Centre de Bioingénierie Gilbert Durand, UMR-CNRS 5504, UR-INRA 792, Département de Génie Biochimique et Alimentaire, Complexe Scientifique de Rangueil, 31077 Toulouse, France1
Author for correspondence: Jean François. Tel: +33 5 61 55 94 92. Fax: +33 5 61 55 94 00. e-mail: fran.jm{at}insa-tlse.fr
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ABSTRACT |
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Keywords: glycogen, trehalose, GSY2, respiration, Saccharomyces cerevisiae
Abbreviations: Glc6P, glucose 6-phosphate
a Present address: Department of Genetics and Development, Columbia University, USA.
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INTRODUCTION |
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Biochemical and genetic data accumulated over the last 10 years have unravelled at least two levels of control of glycogen metabolism in yeast. First, structural genes encoding enzymes involved in the biosynthesis and the biodegradation of glycogen are co-ordinately regulated under a wide variety of environmental conditions (Hwang et al., 1989 ; François et al., 1992
; Thon et al., 1992
; Hardy et al., 1994
; Ni & Laporte, 1995
). In particular, the expression of these genes is co-induced when glucose concentration diminishes during diauxic growth by a mechanism which is strongly repressed by the RAS-cAMP pathway (Hardy et al., 1994
; Parrou et al., 1999b
). A second level of control involves covalent modification by reversible phosphorylation of glycogen phosphorylase (Gph1p) and glycogen synthase (Gsy2p) which leads to the activation of the former and the inactivation of the latter. A cluster of three phosphorylation sites at the COOH terminus of Gsy2p has been identified and site-directed mutagenesis of these sites or deletion of a 61 COOH terminus fragment blocks Gsy2p in a hyperactivated form (Hardy & Roach, 1993
). Two protagonists implicated in the control of the Gsy2p phosphorylation state have been characterized: a glycogen synthase kinase consisting of the Pho85pPcl10/Pcl8 complex (Huang et al., 1997
; Wilson et al., 1999
) and a glycogen synthase phosphatase corresponding to the Glc7pGac1p complex (François et al., 1992
; Skroch-Stuart et al., 1994
). In contrast, nothing is known about the protein kinase(s) and protein phosphatase(s) regulating the phosphorylation state of glycogen phosphorylase. Evidence from several experiments demonstrates that glucose 6-phosphate (Glc6P) is a potent activator of the dephosphorylation and an inhibitor of the phosphorylation processes mediated by these protagonists (François & Hers, 1988
; Lin et al., 1996
; Huang et al., 1997
). In addition, Glc6P allosterically stimulates glycogen synthase and inhibits glycogen phosphorylase (reviewed by François et al., 1997
).
The iodine-staining reaction has been widely used to search for mutants affected in glycogen metabolism, since yeast colonies growing on agar plates develop a brown coloration proportional to their glycogen content upon exposure to iodine crystal vapour (Chester, 1968 ). This method led to the identification of mutants with a strong glycogen phenotype, e.g. no glycogen or hyperaccumulation of this polymer. Hence, mutations in components of the Ras-cAMP signalling pathway, in SNF1 or PHO85 genes encoding key protein kinases directly or indirectly implicated in carbon metabolism, were often uncovered (Thompson-Jaeger et al., 1991
; Wek et al., 1992
; Cannon et al., 1994
; Huang et al., 1996
; Timblin et al., 1996
). Interestingly, some isolated glycogen-deficient mutants were also defective in respiration and presented a petite phenotype (Chester, 1968
; Filipak et al., 1992
; Yang et al., 1998
). This linkage between mitochondrial function and glucose storage was recently investigated at the genetic level by Yang et al. (1998)
. These authors suggested that the inability of mitochondrial respiratory mutants to synthesize glycogen was due to the inactivation of glycogen synthase by a Ras-cAMP-dependent, Pho85p-independent mechanism.
As part of our work aiming to identify the mechanism of the early induction of glycogen synthesis during diauxic growth on glucose, we sought mutants defective in this process using a double genetic screen based on the lack of iodine staining of colonies and loss of ß-galactosidase activity from a GSY2lacZ construct. Among the isolated mutants, we observed that one of them, which was respiration-deficient, accumulated glycogen during growth on glucose despite its negative iodine-staining reaction. Hence, the discrepancy between the qualitative iodine-staining assay and the quantitative kinetic analysis of glycogen was further investigated.
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METHODS |
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Biochemical and analytical procedures.
Preparation of extracts and assay of glycogen phosphorylase and glycogen synthase were carried out as described by François et al. (1988) . For glycogen synthase, the assay was done using 0·25 mM UDP-Glc as the substrate, in the absence or presence of 20 mM Glc6P. The activation state (or non-phosphorylated state) of this enzyme is estimated by the±Glc6P activity ratio of glycogen synthase. Preparation of extracts, measurement of ß-galactosidase activity and determination of glycogen and trehalose were performed as described by Rose & Botstein (1993)
and Parrou & François (1997)
. Qualitative assessment of glycogen was carried out by using the iodine-staining method (Chester, 1968
) with cells spotted on YEPD (rich) or yeast nitrogen base complemented with auxotrophic requirement (minimal) agar plates. At different times, the plates were inverted over iodine crystals for 1 min, removed for 15 s and exposed again for 2 min. To obtain reliable results, a different plate was used for each staining to avoid any possible growth interference induced by the iodine vapour. To assay Glc6P and nucleotides, the rapid quenching method of yeast cells and extraction of metabolites in boiling buffered ethanol was followed (Gonzalez et al., 1997
). Determination of Glc6P, ATP and ADP was performed by coupling with NADH production or consumption as described by Bergmeyer (1986)
. Results reported in Figs 1
, 2
and 4
are from one set of experiments repeated independently two times.
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RESULTS |
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This unexpected result prompted us to investigate whether the pattern of glycogen accumulation was specific to the pet309 mutation or was a general feature of cells defective in mitochondrial respiration. For this purpose, we used petite cells lacking mitochondrial DNA (rho° mutant) which accumulated even more glycogen than wild-type cells at the end of growth on glucose (Fig. 2). At the onset of glucose exhaustion, glycogen started to be mobilized in both wild-type and petite cells. This mobilization was only transient in wild-type cells, whereas it was sustained in the rho° mutant such that it contained four times less glycogen than the wild-type after 3 d in glucose-starved medium. Fig. 2
also shows that the activity of ß-galactosidase from the GSY2lacZ construct in the rho° strain was 30% lower than that in the wild-type at the end of growth on glucose. This activity remained unchanged during prolonged incubation in the glucose-depleted medium, while it increased in the wild-type by about two times, indicating a resumption of the transcriptional activity during the respiratory phase of growth on ethanol. Another notable difference between wild-type and respiration-deficient cells was that the latter contained very low levels of trehalose. In both types of cells, the accumulation of trehalose was initiated at the moment when a minute amount of glucose remained in the growth medium (Parrou et al., 1999a
; Fig. 2
). Unlike wild-type cells, which accumulated the disaccharide during the respiratory growth phase on ethanol, trehalose synthesis was stopped in the rho° mutant as soon as exogenous glucose was consumed.
Time-course iodine staining of respiratory mutants correlates with glycogen content in liquid cultures
As shown in Figs 1 and 2
, the high level of glycogen accumulation in mitochondrial respiratory mutants during growth on glucose contrasted with their reported lack of iodine staining on agar plates (Chester, 1968
; Wek et al., 1992
; Cannon et al., 1994
; Yang et al., 1998
). To test the reliability of the iodine-staining method, we performed time-course coloration of cell patches of wild-type and respiratory mutants altered in genes involved in respiratory function (Tzagaloff & Dieckmann, 1990
). In close correlation with quantitative glycogen assays, pet309 and rho° mutants turned even more brown than isogenic wild-type when the staining was performed 1 d after spotting on agar plates (Fig. 3
). Similar results were obtained with strains bearing mutations in QCR9, encoding a subunit of cytochrome bc1, and HAP2, encoding a nuclear transcriptional factor involved in global regulation of respiratory genes. In contrast, the mitochondrial respiratory mutants exhibited a lower iodine staining after 3 d growth and this reduced coloration was even more severe after 6 d. This feature was observed on both rich and minimal agar plates, despite better iodine staining, and hence provides a contrast between wild-type and respiratory mutant strains on minimal plates. As a control, the gsy1
gsy2
double mutant which is defective in glycogen synthase activity (Farkas et al., 1991
) remained yellow throughout the experiment. Quantitative measurement confirmed the iodine-staining results since glycogen content was three- to fivefold lower in the mitochondrial respiratory mutants than in their isogenic control after 2 d growth in liquid medium (Table 2
). We conclude from these experiments that a time-course iodine staining of yeast colonies is absolutely required to obtain confident data on mutations affecting glycogen metabolism.
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Defect in respiration stimulates glycogen mobilization by a lowering of Glc6P
The sustained degradation of glycogen in mitochondrial respiratory mutants, particularly in a rho° strain expressing a hyperactivated form of glycogen synthase in minimal medium, supports the idea that the glycogen degradation pathway is stimulated in addition to the inhibition of Gsy2p-linked biosynthesis. In agreement with this suggestion, it is shown in Table 2 that the glycogen phosphorylase activity was 1·5-to 3-fold higher in the respiratory mutants. The activation state of glycogen synthase in respiratory mutants was three to six times lower than that measured in wild-type cells (Yang et al., 1998
; Table 2
). These enzymic changes were correlated with a sharp drop in Glc6P pools measured at the onset of glucose depletion (Table 3
) and remained at low levels during prolonged incubation (not shown). That the Glc6P pool was very low may be due to the fact that gluconeogenesis is not operative in these mutants. The intracellular concentrations of ATP and ADP were also lower than in wild-type cells, presumably due to the failure of these cells to shift to a respiratory mode of growth. Since Glc6P is a key effector in the control of the phosphorylation state of the two enzymes (François & Hers, 1988
; Lin et al., 1997; Huang et al., 1997
), it is suggested that glycogen mobilization may be triggered by the drop in the level of this sugar phosphate observed in the respiratory mutants after glucose depletion.
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DISCUSSION |
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Our quantitative kinetic glycogen analysis showed that, contrary to previous studies (Chester, 1968 ; Filipak et al., 1992
; Wek et al., 1992
; Yang et al., 1998
), mitochondrial respiratory mutants accumulated 2050% more glycogen than their isogenic wild-type cells during the fermentative growth phase on glucose. It is suggested that this enhanced glycogen deposition could arise from glucose that has been energetically spared by these mutants due to their inability to derepress mitochondrial functions during growth. The mechanism of this glucose flux readjustment is, however, totally unclear. Our kinetic analysis also revealed that respiratory mutants readily degraded their glycogen stores when they became starved for glucose. Hence, in contrast to the claim that a defect in respiration blocks glycogen synthesis, we showed that mitochondrial defects stimulate glycogen mobilization. Based on time-course iodine-staining experiments, the rate and the extent of glycogen degradation was apparently more potent in respiration-deficient cells cultivated on glucose minimal medium, as illustrated in rho° cells expressing a hyperactivated form of glycogen synthase. From a physiological point of view, the mobilization of glycogen is the sole means for respiratory mutants to obtain carbon and energy in carbon-starved media as they cannot reassimilate either by-products derived from glucose fermentation or exogenous amino acids present in rich media. We also demonstrated that the capacity of respiration-deficient cells to mobilize glycogen was not suppressed by mutations in the Ras-cAMP pathway or in cyclin-dependent Pho85p kinase, known to negatively control glycogen metabolism (Huang et al., 1996
; Timblin et al., 1996
; Timblin & Bergman, 1997
; Parrou et al., 1999b
). It is, however, worthy note that the mutation in these two essential nutrient-sensing pathways clearly interfered with the ability of respiration-deficient cells to efficiently degrade glycogen during prolonged incubation on minimal medium, but not on rich medium. This might be interpreted as a higher dependency of metabolic adaptations of yeast cells to these signalling pathways when the composition of the culture medium is more strict.
The sustained mobilization of glycogen during prolonged incubation of respiratory mutants in a glucose-depleted medium could not solely be explained by a less active glycogen synthase (Yang et al., 1998 ), since this was still observed in mutant cells expressing a constitutively activated form of this enzyme. Accordingly, a more active glycogen phosphorylase was measured in the respiration-deficient cells. In search of the mechanism which stimulates this process, it was observed, in agreement with Yang et al. (1998)
, that the levels of ATP and Glc6P measured at the onset of glycogen degradation dropped in respiration-deficient cells. Taking into account the fact that Glc6P is a potent activator of the dephosphorylation reaction catalysed by protein phosphatases and an inhibitor of the phosphorylation reaction catalysed by protein kinases (François & Hers, 1988
; Lin et al., 1995
, 1996
; Huang et al., 1997
), we propose that the dramatic drop of this effector in respiration-deficient cells triggers glycogen mobilization by the activation of glycogen phosphorylase and inactivation of glycogen synthase. Hence, defects in respiration provide an additional illustration of the physiological function of Glc6P in the control of glycogen metabolism.
Our study on the linkage between mitochondrial function and storage carbohydrates in yeast confirmed that mitochondrial respiratory mutants were defective in trehalose synthesis (Filipak et al., 1992 ). Despite its inability to grow on a non-fermentable source, a gluconeogenic mutant can accumulate trehalose from glycogen mobilization because, unlike respiratory mutants, it is still able to obtain energy from the reoxidation of ethanol which is slowly taken up from the medium (François et al., 1991
). Therefore, an open question is to understand how the gate for trehalose synthesis is closed to the glucosyl units derived from glycogen in respiration-deficient cells.
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ACKNOWLEDGEMENTS |
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Received 6 April 2000;
revised 12 June 2000;
accepted 5 July 2000.