Laboratory of Molecular Genetics, School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai 400 076, India
Correspondence
Paike Jayadeva Bhat
jayadeva{at}btc.iitb.ac.in
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ABSTRACT |
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INTRODUCTION |
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MRG19 was isolated as a multi-copy repressor of galactose toxicity (Kabir et al., 2000). It was observed that disruption of MRG19 resulted in the derepression of CYC1 under conditions of carbon limitation (Khanday et al., 2002
). Based on this, it was suggested that in a wild-type strain, the function of MRG19 is to regulate the oxidation of limited carbon so as to channel it for biosynthesis. The protein is localized in the nucleus and its production is repressed in the presence of glucose in an MIG1-dependent fashion (Khanday et al., 2002
). Genome-wide expression analysis revealed that MRG19 transcript levels increase upon nitrogen starvation (Gasch et al., 2000
), even in the presence of glucose. MRG19 is the only gene that escapes MIG1-dependent glucose repression upon nitrogen starvation, suggesting that MRG19 plays a role in the link between carbon and nitrogen metabolism. In this study, we demonstrate that MRG19 regulates the expression of NADP-glutamate dehydrogenases, which are the key enzymes of ammonia assimilation, encoded by GDH1/3. Upon nitrogen deprivation, the ratio of steady-state levels of 2-oxoglutarate to glutamate increased in the wild-type strain as compared to the MRG19 disruptant. The MRG19 disruptant was defective in the formation of pseudohyphae. The implications of these results are discussed in the context of the metabolic basis of development.
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METHODS |
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Strains.
DNA manipulation was carried out in Escherichia coli strain XL-1 Blue, as described by Sambrook et al. (1989). Yeast strains are listed in Table 1
. The MRG19-disrupted yeast strain YO5449 was obtained from Euroscarf (Institute of Microbiology, Johann Wolfgang Goethe University, Frankfurt, Germany) and its isogenic wild-type strain YO5449WT was obtained by crossing Y05449 with Y10345, followed by screening for geneticin-sensitive haploids. The wild-type and its isogenic MRG19-disrupted strains were crossed with isogenic ScPJB644-L (wild-type) and ScPJBB644-19
(MRG19-disrupted) to obtain YO5449WT/ScPJB644-L and YO5449/ScPJBB644-19
, respectively.
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Quantification of pseudohyphal cells.
Pseudohyphal cells were quantified as described by Mosch et al. (1996). Strains were grown on SLAD medium at 30 °C for 3 days. The noninvasive cells were removed by washing the agar plates with water, and the invasive cells were scraped off the agar. The cells were then suspended in 50 µl water, and analysed for cell shape by light microscopy. The length to width ratio of the cells was determined, and the cells were classified into two groups: yeast-form cells with a length to width ratio less than two, and pseudohyphal cells with a length to width ratio greater than two. A minimum number of 50 cells was analysed in each experiment, and the percentage of pseudohyphal cells in the population studied was determined. The results presented are means of three individual experiments, and the level of significance (t=29·029, d.f.=2 and P value=0·0012) was estimated by calculating paired t-test and P values as described by Freund & Wilson (1993)
.
Image analysis.
A Leica DM LM microscope was used for all microscopic analyses. The images were analysed using Image Analysis Software from Soft Imaging Systems.
Western blot analyses.
Cells were harvested and extracts were prepared as described previously (Khanday et al., 2002). Western blot analyses were carried out as described previously (Khanday et al., 2002
). Protein was estimated as described by Bradford (1976)
. All the experiments were repeated at least three times. As a control, Western blots of the cell extracts were also carried out using glucose-6-phosphate dehydrogenase (Zwf1p) antiserum (Sigma).
-Galactosidase assays.
-Galactosidase activity was assayed in cell extracts, as described by Adams et al. (1997)
. Duplicate samples were taken for each determination. Experiments were repeated at least three times with a different transformant each time. Specific activities are represented as nmol product min1 (mg protein)1.
Extraction of intracellular metabolites.
For the estimation of intracellular metabolites, 1 l cultures were rapidly chilled, sedimented by centrifugation, immediately resuspended in 23 ml distilled water in a thin-walled test tube, and placed in a boiling water bath for 10 min. Samples were cooled in an ice bath, and cell debris was removed by centrifugation at 5000 g for 5 min. The clear supernatant was transferred to a fresh tube, and stored at 20 °C for later analysis (Kang et al., 1982).
Estimation of metabolites.
2-Oxoglutarate was estimated enzymically by using commercial beef glutamate dehydrogenase (Sigma), and following the oxidation of NADPH spectophotometrically at 340 nm. A 1 ml volume of reaction mixture contained 20 mM Tris/HCl pH 8, 2·5 mM NH4Cl, 0·015 units glutamate dehydrogenase µl1 and 400 µl sample. Auto zero was set at 340 nm and 100 µl 1 mM NADPH was added. The decrease in A340 was measured over a period of 2 min. A 2-oxoglutarate standard ranging from 0·025 to 0·1 mM was used (Burlina, 1985). Glutamate was estimated as described by Der Garabedian (1986)
. The protein-free cell extract was first treated to remove interfering and reducing agents by the method described by Beutler (1985)
.
Estimation of sporulation efficiency.
Sporulation efficiency was quantified by growing cells in sporulation medium and incubating at 30 °C for 710 days. At least 300 cells were scored by microscopic examination, and the percentage of spores with respect to the total number of cells was calculated as the sporulation efficiency.
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RESULTS |
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DISCUSSION |
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The ratio of 2-oxoglutarate to glutamate in the wild-type (1·85) and the MRG19 disruption strain (1·35) grown in glycerol/lactate with sufficient ammonium sulphate did not change significantly (although the absolute amounts increased approximately twofold). The levels of 2-oxoglutarate reported here are comparable with those reported elsewhere (DeLuna et al., 2001). In SLAD medium, the ratio of 2-oxoglutarate to glutamate in the wild-type (8·13) is higher than that in the MRG19-disrupted strain (4·4). This is mainly due to a decrease in 2-oxoglutarate [from 30 to 18·6 nmol (mg protein)1] rather than to an increase in the glutamate level [3·69 to 4·2 nmol (mg protein)1] in the MRG19 disruptant, as compared to the wild-type. Therefore, the only discernible difference is in the level of 2-oxoglutarate in the MRG19 disruptant as compared to the wild-type strain in SLAD mediium. This increase, however, does not agree with the observation that GDH1/3 levels showed a decrease in the MRG19-disrupted strain under this condition. It is possible that under nitrogen-insufficient conditions, the GSGOGAT pathway could be more active in MRG19 deletion, thereby reducing the level of 2-oxoglutarate. In addition to this, the retrograde (RTG) system is responsible for providing 2-oxoglutarate for glutamate synthesis in the presence of glucose at low glutamate levels (Parikh et al., 1987
; Liao et al., 1991
; Liu & Butow, 1999
). It is possible that in an MRG19-deletion strain, the RTG system may not be as active as in a wild-type strain. These observations suggest that in a wild-type strain, the ratio of 2-oxoglutarate to glutamate in the nitrogen-depleted condition is significantly higher than that in the nitrogen-sufficient condition. It is clear from our data that this ratio decreases in an MRG19-disrupted strain as compared to the wild-type upon nitrogen depletion. From this, it appears that the MRG19-disrupted strain tends to mimic the nitrogen-sufficient condition upon nitrogen depletion. This difference between the strains in the nitrogen-depleted condition correlates with the decreased ability of the MRG19-disrupted strain to put forth pseudohyphae. Studies have shown that Mrg19p forms a complex with Bmh1/2p (Ho et al., 2002
), which is known to be essential for pseudohyphal formation (Roberts et al., 1997
), suggesting that it could also modulate the signal transduction pathway for pseudohyphal formation.
It is interesting to note that MRG19 expression is switched off when the cell encounters sufficient carbon and nitrogen, and is activated upon carbon or nitrogen starvation. However, when both carbon and nitrogen are depleted, expression of MRG19 is repressed. In the light of the observation that MRG19 is repressed upon sporulation, and our results that disruption of MRG19 increases sporulation, we suggest that MRG19 acts as a suppressor of sporulation. However, the effects of MRG19 disruption on different parameters are moderate, suggesting that redundant pathways regulate the response to limited nutrients. This makes it difficult to assign a specific role to MRG19. Our studies do not provide a mechanistic insight into whether the metabolic perturbation and phenotypic changes observed with respect to MRG19 have a cause and effect relationship, but unravel a hitherto unknown link between the metabolic status of the cell and the corresponding developmental pathway. These observations provide an avenue to further explore the connection between carbon and nitrogen metabolism.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 20 May 2004;
revised 22 July 2004;
accepted 1 October 2004.
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