Yeast Glycogen Synthase Research Articles

Response of a yeast glycogen synthase gene to stress

In the yeast Saccharomyces cerevisiae, glycogen synthase is encoded by two genes: GSY1 and GSY2. The activity of the enzymes increases as cultures enter the stationary phase of growth. Using a GSY2::lacZ fusion gene, we have demonstrated that the increase in glycogen synthase activity resulted, at least in part, from an increase in the level of the protein rather than simply from a change in its phosphorylation state. Northern blot analysis showed a parallel increase in the level of the GSY2 mRNA, which is consistent with transcriptional activation of GSY2. Deletion analysis identified three regions upstream of GSY2 which are involved in GSY2 expression: regions A (-390 to -347 relative to the start of translation), B (-252 to -209) and C (-209 to -167). Region A or C independently activated expression of GSY2. In contrast, region B alone yielded only modest expression. Expression of GSY2 is induced by growth to stationary phase, heat shock or nitrogen starvation. Response to these stressors is mediated by elements within regions A and C. These elements appear to be related to the stress-response elements found in other stress-responsive genes.

Control of yeast glycogen synthase-2 by COOH-terminal phosphorylation.

The budding yeast Saccharomyces cerevisiae expresses two isoforms of glycogen synthase, of which glycogen synthase-2 (GS-2) appears to be the most important determinant of glycogen accumulation (Farkas, I., Hardy, T. A., Goebl, M. G., and Roach, P. J. (1991) J. Biol. Chem. 266, 15602-15607). Partial proteolysis of purified yeast glycogen synthase activated the enzyme, mimicking the effects of dephosphorylation. The cleavage was localized to the COOH terminus of the molecule and trypsin treatment released 32P from enzyme labeled in vivo with 32P or in vitro by cyclic AMP-dependent protein kinase. Similarly, when cells were labeled with 32P, no radioactivity was incorporated into a mutant form of GS-2 truncated at residue 643 while the wild type enzyme was phosphorylated at both Ser and Thr residues. The 9 Ser and Thr residues COOH-terminal to position 643 were mutated individually to Ala, and the GS-2 mutants were expressed from a low copy plasmid in yeast that lacked functional chromosomal copies of the two glycogen synthase genes. Mutations at Ser-650, Ser-654, and Thr-667 resulted in significant activation of yeast glycogen synthase and elevation in the level of accumulated glycogen as compared with wild type. Likewise, expression of the truncated GS-2 resulted in hyperactive enzyme and the overaccumulation of glycogen. None of the other Ser or Thr mutations substantially affected glycogen synthase activity and glycogen storage. We conclude that Ser-650, Ser-654, and Thr-667 are regulatory phosphorylation sites in vivo. However, in vitro, cyclic AMP-dependent protein kinase modified Ser residue(s) COOH-terminal to position 659, and so the identity of the physiological GS-2 kinases is unclear. Yeast strains bearing glc7 and gac1 mutations are defective in genes encoding type 1 protein phosphatase components and are impaired in their ability to accumulate glycogen. Expression of the truncated GS-2 in these strains restored glycogen accumulation, as did the presence of GS-2 mutated at Ser-650, Ser-654, or Thr-667. These data are consistent with the hypothesis that type 1 phosphatase regulates GS-2 by controlling its phosphorylation state.

Two glycogen synthase isoforms in Saccharomyces cerevisiae are coded by distinct genes that are differentially controlled

In previous work, we identified a Saccharomyces cerevisiae glycogen synthase gene, GSY1, which codes for an 85-kDa polypeptide present in purified yeast glycogen synthase (Farkas, I., Hardy, T.A., DePaoli-Roach, A.A., and Roach, P.J. (1990) J. Biol. Chem. 265, 20879-20886). We have now cloned another gene, GSY2, which encodes a second S. cerevisiae glycogen synthase. The GSY2 sequence predicts a protein of 704 residues, molecular weight 79,963, with 80% identity to the protein encoded by GSY1. Amino acid sequences obtained from a second polypeptide of 77 kDa present in yeast glycogen synthase preparations matched those predicted by GSY2. GSY1 resides on chromosome VI, and GSY2 is located on chromosome XII. Disruption of the GSY1 gene produced a strain retaining about 85% of wild type glycogen synthase activity at stationary phase, while disruption of the GSY2 gene yielded a strain with only about 10% of wild type enzyme activity. The level of glycogen synthase activity in yeast cells disrupted for GSY1 increased in stationary phase, whereas the activity remained at a constant low level in cells disrupted for GSY2. Disruption of both genes resulted in a viable haploid that totally lacked glycogen synthase activity and was defective in glycogen deposition. In conclusion, yeast expresses two forms of glycogen synthase with activity levels that behave differently in the growth cycle. The GSY2 gene product appears to be the predominant glycogen synthase with activity linked to nutrient depletion.

Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A

A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae. The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis. This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C. The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer. On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration. The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid. Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines. Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50%. The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A. An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not. These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.

Glycogen metabolism and signal transduction in mammals and yeast

Mammalian glycogen synthase, with its complex multisite phosphorylation mechanisms, continues to provide interesting and novel examples of the regulation of protein function. The mammalian enzyme is phosphorylated in a hierarchal manner such that modification of certain sites requires the prior phosphorylation of other sites. Yeast contains two glycogen synthases that have extensive similarities to their mammalian counterpart but the greatest divergence in amino acid sequence is seen precisely in the regions likely to be involved in covalent control. We hope that examination of the control of the yeast glycogen synthase will be as informative as study of the mammalian enzymes, whether by revealing important parallels with the mammalian system or by uncovering major differences in mechanism.

Isolation of the GSY1 gene encoding yeast glycogen synthase and evidence for the existence of a second gene.

Glycogen synthase preparations from Saccharomyces cerevisiae contained two polypeptides of molecular weights 85,000 and 77,000. Oligonucleotides based on protein sequence were utilized to clone a S. cerevisiae glycogen synthase gene, GSY1. The gene would encode a protein of 707 residues, molecular mass 80,501 daltons, with 50% overall identity to mammalian muscle glycogen synthases. The amino-terminal sequence obtained from the 85,000-dalton species matched the NH2 terminus predicted by the GSY1 sequence. Disruption of the GSY1 gene resulted in a viable haploid with glycogen synthase activity, and purification of glycogen synthase from this mutant strain resulted in an enzyme that contained the 77,000-dalton polypeptide. Southern hybridization of genomic DNA using the GSY1 coding sequence as a probe revealed a second weakly hybridizing fragment, present also in the strain with the GSY1 gene disrupted. However, the sequences of several tryptic peptides derived from the 77,000-dalton polypeptide were identical or similar to the sequence predicted by the GSY1 gene. The data are explained if S. cerevisiae has two glycogen synthase genes encoding proteins with significant sequence similarity The protein sequence predicted by the GSY1 gene lacks the extreme NH2-terminal phosphorylation sites of the mammalian enzymes. The COOH-terminal phosphorylated region of the mammalian enzyme over-all displayed low identity to the yeast COOH terminus, but there was homology in the region of the mammalian phosphorylation sites 3 and 4. Three potential cyclic AMP-dependent protein kinase sites are located in this region of the yeast enzyme. The region of glycogen synthase likely to be involved in covalent regulation are thus more variable than the catalytic center of the molecule.

Purification, characterization and partial amino acid sequence of glycogen synthase from Saccharomyces cerevisiae

Glycogen synthase from Saccharomyces cerevisiae was purified to homogeneity. The enzyme showed a subunit molecular mass of 80 kDa. The holoenzyme appears to be a tetramer. Antibodies developed against purified yeast glycogen synthase inactivated the enzyme in yeast extracts and allowed the detection of the protein in Western blots. Amino acid analysis showed that the enzyme is very rich in glutamate and/or glutamine residues. The N-terminal sequence (11 amino acid residues) was determined. In addition, selected tryptic-digest peptides were purified by reverse-phase h.p.l.c. and submitted to gas-phase sequencing. Up to eight sequences (79 amino acid residues) could be aligned with the human muscle enzyme sequence. Levels of identity range between 37 and 100%, indicating that, although human and yeast glycogen synthases probably share some conserved regions, significant differences in their primary structure should be expected.