2.1.1Nucleation by glycogenin (Glg1p/Glg2p)
As in the case of other high molecular mass biopolymers, the synthesis of glycogen involves initiation, elongation, and ramification steps. The initiation step is carried out by a protein designated ‘glycogenin’ which bears a self-autoglucosylating activity and produces from UDP-glucose (UDP-Glc) a short α (1,4)-glucosyl chain that is covalently attached to a tyrosine residue (Fig. 1). This protein was originally identified as a 38-kDa ‘contaminant’ of a purified preparation of glycogen synthase in skeletal muscle [9,10]. Roach and colleagues took advantage of this interesting property to isolate, by a two-hybrid screen using glycogen synthase encoded by GSY2 as the bait, a yeast homologue of the mammalian glycogenin. This gene, designated GLG2, is located on chromosome X and is highly homologous to another open reading frame identified on chromosome XI and named GLG1. Loss of either GLG genes does not result in apparent glycogen defect, whereas the lack of glycogen in glg1glg2 cells would indicate an absolute requirement for a self-glucosylating initiator protein in the biosynthesis of glycogen . However, preliminary results suggest that a glg1glg2 mutant strain recovers about 30% of the wild-type glycogen levels when it is additionally disrupted for TPS1 encoding the Tre6P synthase catalytic subunit (unpublished).
Figure 1. Glycogen and trehalose metabolic pathways in the yeast S. cerevisiae. A two-step reaction catalyzed by phosphoglucomutase (two isoforms, Pgm1p and Pgm2p) and UDP-Glc pyrophosphorylase (Ugp1p) leads to the synthesis of UDP-Glc. Glycogen synthesis is initiated by glycogenin (two isoforms, Glg1p and Glg2p), that produces a short α (1,4)-glucosyl chain that is elongated by glycogen synthase (two isoforms, Gsy1p and Gsy2p). The chains are ramified by the branching enzyme (Glc3p) which transfers a block of 6–8 residues from the end of a linear chain to an internal glucosyl unit and creates an α (1,6)-linkage. Glycogen degradation occurs by the combined action of glycogen phosphorylase (Gph1p) which releases glucose-1-P, and a debranching enzyme (Gdb1p) which transfers a maltosyl unit to the end of an adjacent linear α (1,4)-chain and releases glucose by cleaving the remaining α (1,6)-linkage. Trehalose biosynthesis is catalyzed by the trehalose synthase complex composed of four subunits. The trehalose-6-phosphate (Tre6P) synthase subunit (Tps1p) produces Tre6P from UDP-Glc and glucose-6-phosphate (Glc6P), which is dephosphorylated in trehalose by the Tre6P phosphatase subunit (Tps2p). Tps3p and Tsl1p are two regulatory subunits that stabilize the complex. Trehalose is degraded by the neutral (Nth1p) or the acid (Ath1p) trehalase. The role of Nth2p in this degradation process is not yet clarified.
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Glg1p differs from Glg2p in being higher in molecular mass (67 kDa versus 43 kDa). They exhibit 55% sequence identity over a 260 residues NH2-terminal segment and 33% identity with the mammalian glycogenin in this region. All eukaryotic glycogenins display a high affinity (Km around 5–10 μM) for UDP-Glc and exist in vivo as oligomers of unknown stoichiometry . Unlike mammalian glycogenins, yeast Glg proteins possess multiple Tyr residues that are needed for sustained glycogen accumulation and harbor a COOH-terminal domain that interacts with the yeast glycogen synthase. The truncation of this domain reduces glycogen synthase activity and severely impairs glycogen accumulation . The observation that a glg1 mutant strain which additionally carries a Tyr-230/Phe and Tyr-232/Phe substitution in Glg2p still accumulates 10% of the wild-type level of glycogen suggested that these substitutions had forced another tyrosine residue (e.g. Tyr-237) at the C-terminus to be a glucosyl acceptor. Alternatively glycogen synthase may elongate short primers (maltose, maltotriose) produced by the intrinsic transglucosidase activity of Glg proteins. In either case, disruption of the interaction with glycogen synthase by removal of the C-terminal domain of the mutant Glg proteins abolished glycogen synthesis . Surprisingly, glycogen levels are not increased in yeast cells overexpressing GLG genes, suggesting that either yeast glycogenins are in molar excess over glycogen synthase, or that these proteins could be reiteratively used. However, this reiteration would require the existence of an additional enzyme to release the glycogenin from the growing glucosyl chains, but such an enzyme has not been found yet.
2.1.2Elongation by glycogen synthases (Gsy1p/Gsy2p)
Glycogen synthase catalyzes the formation of α (1,4)-glucosidic bonds from UDP-Glc to the non-reducing end of linear α (1,4)-chains of glycogen (Fig. 1). S. cerevisiae contains two genes, GSY1 and GSY2, encoding 80 501 and 79 963 Da polypeptides that correspond respectively to glycogen synthase isoform I and II . The two proteins are 80% identical and share 50% similarity with mammalian muscle and rat liver glycogen synthase. Deletion analysis indicated that GSY2 encodes the predominant glycogen synthase since loss of its function resulted in a 90% reduction in both enzyme activity and glycogen levels. Deletion of both genes produced cells with no other apparent phenotype than glycogen synthesis deficiency .
The original work of Cabib and colleagues [14–16], extended by others [13,17–20], established that yeast glycogen synthase shares many properties with its mammalian counterpart, including allosteric stimulation by Glc6P and reversible covalent phosphorylation. As the active – non-phosphorylated – form of glycogen synthase is almost insensitive to the sugar phosphate, the ratio of the activities assayed in the absence and in the presence of Glc6P (−/+ Glc6P activity ratio) can provide a good estimate of the phosphorylation state of the glycogen synthase [15,17,19]. Partial proteolysis of a purified preparation of glycogen synthase (both isoforms) resulted in its Glc6P insensitivity and provided the first evidence that the phosphorylation occurred at the COOH-terminus [15,16]. A cluster of three putative sites for cAMP-dependent phosphorylation was identified at the C-terminus of the two isoforms [13,21], in agreement with the finding of a maximum of 3 mol phosphate per mol subunit in the purified inactive glycogen synthase . Moreover, it was demonstrated that the in vivo phosphorylated sites are Ser-650, Ser-654 and Thr-667. While a change of one of the three amino acid residues to Ala only caused a 35% increase in the activity ratio, full activation of Gsy2p can be obtained either by mutation of the three amino acids, or by removal of a 60-amino acids fragment of the C-terminus where the amino acids were located .
The next question was to identify the protein kinases implicated in glycogen synthase phosphorylation. The cAMP-dependent protein kinase (PKA) is likely involved in the control of the glycogen synthase phosphorylation state as indicated by an inverse relationship between the −/+ Glc6P activity ratio of this enzyme and the PKA activity in yeast strains altered in the cAMP-PKA pathway ([19,22,23], unpublished results). These in vivo results contrast with the fact that none of the three sites on Gsy2p could be phosphorylated in vitro by PKA . This indicates that this kinase can act, most probably indirectly, in the control of glycogen synthase phosphorylation (Fig. 2). A partial purification of a yeast extract on phenyl-Sepharose column revealed two distinct peaks, termed Gpk1p and Gpk2p, that phosphorylated a recombinant Gsy2p. Gpk1p, whose corresponding gene is not yet known, phosphorylates Gsy2p in a cAMP-, Ca2+- and calmodulin-independent way, but the phosphorylated amino acid residues have not been identified . Gpk2p is identical to the protein kinase Pho85, a member of the cyclin-dependent protein kinase family . The implication of this kinase in glycogen synthesis has been shown by two other independent approaches. Roach and coworkers  isolated PHO85 as a second site suppressor of the glycogen storage defect of snf1 cells, whereas Bergman's group  found that disruption of PHO85 resulted in pleiotropic phenotypes, including a hyperaccumulation of glycogen. In addition, Huang et al.  identified the cyclin-like Pcl8p and Pcl10p as targeting subunits of Pho85p to phosphorylate Gsy2p at Ser-654 and Thr-667 (Fig. 2). It remains to determine whether the Gpk1p may phosphorylate Ser-650. The protein kinase responsible for the inactivation of glycogen synthase in response to the treatment of MATa haploid cells with the pheromone α-factor  has not been identified yet. Since the pheromone-induced MAP kinase Fus3p cannot directly phosphorylate Gsy2p (P. Roach, personal communication), this inactivation may result from a Fus3p-dependent stimulation of either Pho85p or Gpk1p. Alternatively, the mating pheromone may inhibit specific glycogen synthase phosphatases.
Figure 2. Metabolic control of glycogen metabolism in S. cerevisiae. PKA and Snf1p antagonistically control the phosphorylation state of Gsy2p and Gph1p. Pho85p, in association with Pcl8p and Pcl10p, phosphorylates and inactivates Gsy2p. The reverse reaction is catalyzed by the phosphatase Glc7p and its targetting subunits Gac1p and Pig1p. The existence of other kinases and phosphatases is still hypothetic. Kinase(s) and phosphatase(s) that control the phosphorylation state of Gph1p have not been identified yet. The main effector of this process is Glc6P which acts as a potent stimulator of the dephosphorylation and inhibitor of the phosphorylation processes. Pi is also another important effector in glycogen control, being the substrate of Gph1p and an inhibitor of glycogen synthase. (Arrows) positive interaction; (bars) negative interaction; (dashed lines) direct interaction not yet determined.
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Protein phosphatases play critical roles in cellular activities [29,30] and in glycogen metabolism in particular [31,32]. Yeast possesses the four different classes of Ser/Thr protein phosphatases catalytic subunit designated PP1, 2A, 2B and 2C, and a large number of targeting subunits that direct the catalytic subunits to particular locations within the cell and selectively enhance their activity towards specific substrates [29,33]. The first targeting subunit identified in yeast was the product of GAC1 which was isolated as a dosage-dependent suppressor of the glycogen storage defect of a mutant with high adenylate cyclase activity. This gene encodes a 88-kDa polypeptide 30% identical to the mammalian regulatory subunit RGI of the PP1 [33,34]. Using the two-hybrid system, Skroch-Stuart et al.  demonstrated that Gac1p physically interacts with the catalytic subunit of PP1 encoded by GLC7 and that this interaction is defective in the glycogen-deficient glc7-1 mutant cells. These authors also suggested that the productive Glc7p–Gac1p interaction could be negatively affected by the phosphorylation of Gac1p, which is reminiscent of the negative cAMP-dependent control of RGI–PP1 association . All glc7 alleles isolated conferred glycogen deficiency or glycogen hyperaccumulation, likely due to an alteration of Glc7p–Gac1p interaction [37,38]. Two additional genes, PIG1 and PIG2, were isolated using a two-hybrid screen with Gsy2p as bait . PIG1 encodes a protein 38% identical over a 230-amino acids segment to Gac1p, whereas PIG2 has 30% identity with GIP2 which was isolated using Glc7p as bait . Sequence analysis indicated that Gac1p, Pig1p, Pig2p and the product of YER054c (unknown function) are the only four proteins of the yeast genome sharing a common 25-amino acid stretch designated ‘GVNK’. This stretch is also identifiable in the mammalian RGI and may be required for interaction with either glycogen particles, PP1 catalytic subunits and/or Gsy2p. Although the Gac1p–Glc7p complex appears to be the most potent glycogen synthase phosphatase in yeast as the loss of GAC1 function strongly reduces glycogen accumulation and leaves glycogen synthase in an inactive form  (Fig. 2), PIG1 may participate in concert with GAC1 in the control of glycogen synthesis since a gac1pig1 double mutant shows more severe glycogen defect than a gac1 single mutant . However, the pig1 single mutant had no phenotype and the role of PIG2 in glycogen metabolism is still unknown. In a screen for suppressors of the glycogen accumulation defect in glc7-1 cells, Huang et al.  uncovered a mutant form of the REG1 which encodes another PP1 targeting subunit implicated in glucose repression . The mechanism by which REG1 influences glycogen synthesis is unclear, and it is independent on its role in glucose repression . Besides the major role of PP1 in controlling glycogen synthase, type 2A protein phosphatase also appears to exert a control, albeit minor, on this enzyme, as illustrated by two experimental results. On the one hand, Reimann and colleagues  purified a yeast glycogen synthase phosphatase which exhibits typical enzymological properties of PP2A, and which was able to increase in vitro the −/+ Glc6P activity ratio of glycogen synthase from 12 to 30%. On the other hand, the progressive depletion of major PP2A activities, which are encoded by PPH21 and PPH22, resulted in a concomitant 30% decrease of glycogen synthase activity . Moreover, the yeast genome encodes other Ser/Thr protein phosphatases 2A closely related to, but different from, the catalytic subunits of PP1 and PP2A previously mentioned. The loss of one of them encoded by SIT4 caused a 25% reduction in the activity ratio of glycogen synthase , whereas disruption of another PP2A homologue encoded by PPG led to a 30% decrease in the amount of total glycogen synthase with no change in the activity ratio . In both cases, a slight 30% reduction in glycogen levels was measured, indicating that these two PP2A-related protein phosphatases exert a minor influence on the control of glycogen metabolism. The action of these type 2A phosphatases on glycogen metabolism is likely due to their role in the regulation of cell growth .
To summarize, the activation state of glycogen synthase is ultimately dependent on the relative activity of protein kinases and protein phosphatases (Fig. 2). Biochemical data indicate that this phosphorylation–dephosphorylation ‘equilibrium’ is antagonistically controlled by the levels of Glc6P and cAMP, which are themselves affected by external stimuli. While the mechanism by which high levels of cAMP favor glycogen synthase phosphorylation is not yet clarified, several data indicate that the major role of Glc6P is to act as a stimulator of the dephosphorylation and as an inhibitor of the phosphorylation processes  (Fig. 2). Such a role of Glc6P, which has been recognized for years in mammals [31,32], has now been highlighted by a genetic approach in yeast. Looking for second site suppressor mutations of glycogen deficiency in snf1 cells, Huang et al.  identified a mutation in PFK2 which encodes the β-subunit of 6-phosphofructo-1-kinase. This mutation leads to the hyperaccumulation of glycogen associated with a 10-fold higher intracellular pool of Glc6P. Similar phenotypic traits have previously been observed in phosphoglucose isomerase (PGI) defective strains . In addition, the glycogen synthase kinase present in a cell-free extract obtained from a pfk2 or a pgi1 mutant was less efficient than that of a wild-type to phosphorylate a recombinant Gsy2p. However, a gel filtration on Sephadex G-25 of extracts from these mutant strains, which removed small metabolites including Glc6P, resulted in the recovery of a ‘wild-type’ glycogen synthase kinase activity . Hence, it remains to determine whether Glc6P mediates these effects through its binding to glycogen synthase or to some modifying enzymes.
2.1.3Branching by amylo (1,4)(1,6)-transglucosidase (Glc3p)
After initiation by glycogenin and elongation by glycogen synthase, the linear α (1,4)-glucosyl chains are ramified by the action of amylo (1,4)(1,6)-transglucosidase (branching enzyme) which transfers a block of 6–8 residues from the end of a linear chain to create an α (1,6)-linkage to an internal glucosyl unit on an adjacent chain  (Fig. 1). It is estimated that yeast glycogen particles contain 7–10% of α (1,6)-glucosidic linkages, which is 2–3 times higher than the proportion of such linkages found in the amylopectin structure of starch [51,52]. The highly branched structure of glycogen is responsible for the brown staining of yeast cells upon exposure to iodine crystals vapor, whereas a lower branching gives rise to a green–purple color of the cells. GLC3 was cloned by complementation of iodine staining-deficient mutants. This gene is located on chromosome V and encodes a protein 42% and 67% similar to prokaryotic and human glycogen branching enzyme, respectively. Disruption of GLC3 showed that the branching activity is essential for efficient glycogen accumulation since a glc3 null mutant accumulated only ca. 10% of the wild-type glycogen [53,54].