Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands
Correspondence
Joost Teixeira de Mattos
teixeira{at}science.uva.nl
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ABSTRACT |
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Present address: University of Warsaw, Warsaw, Poland.
Present address: EMBO, Heidelberg, Germany.
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INTRODUCTION |
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The glucose repression pathway regulates a large number of genes, including those involved in metabolism of sugars other than glucose (SUC/MAL/GAL genes) and genes encoding proteins required for respiration (Gancedo, 1998). One of the downstream effectors of the glucose repression pathway is Mig1p, a transcriptional repressor that is translocated from the cytoplasm to the nucleus when glucose is added to the medium (De Vit et al., 1997
). Mig2p is a homologue of Mig1p, which remains localized in the nucleus in response to glucose and which probably performs a less important role in glucose repression than Mig1p (Lutfiyya & Johnston, 1996
). Another component of the glucose repression pathway is Hxk2p. This glycolytic enzyme also acts as a transcription regulatory protein by binding to the glucose-repressed SUC2 promoter as a complex with other proteins (Herrero et al., 1998
).
Since overexpression of the HAP4 gene enables mitochondrial biogenesis to escape from glucose repression (Lascaris et al., 2002), the question arises whether an additional defect in the glucose repression pathway may modify the effect of HAP4 overexpression. Since carbon source control is for a large part mediated by the transcription repressor Mig1p which is essential for a functional glucose repression pathway we analysed whether deletion of the glucose repressor gene MIG1 could influence the effect of overexpression of HAP4.
To analyse the double mutant mig1HAP4
we used plate assays that provided information on cellular physiology. Subsequent analysis of the transcriptional changes did not show an effect of deletion of MIG1 on targets of Hap4p, but surprisingly demonstrated a strong feedback control of HAP4 overexpression on several Mig1p-targeted genes. Our results indicate that metabolic pathways that feed glycolysis in glucose-derepressed conditions are under the control of respiration.
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METHODS |
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Growth conditions and 2-deoxyglucose (2-DOG) sensitivity plate assay.
Cells were grown aerobically in YPD (1 % yeast extract, 1 % Bacto-peptone and 2 or 3 % D-glucose), YPRaff (YP containing 3 % raffinose) or YPEG (YP containing 2 % ethanol and 2 % glycerol). In all experiments cells were harvested at early exponential phase. In 2-DOG sensitivity plate assays, cells were spotted at different dilutions (10105) on rich medium plates that contained a carbon source (2 % glucose, 3 % raffinose, 3 % melibiose, 3 % galactose or 2 %/2 % ethanol/glycerol) and 200 µg 2-DOG ml1 (50 µg ml1 for ethanol/glycerol plates).
Northern analysis and microarray experiments.
RNA isolation, Northern blotting, (pre-)hybridizations, washing and visualization were performed as described previously (Lascaris et al., 2000). Probes to detect CYT1, SUC2 and the loading control ACT1 were obtained by 32P[ATP] labelling of PCR-generated DNA fragments. The JEN1 probe was an 844 bp NcoIPstI fragment from the pH3 (pUC18 : : JEN1) plasmid that was kindly provided by Raquel Pego de Andrade (Andrade & Casal, 2001
). Other probes derived from DNA fragments are as described by van Maris et al. (2001)
.
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RESULTS |
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First, we analysed to what extent growth was affected by the glucose analogue 2-DOG (Zimmermann & Scheel, 1977). This glucose analogue triggers signalling of glucose repression, but cannot be metabolized via glycolysis, causing strong inhibition of growth on carbon sources other than glucose. A strain with a defect in the glucose repression system, i.e. a
mig1 deletion strain, has been shown to be less sensitive to 2-DOG as confirmed by our assays (see Fig. 1
). Similar to the
mig1 deletion strain, overexpression of the HAP4 gene also enables cells to escape from glucose repression since they grow better than the corresponding wild-type cells on raffinose, melibiose (Fig. 1
) and galactose (data not shown) medium containing 2-DOG. Importantly, cells in which both the Mig1p repressor is absent and the gene encoding the Hap4p activator is overexpressed grow best in the presence of 2-DOG. This shows that both mutations act additively to relieve glucose repression and indicates that both mutations affect different parts of the glucose repression system.
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Deletion of MIG1 and overproduction of HAP4 do not act additively on the respiratory chain reporters CYT1 and QCR8
We looked at whether the combined deletion of MIG1 and the overexpression of HAP4 result in increased expression of respiratory chain components under glucose-growth conditions. The expression of the respiratory genes CYT1 (Fig. 2) and QCR8 (data not shown) was not strongly derepressed in the glucose-grown
mig1 deletion strain itself. Minor derepression of CYT1, however, can be observed, which is likely to be due to a minor derepression of the endogenous HAP4 gene. Importantly, if the MIG1 gene is deleted in the HAP4 overexpressing strain, no additional increase in transcription can be observed.
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HAP4 overproduction causes strong repression of the derepressed SUC2 gene
Although a mig1 deletion apparently does not contribute significantly in HAP4
-mediated up-regulation of genes involved in mitochondrial function, the converse possibility was also investigated: does overexpression of HAP4 contribute to up-regulation/derepression of Mig1p-regulated genes that are involved in metabolism of sugars other than glucose? For this reason we analysed whether overexpression of HAP4 affects the expression of SUC2, one of the prime targets of Mig1p. As shown in Fig. 3
, overexpression of HAP4 in a
mig1 deletion background does not contribute to derepression of SUC2, but surprisingly restores the repressed state of the SUC2 promoter (compare lanes 2 and 4 in Fig. 3
).
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The Mig1p-regulated genes MAL61, encoding maltose permease, and HXT2, encoding a high-affinity glucose transporter, also exhibit HAP4-mediated repression. Furthermore, from whole-genome expression profiles of glucose-grown
mig1
mig2 and the
hxk2 strains with and without a HAP4 overexpression cassette (J. M. Schuurmans & M. J. Teixeira de Mattos, unpublished data), only nine derepressed genes were found to show a twofold decrease due to overexpression of HAP4: SUC2, HXK1, HSP12, HXT16, MAL11, MAL32, YER067W and the MALx2 homologues FSP2 and YGR287C. This indicates that a small, specific group of genes is affected by HAP4
-mediated repression. HAP4
-mediated repression, however, does not extend to all genes regulated by Mig1p and/or Mig2p. As shown in Fig. 3
, the JEN1 gene, which encodes lactate permease and which is regulated by Mig1p and the co-repressor Mig2p (Bojunga & Entian, 1999
), was not repressed under conditions of HAP4 overexpression.
HAP4-mediated repression is also evident under SUC2-inducing conditions
We further studied whether HAP4-mediated repression also occurs under conditions where the SUC2 gene is required for growth and is fully induced. Wild-type and mutant cells grown on raffinose, a sugar that Suc2p (invertase) hydrolyses to fructose and melibiose, indeed show the repressive action of HAP4 overexpression (Fig. 4
a). This is of interest, since it demonstrates that the repressive effect of HAP4 overexpression also occurs if the glucose repression system is not functional (compare HAP4
and wild-type in Fig. 4a
). Consistently, activating the glucose repression pathway by addition of 2-DOG to raffinose-growing cells results in strong repression of SUC2 expression (Fig. 4b
) in the wild-type (not shown) and HAP4 overexpression strains, but not in the
mig1HAP4
strain. Glucose repressors other than Mig1p apparently play a minor role relative to the repressive effects of Mig1p and of overexpression of HAP4. Taken together, these findings show that the strong repression mediated by overexpression of HAP4 does not require glucose metabolism and is independent of glucose signalling.
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DISCUSSION |
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The question arises why a system has evolved in which the expression of SUC2 follows the respiro-fermentative state of the cell. To answer this question we have to bear in mind that Suc2p (invertase) is required and expression of the SUC2 gene is induced during respiro-fermentative growth on raffinose or sucrose. The respiratory capacity in these conditions is high and the fermentative capacity low. Nevertheless, part of these sugars is fermented to ethanol. Overexpression of HAP4 is likely to enhance the respiratory capacity further so that less sugar will be fermented. This can be expected to result in a decrease in the glycolytic flux and a subsequent increase in glucose concentration in the periplasmic space. It should be noted that relatively minor changes in the balance between respiration and fermentation would have severe effects due to large differences in energy yield between fermentation and respiration. Hence, it is hypothesized that respiratory feedback control may down-regulate invertase activity in order to decrease the periplasmic glucose concentration. Two unfavourable effects of increased periplasmic glucose may be prevented by this control mechanism. First, it may prevent the activation of several signal transduction pathways, i.e. the glucose repression pathway, cAMP signalling and the Snf3/Rtg2 system, stabilizing the balance between respiration and fermentation. Second, it may prevent glucose and also fructose molecules being lost by diffusion from the periplasmic space into the extracellular medium. Loss of fructose has been observed to occur when wild-type cells were grown until near glucose depletion in medium containing a mixture of sucrose and glucose (Raamsdonk, 2000). We suggest that respiratory feedback control is the regulatory mechanism that maintains periplasmic glucose concentrations at a low level and hence stabilizes the balance between respiration and fermentation.
In this system, low periplasmic glucose concentrations may be sensed by the glucose sensor Snf3p, an HXT homologue that acts at low glucose concentrations (Ozcan & Johnston, 1999). Interestingly, Snf3p interacts specifically with Mth1p, a factor involved in glucose repression (Gamo et al., 1994
; Schmidt et al., 1999
). The origin of the signal provided by overexpression of HAP4 increased respiratory activity may be, for instance, the ADP/ATP ratio, the redox state of the cell or oxidative stress formed by mitochondrial activity. Which pathway/signalling mechanism(s) actually is (are) involved is not known.
Glucose-induced mRNA turnover of SUC2 may also be important for the apparently fast degradation of all SUC2 mRNA (Cereghino & Scheffler, 1996) after glucose has been added to the culture. The repression that is mediated by HAP4
on the other hand is unlikely to be due to increased mRNA turnover, since the JEN1 gene does not show HAP4
-mediated repression (see Fig. 3
), whereas the mRNA of JEN1, similar to SUC2 mRNA, is degraded by a glucose-induced mechanism (Andrade & Casal, 2001
).
Glucose repression kinetics of SUC2 in the mig1 strain consists of normal derepression starting at least 1 h after glucose addition, which is unexpectedly slow. When the HAP4 gene is overexpressed, derepression does not occur. However, derepression in this strain can be triggered by blocking mitochondrial function with antimycin A (data not shown). Previously, we have shown that overexpression of HAP4 results in a marked increase in mitochondrial biogenesis (Lascaris et al., 2002
). Since mitochondrial biogenesis in the
mig1 deletion strain is glucose-repressed, similar to the wild-type strain, it may be that the changes in expression of the SUC2 gene correlate with slow repression by glucose of mitochondrial biogenesis and/or function.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 29 August 2003;
revised 19 December 2003;
accepted 7 January 2004.
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