Functional analysis of the yeast Glc7-binding protein Reg1 identifies a protein phosphatase type 1-binding motif as essential for repression of ADH2 expression.

Protein phosphatase type 1 (PP1) plays a key role in regulating a diverse variety of processes in eukaryotic cells (3, 48). The amino acid sequences of the mammalian and yeast homologues of the PP1 catalytic subunit (PP1c) are more than 80% identical, suggesting that their function and the regulatory mechanisms that control their activity have been conserved throughout evolution. The gene coding for the Saccharomyces cerevisiae homologue of PP1c is GLC7. Glc7 is required for the appropriate regulation of a number of cellular processes, including glycogen biosynthesis; translation; cell cycle progression; chromosome segregation, meiosis, and sporulation; and repression of many glucose-regulated genes (54). Unlike protein kinase catalytic subunits which can recognize a window of amino acid sequence surrounding the phosphorylation site (39), PP1c exhibits little inherent substrate specificity in vitro (11, 39). There is now a large body of evidence suggesting that specificity is conferred by regulatory subunits. When complexed with PP1c, they target it to specific substrates (11, 22). In S. cerevisiae, a number of Glc7-binding proteins affecting specific cellular processes have been identified (54). These potential regulatory subunits include Gac1 and Pig1, which affect glycogen accumulation (9, 55); Reg2 and Sds22, which affect growth and cell cycle progression (24, 33, 41); Gip1, which is required for completion of meiosis and sporulation (59); Scd5, which affects the vesicular secretory pathway (59); Pig2, whose function is unknown (9); and Reg1, which affects glucose repression, growth, and glycogen accumulation (23, 24, 35, 42, 44). In S. cerevisiae, glucose repression is the major mechanism through which the expression of genes involved in the utilization of alternative or fermentable carbon sources is coordinately regulated (25). In the presence of high concentrations of glucose, the expression of glucose-regulated genes is low or repressed. When the concentration of glucose drops below 0.2%, expression of these genes is activated or derepressed. For glucose-repressible genes like SUC2, GAL1, and ADH2, this change in the level of expression can be 200-fold or greater (7, 20, 27). A number of genes have been identified as playing integral roles in glucose repression (25). Among these are REG1, GLC7, and SNF1, the yeast homologue of the catalytic subunit of AMP-activated protein kinase (30). SNF1 is required for derepression of gene expression in glucose-limited cells (4, 10, 67), while REG1 and GLC7 are required for the maintenance of the fully repressed state (23, 42, 44). A combination of genetic, two-hybrid, and coimmunoprecipitation experiments have indicated that Snf1 is complexed with Snf4 and one member of the Sip/Gal83 class of proteins (7, 65). Snf1 is thought to be anchored in the complex by its C-terminal regulatory domain to the centrally located KIS domain of the Sip/Gal83 protein (38). Snf4 is also anchored in the complex by interacting with the Sip/Gal83 protein; however, this interaction is with the C-terminal ACS domain. These interactions do not appear to be carbon source regulated. The interaction of Snf1 with Snf4, however, does appear to be carbon source regulated (37). In repressed cells, the N-terminal kinase domain of Snf1 appears to interact with its C-terminal regulatory domain, which is thought to inhibit kinase activity. Upon depletion of glucose from the growth medium, Snf4 is thought to bind to the kinase domain, displacing the regulatory domain and, thereby, freeing the Snf1 kinase domain from autoinhibition. Two-hybrid and coimmunoprecipitation experiments have also suggested that Reg1 and Glc7 act together as a complex (59). Like interactions with the Sip/Gal83 component of the Snf1 complex, the interaction between Reg1 and Glc7 does not appear to be glucose regulated. Recently, evidence has been presented indicating that Reg1 interacts with the kinase domain of Snf1, altering protein-protein interactions within the kinase complex (40). Two-hybrid experiments have suggested that Reg1 interacts weakly with the kinase domain of Snf1 in repressed cells and strongly in derepressed cells. This interaction required amino acid T210 in the activation loop, which is essential for Snf1 kinase activity and for the interaction with Snf4. Based on these observations, it was proposed that Reg1 targets Glc7 to an active Snf1 complex by binding to the kinase domain. Once bound, Glc7 could then dephosphorylate Snf1, thereby releasing Snf4 from the kinase regulatory domain and returning the complex to an autoinhibited state. Although the Reg1-Glc7 complex has been clearly implicated in the repression of SUC2 expression, surprisingly, only Reg1 has been demonstrated to play a role in repressing ADH2 expression (20). Even though reg1 mutant cells growing under normally repressing conditions have up to 40-fold greater ADH2 expression than wild-type cells, a glc7-T152K mutant, which has a constitutively high level of SUC2 expression, is fully repressed for ADH2 expression (20). The level of this constitutive ADH2 expression is similar to that seen in cells with an ADR1c allele. ADR1c mutations fall within or near the cyclic AMP-dependent protein kinase (cAPK) phosphorylation site at serine 230 of ADR1 (18), the major activator of ADH2 transcription (17). Reg1 appears to act independently of this phosphorylation site, however, since ADR1c alleles synergistically increase ADH2 expression in reg1 mutant cells under normally repressing growth conditions (20). Like activated ADH2 expression in derepressed wild-type cells, constitutive ADH2 expression in reg1 mutant cells under normally repressing growth conditions requires ADR1. Cells with mutations in both ADR1 and REG1 have fully repressed ADH2 expression during growth on a high-glucose medium. Also, a reporter gene with promoter sequences containing UAS1, the Adr1 binding site in the ADH2 promoter (51), is constitutively expressed in reg1 mutant cells (20). The level of Adr1 in reg1 mutant cells under normally repressing growth conditions is nearly the same as that in derepressed wild-type cells. This 3-fold higher level of Adr1, however, cannot fully account for the 40-fold higher level of ADH2 expression, since ADH2 expression appears to increase linearly with the level of ADR1 expression in repressed cells (15). Also, recent data from our lab has shown that repressed cells having the same level of Adr1 as derepressed cells do not constitutively express ADH2 (53). ADH2 expression has the same requirement for Snf1 as SUC2 expression (10), and repression of both ADH2 and SUC2 in a high-glucose medium has the same requirement for Reg1 (4, 20). This raised the question of how Reg1 could function in the apparent absence of a requirement for Glc7. In the study presented here, we provide the first evidence showing that even though repression of ADH2 expression has an apparent differential requirement for Reg1 and Glc7, Glc7 may indeed play a role in repressing ADH2 expression. A sequence similar to the mammalian PP1-binding motif (R/K)(V/I)XF (22) was identified as being essential for repression of both ADH2 and SUC2 expression and for the interaction of Reg1 with Glc7. We also show that the level of Adr1 in glc7 mutant cells is limiting for constitutive ADH2 expression under repressing growth conditions. Subcellular localization of Reg1-green fluorescent protein (GFP) suggested that Reg1 is cytoplasmic and excluded from the nucleus. These findings suggest that the Reg1-Glc7 phosphatase complex is part of the cytoplasmic machinery for resetting Snf1 kinase activity to a basal level and imply that the activated form of the Snf1 complex rapidly cycles between the nucleus and the cytoplasm.