Antagonistic Controls of Autophagy and Glycogen Accumulation by Snf1p, the Yeast Homolog of AMP-Activated Protein Kinase, and the Cyclin-Dependent Kinase Pho85p

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 preserved very late into starvation and are thought to be utilized during sporulation (10). In dividing cells, there is also some evidence for a relationship between accumulation of carbohydrate reserves and the cell cycle (52). Glycogen and trehalose are synthesized in G1 and depleted at the stage of bud emergence. The pathway of glycogen biosynthesis, conserved between yeast and mammals, starts with glycogenin, a self-glucosylating initiator protein that forms an oligosaccharide primer that is the substrate for elongation and branching by glycogen synthase and the branching enzyme, respectively (7, 42). Glycogen synthase is an important site of control in both yeast and mammals. In yeast, glycogen synthase is encoded by two genes, GSY1 and GSY2, of which Gsy2p accounts for 80% of the glycogen synthase activity in the stationary phase (13). GSY2 transcription is induced by stresses, such as nutrient limitation, high salt, or heat shock (13, 43, 46), and the Gsy2p enzyme is also negatively regulated by covalent phosphorylation (25). A third control is by allosteric activation, the most important ligand being Glc-6-P, the binding of which overcomes inactivation due to phosphorylation (25, 49). This property leads to the use of the ratio of the activity in the absence of Glc-6-P divided by that in its presence as a kinetic index of the phosphorylation state of the enzyme (−/+ Glc-6-P activity ratio). Snf1p is, by sequence similarity, the closest yeast homologue of the mammalian AMP-activated protein kinase (23) and has been studied mostly in relation to its role in glucose repression (5, 18, 23). In cells lacking SNF1, glucose-repressed genes cannot be derepressed in the absence of glucose (5, 6). SNF1 also has a role in glycogen metabolism and one of the original set of glycogen-deficient mutant strains (GLC mutants), glc2, carried a mutant allele of SNF1 (4). Hardy et al. (24) showed that the impaired glycogen accumulation in snf1 cells correlated with hyperphosphorylation of glycogen synthase, as evidenced by a low −/+ Glc-6-P activity ratio, rather than control of glycogen synthase expression. In an effort to identify protein kinases that phosphorylate glycogen synthase, an snf1 strain was thus used in a screen for second site suppressors of the glycogen defect (26). We identified PHO85, and the snf1 pho85 double mutants selected in the screen had both a wild-type glycogen synthase activity ratio and normal glycogen levels. PHO85 encodes a cyclin-dependent protein kinase (CDK) catalytic subunit that, like other CDKs (1), acts in concert with cyclin partners, called Pcls, of which 10 are known (40). PHO85 was first identified through its involvement in phosphate metabolism, for which it pairs with the cyclin Pho80p to phosphorylate the Pho4p transcription factor (30a, 56). Subsequent work showed that Pcl8p and Pcl10p link Pho85p to inactivation of glycogen synthase (27). Thus, deletion of PCL8 and PCL10 in a wild-type strain results in a high glycogen synthase activity ratio and hyperaccumulation of glycogen (27). Similarly, loss of PCL8 and PCL10 in an snf1 strain restores glycogen synthase activity, but, in an unexpected result that represents the starting point for this study, did not restore glycogen accumulation (27). Therefore, we proposed that there must be some other factor or process, controlled by Snf1p, that is not corrected by mutation of PCL8 and PCL10 (27). Another response to nutrient starvation by both yeast and mammalian cells is the induction of autophagy (11, 12, 53). Autophagy is a process whereby cells randomly engulf cytosol and organelles to form autophagosomes that are delivered to the vacuole (in yeast) or lysozome (in mammals). There the autophagosomes are degraded to recycle some components, such as amino acids, and to generate energy during starvation. A number of genes have been implicated in this process through different genetic screens (34). One of the first yeast autophagy genes identified was APG1 which encodes a Ser/Thr protein kinase (57), whose kinase activity is required for its function (39). By epistasis, APG1 has been placed downstream of another autophagy gene, APG13 (17), whose sequence matches nothing in the databases. In the course of our efforts to identify novel factors controlled by Snf1p that affected glycogen metabolism, a high-copy suppressor screen with an snf1 pc18 pcl10 host identified APG1 and APG13. We described here how the process of autophagy is related to the ability of cells to maintain glycogen stores, and we propose that autophagy is controlled by Snf1p and Pho85p.