Ptc1 protein phosphatase 2C contributes to glucose regulation of SNF1/AMP-activated protein kinase (AMPK) in Saccharomyces cerevisiae.

The SNF1/AMP-activated protein kinases (AMPKs) function in energy regulation in eukaryotic cells. SNF1/AMPKs are αßγ heterotrimers that are activated by phosphorylation of the activation loop Thr on the catalytic subunit. Protein kinases that activate SNF1/AMPK have been identified, but the protein phosphatases responsible for dephosphorylation of the activation loop are less well defined. For Saccharomyces cerevisiae SNF1/AMPK, Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-related phosphatase function together to dephosphorylate Thr-210 on the Snf1 catalytic subunit during growth on high concentrations of glucose; reg1Δ and sit4Δ single mutations do not impair dephosphorylation when inappropriate glycogen synthesis, also caused by these mutations, is blocked. We here present evidence that Ptc1 protein phosphatase 2C also has a role in dephosphorylation of Snf1 Thr-210 in vivo. The sit4Δ ptc1Δ mutant exhibited partial defects in regulation of the phosphorylation state of Snf1. The reg1Δ ptc1Δ mutant was viable only when expressing mutant Snf1 proteins with reduced kinase activity, and Thr-210 phosphorylation of the mutant SNF1 heterotrimers was substantially elevated during growth on high glucose. This evidence, together with findings on the reg1Δ sit4Δ mutant, indicates that although Reg1-Glc7 plays the major role, all three phosphatases contribute to maintenance of the Snf1 activation loop in the dephosphorylated state during growth on high glucose. Ptc1 has overlapping functions with Reg1-Glc7 and Sit4 in glucose regulation of SNF1/AMPK and cell viability.

Heterotrimer-independent regulation of activation-loop phosphorylation of Snf1 protein kinase involves two protein phosphatases.

The SNF1/AMP-activated protein kinases are αßγ-heterotrimers that sense and regulate energy status in eukaryotes. They are activated by phosphorylation of the catalytic Snf1/α subunit, and the Snf4/γ regulatory subunit regulates phosphorylation through adenine nucleotide binding. In Saccharomyces cerevisiae, the Snf1 subunit is phosphorylated on the activation-loop Thr-210 in response to glucose limitation. To assess the requirement of the heterotrimer for regulated Thr-210 phosphorylation, we examined Snf1 and a truncated Snf1 kinase domain (residues 1-309) that has partial Snf1 function. Snf1(1-309) does not interact with the ß and Snf4/γ regulatory subunits, and its activity was independent of them in vivo. Phosphorylation of both Snf1 and Snf1(1-309) increased in response to glucose limitation in wild-type cells and in cells lacking ß- and Snf4/γ-subunits. These results indicate that glucose regulation of activation-loop phosphorylation can occur by mechanism(s) that function independently of the regulatory subunits. We further show that the Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-like phosphatase are largely responsible for dephosphorylation of Thr-210 of Snf1(1-309). Together, these findings suggest that these two phosphatases mediate heterotrimer-independent regulation of Thr-210 phosphorylation.

Alterations at dispersed sites cause phosphorylation and activation of SNF1 protein kinase during growth on high glucose.

The SNF1/AMP-activated protein kinases are central energy regulators in eukaryotes. SNF1 of Saccharomyces cerevisiae is inhibited during growth on high levels of glucose and is activated in response to glucose depletion and other stresses. Activation entails phosphorylation of Thr(210) on the activation loop of the catalytic subunit Snf1 by Snf1-activating kinases. We have used mutational analysis to identify Snf1 residues that are important for regulation. Alteration of Tyr(106) in the αC helix or Leu(198) adjacent to the Asp-Phe-Gly motif on the activation loop relieved glucose inhibition of phosphorylation, resulting in phosphorylation of Thr(210) during growth on high levels of glucose. Substitution of Arg for Gly(53), at the N terminus of the kinase domain, increased activation on both high and low glucose. Alteration of the ubiquitin-associated domain revealed a modest autoinhibitory effect. Previous studies identified alterations of the Gal83 (ß) and Snf4 (γ) subunits that relieve glucose inhibition, and we have here identified a distinct set of Gal83 residues that are required. Together, these results indicate that alterations at dispersed sites within each subunit of SNF1 cause phosphorylation of the kinase during growth on high levels of glucose. These findings suggest that the conformation of the SNF1 complex is crucial to maintenance of the inactive state during growth on high glucose and that the default state for SNF1 is one in which Thr(210) is phosphorylated and the kinase is active.

Roles of two protein phosphatases, Reg1-Glc7 and Sit4, and glycogen synthesis in regulation of SNF1 protein kinase.

The SNF1 protein kinase of Saccharomyces cerevisiae is a member of the SNF1/AMP-activated protein kinase family, which is essential for metabolic control, energy homeostasis, and stress responses in eukaryotes. SNF1 is activated in response to glucose limitation by phosphorylation of Thr210 on the activation loop of the catalytic subunit Snf1. The SNF1 ß-subunit contains a glycogen-binding domain that has been implicated in glucose inhibition of Snf1 Thr210 phosphorylation. To assess the role of glycogen, we examined Snf1 phosphorylation in strains with altered glycogen metabolism. A reg1Δ mutant, lacking Reg1-Glc7 protein phosphatase 1, exhibits elevated glycogen accumulation and phosphorylation of Snf1 during growth on high levels of glucose. Unexpectedly, mutations that abolished glycogen synthesis also restored Thr210 dephosphorylation in glucose-grown reg1Δ cells, indicating that elevated glycogen synthesis contributes to activation of SNF1 and that another phosphatase acts on Snf1. We present evidence that Sit4, a type 2A-like protein phosphatase, contributes to dephosphorylation of Snf1 Thr210. Finally, evidence that the effects of glycogen are not mediated by binding to the ß-subunit raises the possibility that elevated glycogen synthesis alters glucose metabolism and thereby reduces glucose signaling to the SNF1 pathway.

Interaction of SNF1 protein kinase with its activating kinase Sak1.

The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change.

Biochemical and functional studies on the regulation of the Saccharomyces cerevisiae AMPK homolog SNF1.

AMP-activated protein kinase (AMPK) is a master metabolic regulator for controlling cellular energy homeostasis. Its homolog in yeast, SNF1, is activated in response to glucose depletion and other stresses. The catalytic (alpha) subunit of AMPK/SNF1, Snf1 in yeast, contains a protein Ser/Thr kinase domain (KD), an auto-inhibitory domain (AID), and a region that mediates interactions with the two regulatory (beta and gamma) subunits. Previous studies suggested that Snf1 contains an additional segment, a regulatory sequence (RS, corresponding to residues 392-518), which may also have an important role in regulating the activity of the enzyme. The crystal structure of the heterotrimer core of Saccharomyces cerevisiae SNF1 showed interactions between a part of the RS (residues 460-498) and the gamma subunit Snf4. Here we report biochemical and functional studies on the regulation of SNF1 by the RS. GST pulldown experiments demonstrate strong and direct interactions between residues 450-500 of the RS and the heterotrimer core, and single-site mutations in the RS-Snf4 interface can greatly reduce these interactions in vitro. On the other hand, functional studies appear to show only small effects of the RS-Snf4 interactions on the activity of SNF1 in vivo. This suggests that residues 450-500 may be constitutively associated with Snf4, and the remaining segments of the RS, as well as the AID, may be involved in regulating SNF1 activity.

Roles of the glycogen-binding domain and Snf4 in glucose inhibition of SNF1 protein kinase.

The SNF1/AMP-activated protein kinase (AMPK) family is required for adaptation to metabolic stress and energy homeostasis. The gamma subunit of AMPK binds AMP and ATP, and mutations that affect binding cause human disease. We have here addressed the role of the Snf4 (gamma) subunit in regulating SNF1 protein kinase in response to glucose availability in Saccharomyces cerevisiae. Previous studies of mutant cells lacking Snf4 suggested that Snf4 counteracts autoinhibition by the C-terminal sequence of the Snf1 catalytic subunit but is dispensable for glucose regulation, and AMP does not activate SNF1 in vitro. We first introduced substitutions at sites that, in AMPK, contribute to nucleotide binding and regulation. Mutations at several sites relieved glucose inhibition of SNF1, as judged by catalytic activity, phosphorylation of the activation-loop Thr-210, and growth assays, although analogs of the severe human mutations R531G/Q had little effect. We further showed that alterations of Snf4 residues that interact with the glycogen-binding domain (GBD) of the beta subunit strongly relieved glucose inhibition. Finally, substitutions in the GBD of the Gal83 beta subunit that are predicted to disrupt interactions with Snf4 and also complete deletion of the GBD similarly relieved glucose inhibition of SNF1. Analysis of mutant cells lacking glycogen synthase showed that regulation of SNF1 is normal in the absence of glycogen. These findings reveal novel roles for Snf4 and the GBD in regulation of SNF1.

SNF1/AMPK pathways in yeast.

The SNF1/AMPK family of protein kinases is highly conserved in eukaryotes and is required for energy homeostasis in mammals, plants, and fungi. SNF1 protein kinase was initially identified by genetic analysis in the budding yeast Saccharomyces cerevisiae. SNF1 is required primarily for the adaptation of yeast cells to glucose limitation and for growth on carbon sources that are less preferred than glucose, but is also involved in responses to other environmental stresses. SNF1 regulates transcription of a large set of genes, modifies the activity of metabolic enzymes, and controls various nutrient-responsive cellular developmental processes. Like AMPK, SNF1 protein kinase is heterotrimeric. It is phosphorylated and activated by the upstream kinases Sak1, Tos3, and Elm1 and is inactivated by the Reg1-Glc7 protein phosphatase 1. Further regulation of SNF1 is achieved through autoinhibition and through control of its subcellular localization. Here we review the current understanding of SNF1 protein kinase pathways in Saccharomyces cerevisiae and other yeasts.

Regulation of snf1 protein kinase in response to environmental stress.

The Saccharomyces cerevisiae Snf1 protein kinase, a member of the Snf1/AMPK (AMP-activated protein kinase) family, has important roles in metabolic control, particularly in response to nutrient stress. Here we have addressed the role of Snf1 in responses to other environmental stresses. Exposure of cells to sodium ion stress, alkaline pH, or oxidative stress caused an increase in Snf1 catalytic activity and phosphorylation of Thr-210 in the activation loop, whereas treatment with sorbitol or heat shock did not. Inhibition of respiratory metabolism by addition of antimycin A to cells also increased Snf1 activity. Analysis of mutants indicated that the kinases Sak1, Tos3, and Elm1, which activate Snf1 in response to glucose limitation, are also required under other stress conditions. Each kinase sufficed for activation in response to stress, but Sak1 had the major role. In sak1Delta tos3Delta elm1Delta cells expressing mammalian Ca(2+)/calmodulin-dependent protein kinase kinase alpha, Snf1 was activated by both sodium ion and alkaline stress, suggesting that stress signals regulate Snf1 activity by a mechanism that is independent of the upstream kinase. Finally, we showed that Snf1 protein kinase is regulated differently during adaptation of cells to NaCl and alkaline pH with respect to both temporal regulation of activation and subcellular localization. Snf1 protein kinase becomes enriched in the nucleus in response to alkaline pH but not salt stress. Such differences could contribute to specificity of the stress responses.

Structure of the Bateman2 domain of yeast Snf4: dimeric association and relevance for AMP binding.

AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis in mammals. AMP is believed to control the activity of AMPK by binding to the gamma subunit of this heterotrimeric enzyme. This subunit contains two Bateman domains, each of which is composed of a tandem pair of cystathionine beta-synthase (CBS) motifs. No structural information is currently available on this subunit, and the molecular basis for its interactions with AMP is not well understood. We report here the crystal structure at 1.9 Angstrom resolution of the Bateman2 domain of Snf4, the gamma subunit of the yeast ortholog of AMPK. The structure revealed a dimer of the Bateman2 domain, and this dimerization is supported by our light-scattering, mutagenesis, and biochemical studies. There is a prominent pocket at the center of this dimer, and most of the disease-causing mutations are located in or near this pocket.

Regulation of the nucleocytoplasmic distribution of Snf1-Gal83 protein kinase.

Snf1 protein kinase containing the beta subunit Gal83 is localized in the cytoplasm during growth of Saccharomyces cerevisiae cells in abundant glucose and accumulates in the nucleus in response to glucose limitation. Nuclear localization of Snf1-Gal83 requires activation of the Snf1 catalytic subunit and depends on Gal83, but in the snf1Delta mutant, Gal83 exhibits glucose-regulated nuclear accumulation. We show here that the N terminus of Gal83, which is divergent from those of the other beta subunits, is necessary and sufficient for Snf1-independent, glucose-regulated localization. We identify a leucine-rich nuclear export signal in the N terminus and show that export depends on the Crm1 export receptor. We present evidence that catalytically inactive Snf1 promotes the cytoplasmic retention of Gal83 in glucose-grown cells through its interaction with the C terminus of Gal83; cytoplasmic localization of inactive Snf1-Gal83 maintains accessibility to the Snf1-activating kinases. Finally, we characterize the effects of glucose phosphorylation on localization. These studies define roles for Snf1 and Gal83 in determining the nucleocytoplasmic distribution of Snf1-Gal83 protein kinase.

Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro.

The Snf1/AMP-activated protein kinase (AMPK) family is important for metabolic regulation and is highly conserved from yeast to mammals. The upstream kinases are also functionally conserved, and the AMPK kinases LKB1 and Ca2+/calmodulin-dependent protein kinase kinase activate Snf1 in mutant yeast cells lacking the native Snf1-activating kinases, Sak1, Tos3, and Elm1. Here, we exploited the yeast genetic system to identify members of the mammalian AMPK kinase family by their function as Snf1-activating kinases. A mouse embryo cDNA library in a yeast expression vector was used to transform sak1Delta tos3Delta elm1Delta yeast cells. Selection for a Snf+ growth phenotype yielded cDNA plasmids expressing LKB1, Ca2+/calmodulin-dependent protein kinase kinase, and transforming growth factor-beta-activated kinase (TAK1), a member of the mitogen-activated protein kinase kinase kinase family. We present genetic and biochemical evidence that TAK1 activates Snf1 protein kinase in vivo and in vitro. We further show that recombinant TAK1, fused to the activation domain of its binding partner TAB1, phosphorylates Thr-172 in the activation loop of the AMPK catalytic domain. Finally, expression of TAK1 and TAB1 in HeLa cells or treatment of cells with cytokines stimulated phosphorylation of Thr-172 of AMPK. These findings indicate that TAK1 is a functional member of the Snf1/AMPK kinase family and support TAK1 as a candidate for an authentic AMPK kinase in mammalian cells.

A specific catalytic subunit isoform of protein kinase CK2 is required for phosphorylation of the repressor Nrg1 in Saccharomyces cerevisiae.

Protein kinase CK2 is highly conserved in eukaryotes and plays roles in many different cellular processes. CK2 is a tetramer comprising two catalytic and two regulatory subunits. Most organisms have two major isoforms of the catalytic subunit, and evidence suggests strongly overlapping function. In the yeast Saccharomyces cerevisiae, CK2 is essential for viability, and either catalytic subunit isoform, Cka1 or Cka2, suffices, but previous genetic evidence suggests that the isoforms have some distinct roles. In this work, we present evidence that the transcriptional repressor Nrg1, which regulates various stress-responsive genes, is a downstream target of CK2 containing the Cka1 isoform. We found that Nrg1 is phosphorylated in response to stress and that its phosphorylation was defective in cka1Delta, but not cka2Delta, mutants. Thus, the Cka1 catalytic subunit isoform is specifically required for phosphorylation of Nrg1 in vivo. The CK2 regulatory subunits were also required, indicating that the CK2 holoenzyme is involved. Both yeast and recombinant human CK2 phosphorylated recombinant Nrg1 in vitro. This identification of a protein whose phosphorylation requires a specific CK2 catalytic subunit isoform supports the view that the two isoforms exhibit functional specificity in vivo.

Repressors Nrg1 and Nrg2 regulate a set of stress-responsive genes in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae responds to environmental stress by rapidly altering the expression of large sets of genes. We report evidence that the transcriptional repressors Nrg1 and Nrg2 (Nrg1/Nrg2), which were previously implicated in glucose repression, regulate a set of stress-responsive genes. Genome-wide expression analysis identified 150 genes that were upregulated in nrg1Delta nrg2Delta double mutant cells, relative to wild-type cells, during growth in glucose. We found that many of these genes are regulated by glucose repression. Stress response elements (STREs) and STRE-like elements are overrepresented in the promoters of these genes, and a search of available expression data sets showed that many are regulated in response to a variety of environmental stress signals. In accord with these findings, mutation of NRG1 and NRG2 enhanced the resistance of cells to salt and oxidative stress and decreased tolerance to freezing. We present evidence that Nrg1/Nrg2 not only contribute to repression of target genes in the absence of stress but also limit induction in response to salt stress. We suggest that Nrg1/Nrg2 fine-tune the regulation of a set of stress-responsive genes.

Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells.

AMP-activated protein kinase (AMPK) is the downstream component of a kinase cascade that plays a pivotal role in energy homeostasis. Activation of AMPK requires phosphorylation of threonine 172 (T172) within the T loop region of the catalytic alpha subunit. Recently, LKB1 was shown to activate AMPK. Here we show that AMPK is also activated by Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK). Overexpression of CaMKKbeta in mammalian cells increases AMPK activity, whereas pharmacological inhibition of CaMKK, or downregulation of CaMKKbeta using RNA interference, almost completely abolishes AMPK activation. CaMKKbeta isolated from rat brain or expressed in E. coli phosphorylates and activates AMPK in vitro. In yeast, CaMKKbeta expression rescues a mutant strain lacking the three kinases upstream of Snf1, the yeast homolog of AMPK. These results demonstrate that AMPK is regulated by at least two upstream kinases and suggest that AMPK may play a role in Ca(2+)-mediated signal transduction pathways.

Role of Tos3, a Snf1 protein kinase kinase, during growth of Saccharomyces cerevisiae on nonfermentable carbon sources.

In Saccharomyces cerevisiae, Snf1 protein kinase of the Snf1/AMP-activated protein kinase family is required for growth on nonfermentable carbon sources and nonpreferred sugars. Three kinases, Pak1, Elm1, and Tos3, activate Snf1 by phosphorylation of its activation-loop threonine, and the absence of all three causes the Snf(-) phenotype. No phenotype has previously been reported for the tos3Delta single mutation. We show here that, when cells are grown on glycerol-ethanol, tos3Delta reduces growth rate, Snf1 catalytic activity, and activation of the Snf1-dependent carbon source-responsive element (CSRE) in the promoters of gluconeogenic genes. In contrast, tos3Delta did not significantly affect Snf1 catalytic activity or CSRE function during abrupt glucose depletion, indicating that Tos3 has a more substantial role in activating Snf1 protein kinase during growth on a nonfermentable carbon source than during acute carbon stress. We also report that Tos3 is localized in the cytosol during growth in either glucose or glycerol-ethanol. These findings lend support to the idea that the Snf1 protein kinase kinases make different contributions to cellular regulation under different growth conditions.

Function of mammalian LKB1 and Ca2+/calmodulin-dependent protein kinase kinase alpha as Snf1-activating kinases in yeast.

The Snf1/AMP-activated protein kinase (AMPK) family is important for metabolic regulation in response to stress. In the yeast Saccharomyces cerevisiae, the Snf1 kinase cascade comprises three Snf1-activating kinases, Pak1, Tos3, and Elm1. The only established mammalian AMPK kinase is LKB1. We show that LKB1 functions heterologously in yeast. In pak1Delta tos3Delta elm1Delta cells, LKB1 activated Snf1 catalytic activity and conferred a Snf(+) growth phenotype. Coexpression of STRADalpha and MO25alpha, which form a complex with LKB1, enhanced LKB1 function. Thus, the Snf1/AMPK kinase cascade is functionally conserved between yeast and mammals. Ca(2+)/calmodulin-dependent kinase kinase (CaMKK) shows more sequence similarity to Pak1, Tos3, and Elm1 than does LKB1. When expressed in pak1Delta tos3Delta elm1Delta cells, CaMKKalpha activated Snf1 catalytic activity, restored the Snf(+) phenotype, and also phosphorylated the activation loop threonine of Snf1 in vitro. These findings indicate that CaMKKalpha is a functional member of the Snf1/AMPK kinase family and support CaMKKalpha as a likely candidate for an AMPK kinase in mammalian cells. Analysis of the function of these heterologous kinases in yeast provided insight into the regulation of Snf1. When activated by LKB1 or CaMKKalpha, Snf1 activity was significantly inhibited by glucose, suggesting that a mechanism independent of the activating kinases can mediate glucose signaling in yeast. Finally, this analysis provided evidence that Pak1 functions in another capacity, besides activating Snf1, to regulate the nuclear enrichment of Snf1 protein kinase in response to carbon stress.

Pak1 protein kinase regulates activation and nuclear localization of Snf1-Gal83 protein kinase.

Three kinases, Pak1, Tos3, and Elm1, activate Snf1 protein kinase in Saccharomyces cerevisiae. This cascade is conserved in mammals, where LKB1 activates AMP-activated protein kinase. We address the specificity of the activating kinases for the three forms of Snf1 protein kinase containing the beta-subunit isoforms Gal83, Sip1, and Sip2. Pak1 is the most important kinase for activating Snf1-Gal83 in response to glucose limitation, but Elm1 also has a significant role; moreover, both Pak1 and Elm1 affect Snf1-Sip2. These findings exclude the possibility of a one-to-one correspondence between the activating kinases and the Snf1 complexes. We further identify a second, unexpected role for Pak1 in regulating Snf1-Gal83: the catalytic activity of Pak1 is required for the nuclear enrichment of Snf1-Gal83 in response to carbon stress. The nuclear enrichment of Snf1 fused to green fluorescent protein (GFP) depends on both Gal83 and Pak1 and is abolished by a mutation of the activation loop threonine; in contrast, the nuclear enrichment of Gal83-GFP occurs in a snf1Delta mutant and depends on Pak1 only when Snf1 is present. Snf1-Gal83 is the only form of the kinase that localizes to the nucleus. These findings, that Pak1 both activates Snf1-Gal83 and controls its nuclear localization, implicate Pak1 in regulating nuclear Snf1 protein kinase activity.

Nrg1 and nrg2 transcriptional repressors are differently regulated in response to carbon source.

The Nrg1 and Nrg2 repressors of Saccharomyces cerevisiae have highly similar zinc fingers and closely related functions in the regulation of glucose-repressed genes. We show that NRG1 and NRG2 are differently regulated in response to carbon source at both the RNA and protein levels. Expression of NRG1 RNA is glucose repressed, whereas NRG2 RNA levels are nearly constant. Nrg1 protein levels are elevated in response to glucose limitation or growth in nonfermentable carbon sources, whereas Nrg2 levels are diminished. Chromatin immunoprecipitation assays showed that Nrg1 and Nrg2 bind DNA both in the presence and absence of glucose. In mutant cells lacking the corepressor Ssn6(Cyc8)-Tup1, promoter-bound Nrg1, but not Nrg2, functions as an activator in a reporter assay, providing evidence that the two Nrg proteins have distinct properties. We suggest that the differences in expression and function of these two repressors, in combination with their similar DNA-binding domains, contribute to the complex regulation of the large set of glucose-repressed genes.