potential transfer of some flux control to other steps in the pathway (Kacser & Burns, 1973
, 1979
; Fell, 1984
, 1997
). This view is reinforced by the general observation that the overexpression of regulatory enzymes subject to feedback inhibition did not significantly increase flux through the corresponding pathways (Fell, 1997
), and the specific observation that the overexpression of individual glycolytic enzymes in yeast did not increase fermentation rates significantly (Schaaff et al., 1989
; Davies & Brindle, 1992)
. Hence, from an MCA perspective, the Pf1k and Pyk1 enzyme levels, controlled in part through PYK1, PFK1 and PFK2 gene regulation, are unlikely to exert a high level of control over glycolytic flux.
To examine these contrasting views of glycolytic regulation in yeast, we have studied the consequences of (a) disturbing the transcriptional and post-transcriptional regulation of the PYK1, PFK1 and PFK2 genes and (b) altering Pyk1 and Pf1k levels upon yeast physiology. The data suggest that gene regulation plays a minor role in the control of glycolytic flux under fermentative growth conditions, and that Pyk1 levels influence both the rate and direction of carbon flux in yeast.
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METHODS |
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Strain construction.
All of the glycolytic mutants constructed in this study were made in S. cerevisiae W303-1B (Thomas & Rothstein, 1989 ). To generate these mutants (Table 1
), promoterless or promoter-containing 3'-truncated versions of the PYK1, PFK1 and PFK2 genes were integrated into the corresponding chromosomal locus (Fig. 1
). To achieve this, nine plasmids were constructed in which the three glycolytic 3'-truncated ORFs lacked a promoter or were fused to the PGK1 or PGK1
767 promoters (Ogden et al., 1986
). All of the necessary PCR amplifications were performed using Pfu polymerase (Stratagene). The nine plasmids were transformed into S. cerevisiae (Gietz & Woods, 1998
) and single-copy integration at the appropriate glycolytic locus confirmed by Southern analysis.
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Southern, Northern and Western blotting.
S. cerevisiae DNA was isolated according to the protocol of Hoffman & Winston (1987) and subjected to Southern blotting (Church & Gilbert, 1984
; Wicksteed et al., 1994
). Standard methods were used for yeast RNA preparation and Northern analysis (Brown, 1994
; Planta et al., 1999
). Signals were quantified by phosphorimaging, and mRNA levels were measured relative to the ACT1 mRNA which acted as an internal control for variations in RNA loading on gels (Moore et al., 1991
; Planta et al., 1999
). Hybridization probes were as follows: PFK1, the 3·2 kb EcoRI fragment from pPFK1 (Heinisch, 1986
); PFK2, the 3 kb EcoRI fragment from pPFK2 (Heinisch, 1986
); PYK1, the 0·89 kb BamHISacII fragment from pPYK1; ACT1, the 1·5 kb HindIIIEcoRI fragment from pSPACT9 (Moore et al., 1991
). Random-primed labelling of DNA fragments was carried out using [
-32P]dCTP with the Pharmacia Ready-To-Go DNA Labelling Kit, which is based on the method of Feinberg & Vogelstein (1983)
.
For extraction of total soluble protein from yeast, 50 ml cells was grown to mid-exponential phase in YPD or SC at 30 °C. Cells were harvested at 3000 g for 5 min, washed twice in 1 M sorbitol and resuspended in 200300 µl extraction buffer [20 mM HEPES, pH 7·5, 0·2 mM EDTA, 1·5 mM MgCl2, 25% (w/v) glycerol, 0·5 mM DTT, Complete protease inhibitor cocktail tablets (Boehringer Mannheim)]. Samples were vortexed with glass beads six times for 1 min with 1 min intervals on ice between vortexing and centrifuged at 14000 g for 15 min at 4 °C. Supernatants were stored at -80 °C in extraction buffer containing 2 mM fructose 2,6-bisphosphate (Reibstein et al., 1986 ). Protein extracts were electrophoresed on SDS-polyacrylamide gels and Western analysis performed using published protocols (Laemmli, 1970
; Towbin et al., 1979
). Nitrocellulose membranes were probed using a polyclonal antiserum against yeast Pfk (Heinisch et al., 1996
). The second antibody was anti-rabbit IgGalkaline phosphatase conjugate (Boehringer Mannheim).
Enzyme assays.
Pyruvate kinase assays were carried out as described previously (Hunsley & Suelter, 1969 ; Yun et al., 1976
). Phosphofructo-1-kinase assays were carried out in duplicate using previously published procedures (Racker, 1947
; Reibstein et al., 1986
). Protein determinations were performed using the Bradford assay (Bradford, 1976
).
Metabolite assays.
Glucose, ethanol and glycerol assays were carried out in triplicate using Boehringer Mannheim kits. Methylglyoxal assays were performed in triplicate using the procedure of Ferguson et al. (1995) . Measurements of intracellular metabolites (glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate and ATP) after a glucose pulse to yeast cells were performed by coupling with NADH production or consumption as described by Bergmeyer (1986)
. Fructose 2,6-bisphosphate was measured according to Van Schaftingen et al. (1982)
. For the glucose-pulse experiment, yeast strains were grown to an OD600 of 2·0 in GYNB, harvested by centrifugation, washed once with water and resuspended at an OD600 of 5·0 in 25 mM potassium phosphate buffer, pH 6·0. After 30 min pre-incubation, glucose was added to 1%, and 5 ml samples were collected at various times and quenched in cold methanol. Metabolites were extracted in buffered ethanol as described in Gonzalez et al. (1997)
. Errors for metabolite assays were between 5 and 10%.
NMR.
Briefly, cells were grown to an OD600 of 0·6 and labelled with 20 mg [1-13C]glucose ml-1 (99 atom%; Sigma) in SC medium for 45 min at 30 °C. The cells were harvested by centrifugation and perchloric acid extracts were prepared for 13C NMR as described previously (Dickinson & Hewlins, 1988 , 1991
). The spectra were recorded as described by Dickinson et al. (1997)
using a Bruker AMX360 spectrometer operating at 90·5 MHz using 32 K data points over 22 kHz with Waltz-16 1H decoupling and by the DEPT method to determine the number of protons attached to each carbon atom. Signals were identified by comparison with spectra of standard compounds recorded under identical conditions. Standard Bruker software (UXNMR) was used throughout. Chemical shifts were reported relative to external tetramethyl silane in C2HCl3. Addition of sodium trimethylsilylpropanesulphonate gave a methyl signal at -2·6 p.p.m. under the conditions used here.
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RESULTS |
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A second aim was to establish the effects of down-regulating Pyk1 and Pf1k levels. To address this aim, a second set of mutants was made in which the PYK1, PFK1 and PFK2 loci were placed under the control of the PGK1767 promoter and 5' leader sequences. This promoter lacks the PGK1 upstream activator sequence required for efficient and regulated expression (-389 to -473 with respect to the ATG; Ogden et al., 1986
; Piper et al., 1988
).
The strategy for the construction of these mutants involved the integration of 3'-truncated ORFs fused to the new promoter region (Fig. 1). Single-copy integration at the correct glycolytic locus was confirmed for each mutant by Southern blotting (e.g. Fig. 1c
). This strategy generated a tandemly repeated locus where the native glycolytic promoter drives a 3'-truncated version of the gene and the new promoter drives the synthesis of the wild-type glycolytic enzyme.
Expression of PYK1, PFK1 and PFK2 in the mutants
For this strategy to be successful, the PYK1, PFK1 and PFK2 genes had to be inactivated by the 3'-truncation event (Fig. 1b). To test this presumption, promoterless mutants were also made. As expected, the promoterless pyk1 and pfk1 pfk2 mutants (YKC21 and YKC24) were unable to grow on glucose. Furthermore, the PYK1 mRNA was not detectable by Northern analysis in the pyk1 mutant, and the PFK1 and PFK2 mRNAs were not detectable in the pfk1 pfk2 double mutant, following growth of these strains on YPL (not shown). Therefore, 3' truncation did inactivate each of these loci, and the synthesis of Pyk1 and Pf1k was dependent on the PGK1 or PGK1
767 promoters in the mutants.
PYK1, PFK1 and PFK2 expression levels were examined in the mutants during growth on glucose. Relative to their congenic parent (W303-1B), PYK1 mRNA levels were slightly elevated in the mutant carrying the PGK1PYK1 fusion (YKC1; 150%) and were reduced in the strain with the PGK1767PYK1 fusion (YKC11; 43%; Table 3
). Analogous observations were made for the corresponding double PFK mutants: PFK1 and PFK2 mRNA levels were elevated in YKC4 (240%) and reduced in YKC14 (40%; Table 4
, Fig. 2b
). Pyk1 and Pf1k assays confirmed that the reductions in mRNA levels had led to roughly equivalent changes in the levels of these enzymes (Tables 3
and 4
). In addition, decreased levels of each Pf1k subunit (Pfk1p and Pfk2p) were confirmed in YKC14 by Western blotting (Fig. 2b
). The decreases in PYK1, PFK1 and PFK2 expression levels observed for the chromosomally integrated PGK1
767 fusions (about 2·5-fold) were not as great as for those observed previously for a multicopy PGK1
767PGK1 gene (Ogden et al., 1986
; Piper et al., 1988
). Nevertheless, mutants containing the PGK1
767 fusions did display decreased Pyk1 and Pf1k levels, and those carrying the PGK1 promoter fusions had slightly elevated PYK1 and PFK expression levels.
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Metabolite levels in PGK1767 mutants
YKC11 metabolism was examined further by measuring the concentrations of intracellular metabolites following a pulse of glucose (Fig. 6). Glucose 6-phosphate, fructose 6-phosphate and ATP levels were increased in YKC11 compared to W303-1B, and fructose 2,6-bisphosphate and trehalose 6-phosphate levels were decreased. Fructose 1,6-bisphosphate and pyruvate levels were not affected significantly. Hence the reduction in Pyk1 levels had altered the levels of some glycolytic intermediates, effectors and ATP. However, the observed reduction in trehalose 6-phosphate levels, the maintenance of normal pyruvate levels and the increase in ATP were not consistent with a simple model involving a metabolic bottleneck at Pyk1.
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13C NMR analysis of glucose utilization
Glucose consumption per unit biomass was normal in YKC11, but ethanol and glycerol production was significantly decreased (Figs 4 and 5
). Therefore, 13C NMR was used to examine the metabolic fate of the carbon utilized by YKC11. YKC11 and W3030-1B cells were labelled with [1-13C]glucose, and cell extracts subjected to NMR (Fig. 7
). In addition to the C-1 peaks for
-D-glucose (
1) and ß-D-glucose (ß1), three prominent signals were observed for W303-1B. Peaks E2 and 1,3 correspond to the expected glycolytic labelling of the C-2 of ethanol and C-1,3 of glycerol. Peak T1 corresponds to C-1,1' of trehalose, labelled through flux from glucose 6-phosphate. Most other peaks are indicative of glutamine and glutamate labelling via flux through the TCA cycle.
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Increased TCA cycling might indicate that, even during growth under fermentative conditions, YKC11 is more dependent upon mitochondrial metabolism than its wild-type parent. Also, Arvantidis & Heinisch (1994) have shown previously that a pfk2 deletion mutant is respiration dependent. Therefore, we compared the effects of ethidium bromide (an inhibitor of mitochondrial function) upon YKC11 and W303-1B. The zone of growth inhibition caused by ethidium bromide was significantly greater for YKC11 (45±7 mm) than for W303-1B (12±1·5 mm), YKC14 (11±1·5 mm) or YKC15 (13±2 mm). Furthermore, unlike these other strains, YKC11 did not yield any petite colonies as a result of ethidium bromide treatment. These observations suggest that mitochondrial metabolism is required for growth of YKC11 on glucose.
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DISCUSSION |
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Placing PYK1, PFK1 and/or PFK2 under the control of the PGK1 promoter did not have any significant effect upon growth rate on glucose or lactate, or glucose or ethanol consumption (Fig. 3). Therefore, the relatively acute genetic regulation of these loci (Moore et al., 1990a
, b
, c
, 1991
) is not essential for growth under fermentative or gluconeogenic conditions. Tight transcriptional and translational control of, for example, PYK1 might increase the competitiveness of yeast under some conditions. Alternatively, controls at the genetic and biochemical levels may be partially redundant with respect to glycolytic regulation.
Pf1k and Pyk1 levels were elevated in YKC1 and YKC4 (Tables 3 and 4
), but no significant increase in glycolytic flux was observed (Fig. 3
). These data are consistent with those of Schaaf et al. (1989
) who reported that glycolytic flux remains unaffected by overexpression of hexokinase, Pf1k, phosphoglycerate kinase, phosphoglycerate mutase, Pyk1, pyruvate decarboxylase or alcohol dehydrogenase. Davies & Brindle (1992)
have also shown that Pf1k overexpression does not affect glycolytic flux significantly. All of these studies have examined engineered yeast cells following a lengthy period of adaptation. Nevertheless, the data all suggest that none of these enzymes exert a high degree of control over glycolytic flux under fermentative conditions. Most of this control is thought to lie at the level of glucose import (Ye et al., 1999
).
Pf1k appears to exert a relatively small amount of control over glycolytic flux under the conditions tested (Figs 3 and 4
; Schaaf et al., 1989
; Davies & Brindle, 1992
), and yet this enzyme is subject to tight allosteric regulation (Bañuelos et al., 1997
; Avigad, 1981
; Bartrons et al., 1982
; Fraenkel, 1982
; Arvantidis & Heinisch, 1994
; Heinisch et al., 1996
). How can this be rationalized? In fact, the principles of MCA (Kacser & Burns, 1973
, 1979
; Fell, 1984
, 1997
) predict that enzymes that display tight allosteric regulation may exert a low degree of flux control with respect to the pathway as a whole. In the case of yeast Pf1k, compensatory changes in fructose 2,6-bisphosphate may act to buffer the system under metabolic duress to maintain essential glycolytic flux (Davies & Brindle, 1992
). A relatively modest reduction in the level of Pf1k was achieved in this study (
40% decrease). The elasticity of this system, achieved by compensatory modulation of fructose 2,6-bisphosphate levels, might have limits. In other words, larger changes in Pf1k level might have more significant effects upon glycolytic flux.
Decreasing Pyk1 levels had unexpected effects upon the physiology of YKC11. Rates of growth and glucose consumption were reduced significantly (approx. 65% of wild-type), indicating that glycolytic flux was reduced in this strain. Hence, Pyk1 exerts some degree of control over glycolytic flux under fermentative growth conditions. Although glucose consumption per unit biomass was normal in YKC11 (Fig. 4), ethanol and glycerol production were reduced (Figs 4
and 5
), and TCA cycling was increased (Fig. 7
). It seems counterintuitive to suggest that a decrease in Pyk1 caused increased TCA cycling. Nevertheless, the data clearly indicate that both the rate and direction of carbon flux was affected in this strain (YKC11).
It is not clear how a decrease in Pyk1 caused this apparent switch from fermentative to respiratory metabolism. The switch from fermentative to respiratory metabolism in yeast has been correlated with decreased growth rate (Postma et al., 1989 ) or glucose concentration (Meijer et al., 1998
). Neither can account for the phenotypic differences observed for YKC11, YKC15 and W303-1B. YKC11 and YKC15 grew more slowly than W303-1B under conditions of glucose excess, yet only YKC11 displayed decreased ethanol production and increased mitochondrial dependence. Whatever the mechanism involved, our data suggest that Pyk1 exerts a significant level of control over both the rate and direction of carbon flux in yeast during growth on glucose.
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ACKNOWLEDGEMENTS |
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Received 7 July 2000;
revised 6 November 2000;
accepted 7 November 2000.