Incubation of rat hepatocytes with glucose induces the translocation of glycogen synthase from soluble fractions to fractions which sediment at 10,000 g. Incubation of the cells with fructose, galactose, 2-deoxyglucose or 5-thioglucose, which activate glycogen synthase, also resulted in the translocation of the enzyme, whereas 3-O-methylglucose, 6-deoxyglucose and 1,5-anhydroglucitol, which do not cause the activation of the enzyme, were ineffective. Adenosine and carbonyl cyanide m-chlorophenylhydrazone, although activating glycogen synthase, did not induce its translocation. Mannoheptulose, which inhibits glucose phosphorylation, impaired the translocation of glycogen synthase induced by glucose. Furthermore, the extent of the translocation of the enzyme triggered by glucose and other sugars showed a high positive correlation with the intracellular concentration of glucose 6-phosphate. Microcystin, which blocks the activation of glycogen synthase by glucose, but not the accumulation of glucose 6-phosphate, did not affect the translocation of the enzyme. These results indicate that glucose 6-phosphate plays a role in the translocation of glycogen synthase in rat hepatocytes.
The yeast homologues of mammalian protein phosphatase 2A (PP2A) are encoded by two genes, PPH21 and PPH22. To evaluate the role of these phosphatases in the control of glycogen metabolism, wild-type cells and mutants carrying deletions of PPH21 or PPH22 were studied. Our results indicate that the lack of a single gene product does not result in significant changes in glycogen content, glycogen synthase, and glycogen phosphorylase activities. Since the double disruption is very detrimental to the cell, the effect of lack of PP2A was evaluated by using strain H336, which carries a deletion of the PPH21 gene and has the PPH22 gene placed under the control of the GAL1 promoter, under conditions that allowed either progressive depletion or overexpression of PPH22. When grown on galactose, H336 cells contain 2-3-fold more PP2A activity than control cells. After 14 h in glucose, however, PP2A activity in strain H336 is markedly reduced. The decrease in PP2A activity correlates with a reduced accumulation of glycogen and a more pronounced inactivation of glycogen synthase while glycogen phosphorylase becomes more resistant to inactivation. These observations suggest a role for PP2A in controlling the activation states of both enzymes. The total amount of phosphorylase was also higher in the PP2A-depleted cells, as determined by both enzymic and immunochemical techniques. However, Northem-blot analysis revealed that this is not due to an increase in the phosphorylase mRNA, which is in fact reduced in these cells. In contrast, overexpression of PP2A causes an increased expression of glycogen phosphorylase and a resulting failure to accumulate glycogen. We conclude that PP2A is involved in regulating both the amounts and the activation states of glycogen synthase and glycogen phosphorylase.Keywords. Protein phosphatase 2A ; glycogen metabolism ; glycogen synthase ; glycogen phosphorylase ; yeast.In yeast cells, glycogen is a major energy reserve [I]. Glycogen breakdown is catalyzed by the enzyme glycogen phosphorylase, encoded by the gene GPHl [2], whereas two genes (GSYl and GSY2) code for Saccharomyces cerevisiae glycogen synthase, the key enzyme in glycogen synthesis [3, 41. GPHl transcription is induced in the late exponential growth phase [2] concomitant with the appearance of glycogen. Approximately at the same time the GSY2 protein, the major isoform of glycogen synthase, is also induced [4]. Genetic and biochemical evidence suggest that, in yeast, glycogen phosphorylase and synthase activities can be modulated, as in other eukaryotes, through phosphorylation-dephosphorylation mechanisms [5 -101. Phosphorylation would inactivate glycogen synthase and activate phosphorylase, whereas dephosphorylation would have the opposite effects. It is generally accepted that phosphorylation of glycogen synthase and phosphorylase is controlled by the levels of CAMP through the activation of the CAMP-dependent protein kinase, although CAMP-independent mechanisms could also be important in the regulation of glycogen metabolism in ...
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.
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