In the yeast Saccharomyces cerevisiae, glycogen is accumulated as a carbohydrate reserve when cells are deprived of nutrients. Yeast mutated in SNF1, a gene encoding a protein kinase required for glucose derepression, has diminished glycogen accumulation and concomitant inactivation of glycogen synthase. Restoration of synthesis in an snf1 strain results only in transient glycogen accumulation, implying the existence of other SNF1-dependent controls of glycogen storage. A genetic screen revealed that two genes involved in autophagy, APG1 and APG13, may be regulated by SNF1. Increased autophagic activity was observed in wild-type cells entering the stationary phase, but this induction was impaired in an snf1 strain. Mutants defective for autophagy were able to synthesize glycogen upon approaching the stationary phase, but were unable to maintain their glycogen stores, because subsequent synthesis was impaired and degradation by phosphorylase, Gph1p, was enhanced. Thus, deletion of GPH1 partially reversed the loss of glycogen accumulation in autophagy mutants. Loss of the vacuolar glucosidase, SGA1, also protected glycogen stores, but only very late in the stationary phase. Gph1p and Sga1p may therefore degrade physically distinct pools of glycogen. Pho85p is a cyclin-dependent protein kinase that antagonizes SNF1 control of glycogen synthesis. Induction of autophagy in pho85 mutants entering the stationary phase was exaggerated compared to the level in wild-type cells, but was blocked in apg1 pho85 mutants. We propose that Snf1p and Pho85p are, respectively, positive and negative regulators of autophagy, probably via Apg1 and/or Apg13. Defective glycogen storage in snf1 cells can be attributed to both defective synthesis upon entry into stationary phase and impaired maintenance of glycogen levels caused by the lack of autophagy.Cells constantly abstract information about their environment and modify their cellular and metabolic programs to cope with the prevailing conditions. For unicellular organisms like the budding yeast, Saccharomyces cerevisiae, much of the information about nutritional status is carried by the nutrients themselves. Depending on the type and availability of carbon, nitrogen, sulfur, and other requirements, the appropriate metabolic and cellular programs are elicited. Exhaustion of a preferred carbon source, like glucose, signals the induction of numerous genes needed to change to other metabolic regimes, in part by derepression of glucose-repressed genes and in part by cyclic AMP (cAMP) pathway control of gene expression (18,30). Another decision linked to nutritional deprivation is the synthesis of storage compounds like glycogen and trehalose, which are the primary carbohydrate reserves of the yeast (15). Glycogen is a branched polymer of glucose units that acts as a reserve of glucose and energy (47). In yeast growing on glucose, glycogen is synthesized late in the logarithmic phase and begins to be utilized when cells enter the stationary phase (7,8,37). However, glycogen stores can be p...

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1 for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 and CLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1 cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable in pho85Delta cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G1 Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1.

Background: The PHO85 gene is a negative regulator of the PHO system in the yeast Saccharomyces cerevisiae and encodes a protein kinase (Pho85p) which is highly homologous to the Cdc28 kinase (Cdc28p). Although the two kinases share a 51% identity and their functional domains are well conserved, PHO85 fails to replace CDC28. Pho85p forms complexes with G 1 -cyclin homologues, including Pcl1p, Pcl2p and Pcl9p, and is thought to be involved in the cell-cycle regulation at G 1 and the end of M. By analysing the genetic and biochemical properties of Pho85p, we studied whether the regulation of Pho85p activity is similar to other cyclin-dependent kinases (Cdks) directly involved in cell cycle regulation.

Pho85p is a yeast cyclin-dependent protein kinase (Cdk) that can interact with 10 cyclins (Pcls) to form multiple protein kinases. The functions of most of the Pcls, including Pc16p and Pc17p, are poorly defined. We report here that Pc16p and Pc17p are involved in the metabolism of the branched storage polysaccharide glycogen under certain conditions and deletion of PCL6 and PCL7 restores glycogen accumulation to a snf1 pcl8 pcl10 triple mutant, paradoxically activating both glycogen synthase and phosphorylase. Pho85p thus affects glycogen accumulation through multiple Cdks composed of different cyclin partners. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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