Hyperactive Glycogen Synthase Mutants of Saccharomyces cerevisiae Suppress the glc7-1 Protein Phosphatase Mutant

The wide distribution of the storage carbohydrate glycogen in nature suggests that it plays important physiological roles. Indeed, genetic, biochemical, and physiological analyses of mammals indicate that glycogen metabolism plays an essential role in energy homeostasis. As one might expect, the synthesis and degradation of glycogen are regulated by multiple signaling pathways. Glycogen also accumulates in fungi; in the budding yeast Saccharomyces cerevisiae, where the pathway has been studied in detail, the metabolism of glycogen is remarkably similar to that in mammals. Most of the enzymatic components (glycogenin, glycogen synthase, phosphorylase, and branching enzyme) are conserved in yeast and mammals (5, 7, 8, 22, 31, 37), but the signaling pathways that regulate glycogen synthase have diverged. In mammals, glycogen metabolism is under hormonal control, whereas in yeast, it is controlled largely by nutrients. Yeast cells normally accumulate glycogen near the end of log-phase growth, due to a decrease in the levels of nutrients such as glucose, phosphate, nitrogen, and sulfate (23). This accumulation is due in part to the increased expression of genes whose products act in the pathway (15, 24, 25, 31) and in part to post translational regulatory mechanisms (10, 16, 21, 27, 30). At least for glucose, the accumulation of glycogen does not seem dependent on the absolute amount present; rather, glycogen accumulates when approximately half the glucose is consumed (23). Although many of the key regulators have been identified, the basic mechanism that controls this accumulation is unknown. Glycogen synthase is regulated at the level of gene expression and by posttranscriptional mechanisms. mRNA levels for the major isoform of glycogen synthase (GSY2 mRNA) increase as cells approach stationary phase when grown in glucose-containing medium (15, 24, 25). Gsy2p is also phosphorylated and negatively regulated by phosphorylation at its COOH terminus (16). Glycogen synthase can be fully activated by glucose-6-phosphate (G6P), even in the fully phosphorylated state (26). Therefore, the ratio of glycogen synthase activities assayed in the absence and presence of G6P (activity ratio) provides a convenient means of assessing the phosphorylation state of the protein. Mammalian glycogen synthase is phosphorylated at its NH2 and COOH termini (28). In contrast, yeast glycogen synthase is phosphorylated only at its COOH terminus. A deletion variant of Gsy2p lacking amino acid residues after amino acid (aa) 643 has a very high activity ratio and is not phosphorylated in vivo (16). Other Gsy2p variants that contain alanine substitutions for Ser-650 (S650A), Ser-654 (S654A), or Thr-667 (T667A) also have high activity ratios, suggesting that these residues are the major sites of phosphorylation (16). Biochemical characterization of glycogen synthase kinase in yeast has revealed at least two distinct activities (18), one of which is cyclin-dependent kinase Pho85p activity. Null mutations in the genes encoding the catalytic subunit, PHO85 (18, 38), or two of its many cyclin subunits, PCL8 and PCL10 (19), result in the hyperaccumulation of glycogen. Pho85p-Pcl10p can phosphorylate Gsy2p in vitro (19, 39). However, Gsy2p variants that lack Ser-654 and Thr-667 are no longer Pho85p-Pcl10p substrates, suggesting that Pho85p phosphorylates Gsy2p at these two sites while another, as-yet-unidentified kinase phosphorylates Gsy2p at Ser-650. Further evidence indicates that at least one other kinase is involved in down-regulating Gsy2p. Yang et al. (40) have recently found that a block to respiration inhibits glycogen accumulation by increasing the phosphorylation state of glycogen synthase. The activity ratio of glycogen synthase is very low in respiration-deficient mutants, and GSY2 mutants that lack any one of the three key phosphorylation sites (Ser-650, Ser-654, or Thr-667) accumulate glycogen in a respiration-deficient background. However, the loss of Pho85 kinase does not restore glycogen accumulation to respiration-deficient cells, indicating that another protein kinase is responsible for the effect. On the reverse side of the regulatory pathway, protein phosphatase type 1 is responsible for dephosphorylating and activating glycogen synthase. Some mutations in GLC7, which encodes the catalytic subunit of protein phosphatase type 1, result in a failure to accumulate glycogen due to retention of glycogen synthase in the inactive, phosphorylated form (11, 27). The phosphatase directly responsible for activating glycogen synthase is thought to be composed of Glc7p and the regulatory subunit Gac1p. Gac1p binds to both Glc7p and glycogen synthase through separate domains and appears to act as a scaffold to target the substrate to the phosphatase (X. Wu, H. Hart, C. Cheng, P. J. Roach, and K. Tatchell, submitted for publication). The related protein Pig1p, first identified in a screen for glycogen synthase binding proteins, may have a role similar to that of Gac1p. The low levels of glycogen in a gac1 mutant are reduced further by deletion of PIG1 (4). The glycogen deficiency of glc7-1 or gac1 mutants can be suppressed either by elimination of the phosphorylation sites in Gsy2p (16) or by inactivation of Pho85p-Pcl8p and Pho85p-Pcl10p kinase (19), furthering the idea that the Glc7-Gac1p phosphatase reverses the phosphorylation and inactivation of Gsy2p by Pho85p-Pcl8p or Pho85p-Pcl10p. To further the understanding of glycogen metabolism, we have isolated revertants of glc7-1 (glycogen defect) mutants. All mutants isolated in this screen are defective in the major isoform of glycogen synthase, Gsy2p, and all suppress the glc7-1 mutation by increasing the activity of glycogen synthase. The mutations in some suppressors are located in the region known to encode the phosphorylated COOH terminus. However, other mutants alter portions of the protein not previously known to have a regulatory role. In addition, we demonstrate for the first time the importance of glycogen accumulation by showing that levels of glycogen play an important role in overall fitness. Strains that accumulate too much or too little glycogen are rapidly outcompeted by wild-type strains.