The Snf1/AMP-activated protein kinase family has broad roles in transcriptional, metabolic, and developmental regulation in response to stress. In Saccharomyces cerevisiae, Snf1 is required for the response to glucose limitation. Snf1 kinase complexes contain the ␣ (catalytic) subunit Snf1, one of the three related ␤ subunits Gal83, Sip1, or Sip2, and the ␥ subunit Snf4. We present evidence that the ␤ subunits regulate the subcellular localization of the Snf1 kinase. Green fluorescent protein fusions to Gal83, Sip1, and Sip2 show different patterns of localization to the nucleus, vacuole, and/or cytoplasm. We show that Gal83 directs Snf1 to the nucleus in a glucose-regulated manner. We further identify a novel signaling pathway that controls this nuclear localization in response to glucose phosphorylation. This pathway is distinct from the glucose signaling pathway that inhibits Snf1 kinase activity and responds not only to glucose but also to galactose and sucrose. Such independent regulation of the localization and the activity of the Snf1 kinase, combined with the distinct localization of kinases containing different ␤ subunits, affords versatility in regulating physiological responses.

In glucose-grown cells, the Mig1 DNA-binding protein recruits the Ssn6-Tup1 corepressor to glucoserepressed promoters in the yeast Saccharomyces cerevisiae. Previous work showed that Mig1 is differentially phosphorylated in response to glucose. Here we examine the role of Mig1 in regulating repression and the role of the Snf1 protein kinase in regulating Mig1 function. Immunoblot analysis of Mig1 protein from a snf1 mutant showed that Snf1 is required for the phosphorylation of Mig1; moreover, hxk2 and reg1 mutations, which relieve glucose inhibition of Snf1, correspondingly affect phosphorylation of Mig1. We show that Snf1 and Mig1 interact in the two-hybrid system and also coimmunoprecipitate from cell extracts, indicating that the two proteins interact in vivo. In immune complex assays of Snf1, coprecipitating Mig1 is phosphorylated in a Snf1-dependent reaction. Mutation of four putative Snf1 recognition sites in Mig1 eliminated most of the differential phosphorylation of Mig1 in response to glucose in vivo and improved the two-hybrid interaction with Snf1. These studies, together with previous genetic findings, indicate that the Snf1 protein kinase regulates phosphorylation of Mig1 in response to glucose.In Saccharomyces cerevisiae the Ssn6 (Cyc8)-Tup1 complex represses transcription of genes regulated by glucose, cell type, oxygen, DNA damage, and other signals (27,34,45,46,48,52,54,56,57,61). Ssn6-Tup1 is recruited to these promoters by specific DNA-binding proteins, including ␣2-Mcm1, a1-␣2, Mig1, Mig2, Rox1, and Rgt1 (1,27,31,39,47,51), and mediates repression by interacting with chromatin (43) and/or the general transcriptional machinery (20,21,41). In this work we have focused on the role of Mig1 in regulating repression by Ssn6-Tup1 in response to the glucose signal.Mig1 is a Cys 2 -His 2 zinc finger protein (36) that binds to the promoters of SUC, GAL, MAL, and other glucose-repressible genes; mutation of Mig1 or its binding sites partially relieves glucose repression (15,17,22,25,35,36,44,53,55). A LexAMig1 fusion protein represses transcription of a CYC1-lacZ reporter containing lexA operators. Such repression requires Ssn6-Tup1 and occurs only in glucose-grown cells (47, 51). Mig1 is differentially phosphorylated in response to glucose availability (11, 47), and the localization of Mig1 to the nucleus requires glucose (11). In contrast, no difference in modification was detected for Ssn6 or Tup1 (46, 57), and Ssn6 resides in the nucleus regardless of glucose availability (46). These findings strongly suggest that the recruitment of Ssn6-Tup1 to a promoter by Mig1 is regulated by glucose. However, it remains possible that other mechanisms also contribute to regulation of repression by the Mig1-Ssn6-Tup1 complex. Here we present evidence that LexA-Mig1 confers glucose-regulated repression to a promoter that is not otherwise glucose repressed, thereby excluding any requirement for other promoter-bound glucoseregulated factors. We also show that repression by LexA-Ssn6 is not glucose regulated, indica...

The Snf1 protein kinase of Saccharomyces cerevisiae is important for many cellular responses to glucose limitation, including haploid invasive growth. We show here that Snf1 regulates transcription of FLO11, which encodes a cell surface glycoprotein required for invasive growth. We further show that Nrg1 and Nrg2, two repressor proteins that interact with Snf1, function as negative regulators of invasive growth and as repressors of FLO11. We also examined the role of Snf1, Nrg1, and Nrg2 in two other Flo11-dependent processes. Mutations affected the initiation of biofilm formation, which is glucose sensitive, but also affected diploid pseudohyphal differentiation, thereby unexpectedly implicating Snf1 in a response to nitrogen limitation. Deletion of the NRG1 and NRG2 genes suppressed the defects of a snf1 mutant in all of these processes. These findings suggest a model in which the Snf1 kinase positively regulates Flo11-dependent developmental events by antagonizing Nrg-mediated repression of the FLO11 gene.

The SSN3 and SSN8 genes of Saccharomyces cerevisiae were identified by mutations that suppress a defect in SNF1, a protein kinase required for release from glucose repression. Mutations in SSN3 and SSN8 also act synergistically with a mutation of the MIG1 repressor protein to relieve glucose repression. We have cloned the SSN3 and SSN8 genes. SSN3 encodes a cyclin-dependent protein kinase (cdk) homolog and is identical to UMES. SSN8 encodes a cyclin homolog 35% identical to human cyclin C. SSN3 and SSN8 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. Using an immune complex assay, we detected protein kinase activity that depends on both SSN3 and SSN8. Thus, the two SSN proteins are likely to function as a cdk-cyclin pair. Genetic analysis indicates that the SSN3-SSN8 complex contributes to transcriptional repression of diversely regulated genes and also affects induction of the GAL] promoter.In the budding yeast Saccharomyces cerevisiae, expression of glucose-repressed genes in response to glucose starvation requires the SNF1 protein-serine/threonine kinase (1). The SNF1 kinase is widely conserved, and its mammalian counterpart, AMP-activated protein kinase, is involved in regulation of lipid metabolism and cellular stress responses (2). In yeast, genetic evidence indicates that one of the functions of SNF1 is to relieve transcriptional repression mediated by the SSN6-TUP1 repressor complex. This complex is tethered to glucose-repressed promoters, including that of SUC2 (encoding invertase), by the DNA-binding protein MIG1 (3-5). Mutations in SSN6 or TUPI completely relieve glucose repression of SUC2 and bypass the requirement for the SNF1 kinase. A migl mutation partially relieves repression, suggesting that other DNA-binding proteins are also involved (6).Mutations in the SSN3 and SSN8 genes were isolated as suppressors of the snfl mutant defect in SUC2 derepression (7). This selection also yielded mutations in six other SSN genes, including MIG1 (= SSNJ) and SSN6. The ssn3 and ssn8 mutations are weak suppressors alone but show strong synergy with migl. Moreover, in strains wild type for SNF1, both ssn3 and ssn8 act synergistically with migl to relieve glucose repression of SUC2 (6). These findings implicate SSN3 and SSN8 in negative regulation of SUC2 expression. Both mutations also cause flocculence.To explore the regulatory roles of SSN3 and SSN8, we have cloned the genes by complementation. Sequence analysis showed homology to the cyclin-dependent protein kinase (cdk) family and cyclin C, respectively. Many protein kinases involved in cell cycle control are composed of a catalytic subunit, the cdk, and an activating/targeting subunit, the cyclin. We present genetic and biochemical evidence that SSN3 and SSN8 constitute a cdk-cyclin pair. Genetic evidence suggests a general role for SSN3-SSN8 in transcriptional control.*

RNA polymerase II holoenzymes respond to activators and repressors that are regulated by signaling pathways. Here we present evidence for a ''shortcut'' mechanism in which the Snf1 protein kinase of the glucose signaling pathway directly regulates transcription by the yeast holoenzyme. In response to glucose limitation, the Snf1 kinase stimulates transcription by holoenzyme that has been artificially recruited to a reporter by a LexA fusion to a holoenzyme component. We show that Snf1 interacts physically with the Srb͞mediator proteins of the holoenzyme in both twohybrid and coimmunoprecipitation assays. We also show that a catalytically hyperactive Snf1, when bound to a promoter as a LexA fusion protein, activates transcription in a glucose-regulated manner; moreover, this activation depends on the integrity of the Srb͞mediator complex. These results suggest that direct regulatory interactions between signal transduction pathways and RNA polymerase II holoenzyme provide a mechanism for transcriptional control in response to important signals.

The RNA polymerase II of Saccharomyces cerevisiae exists in holoenzyme forms containing a complex, known as the mediator, associated with the carboxyl-terminal domain. The mediator includes several SRB proteins and is required for transcriptional activation. Previous work showed that a cyclin-dependent kinase-cyclin pair encoded by SSN3 and SSN8, two members of the SSN suppressor family, are identical to two SRB proteins in the mediator. Here we have identified the remaining SSN genes by cloning and genetic analysis. SSN2 and SSN5 are identical to SRB9 and SRB8, respectively, which encode additional components of the mediator. Genetic evidence implicates the SSN genes in transcriptional repression. Thus, these identities provide genetic insight into mediator and carboxyl-terminal domain function, strongly suggesting a role in mediating transcriptional repression as well as activation. We also show that SSN4 and SSN7 are the same as SIN4 and ROX3, respectively, raising the possibility that these genes also encode mediator proteins.Studies of the Saccharomyces cerevisiae RNA polymerase II have led to the identification of a holoenzyme form containing a large multiprotein complex associated with the carboxylterminal repeat domain (CTD) of the largest subunit and a subset of general transcription factors (22)(23)(24)44). The CTDassociated complex contains many components, including GAL11, SUG1, and SRB proteins. The association of SRB proteins with the CTD is consistent with genetic evidence regarding function: mutations in the SRB genes were isolated as suppressors of cold sensitivity caused by truncation of the CTD (31, 44). Evidence indicates that this complex is required to mediate transcriptional activation, and it has been designated the mediator (15,22,23).Our studies of genes that contribute to glucose repression have unexpectedly led us to two proteins in the mediator. We recently identified a new cyclin-dependent kinase-cyclin pair encoded by SSN3 and SSN8 (25). Sequence comparison indicates that SSN3 and SSN8 are identical to SRB10 (also known as UME5 and ARE1 [43,52]) and SRB11, respectively, which are new members of the SRB suppressor family encoding proteins in the mediator complex (27). Mutations in SSN3 and SSN8 are members of a set of suppressors of snf1, designated ssn1 through ssn8 (5). These ssn mutations suppress growth defects of a mutant lacking the SNF1 protein kinase, which is required to relieve glucose repression of gene expression (6). The identities between these two pairs of SSN and SRB genes link two genetically defined sets of suppressors and provide an unanticipated functional connection between the SSN family and RNA polymerase II.The functions of two genes in the SSN family have been extensively characterized. SSN1, which is the same as MIG1 (51), and SSN6 have been directly implicated in transcriptional repression. The SSN6 protein, together with TUP1, forms a complex (54) that represses transcription of many genes (38, 47). The SSN6-TUP1 complex is tethered to differently regulat...

The Srb10-Srb11 protein kinase of Saccharomyces cerevisiae is a cyclin-dependent kinase (cdk)-cyclin pair which has been found associated with the carboxy-terminal domain (CTD) of RNA polymerase II holoenzyme forms. Previous genetic findings implicated the Srb10-Srb11 kinase in transcriptional repression. Here we use synthetic promoters and LexA fusion proteins to test the requirement for Srb10-Srb11 in repression by Ssn6-Tup1, a global corepressor. We show that srb10⌬ and srb11⌬ mutations reduce repression by DNA-bound LexA-Ssn6 and LexA-Tup1. A point mutation in a conserved subdomain of the kinase similarly reduced repression, indicating that the catalytic activity is required. These findings establish a functional link between Ssn6-Tup1 and the Srb10-Srb11 kinase in vivo. We also explored the relationship between Srb10-Srb11 and CTD kinase I (CTDK-I), another member of the cdk-cyclin family that has been implicated in CTD phosphorylation. We show that mutation of CTK1, encoding the cdk subunit, causes defects in transcriptional repression by LexA-Tup1 and in transcriptional activation. Analysis of the mutant phenotypes and the genetic interactions of srb10⌬ and ctk1⌬ suggests that the two kinases have related but distinct roles in transcriptional control. These genetic findings, together with previous biochemical evidence, suggest that one mechanism of repression by Ssn6-Tup1 involves functional interaction with RNA polymerase II holoenzyme.RNA polymerase II holoenzyme forms purified from the yeast Saccharomyces cerevisiae contain a mediator complex, which functions in transcriptional activation (3,12,18,22,23,53). Components of mediator/holoenzyme forms include Srb proteins, Gal11, Sin4, Rgr1, Rox3, and general transcription factors (16,18,22,23,29,30). Genetic evidence suggests that the mediator/holoenzyme plays a role not only in transcriptional activation but also in repression (for a review, see reference 4). Mutations in the genes encoding Srb8 to Srb11, Gal11, Sin4, Rgr1, and Rox3 appear to relieve negative regulation of diversely regulated genes (6,9,13,20,25,39,44,49,52,60,61).Two of these proteins, Srb10 and Srb11, constitute a cyclindependent kinase (cdk)-cyclin pair (25,30). The connection to RNA polymerase II was first established by the isolation of srb10 and srb11 alleles as suppressors of truncations in the carboxy-terminal repeat domain (CTD) of the largest subunit of polymerase (30). The Srb10-Srb11 kinase was found associated with an RNA polymerase II holoenzyme form and was shown to affect phosphorylation of the CTD in vitro (18,30). Mutations in SRB10 and SRB11 also reduced the activation of GAL promoters (25, 30). Mutations in both genes had previously been isolated in genetic selections for specific effects on gene regulation. Alleles called ssn3 and ssn8 were identified as suppressors of a defect in the Snf1 protein kinase and were shown to affect glucose repression of the SUC2 gene (5, 25, 60). A related selection for suppressors affecting the Snf1-dependent expression of gluconeogeni...

Transmembrane proteins known as G protein-coupled receptors (GPCRs) have been shown to form functional homo- or hetero-oligomeric complexes, although agreement has been slow to emerge on whether homo-oligomerization plays functional roles. Here we introduce a platform to determine the identity and abundance of differing quaternary structures formed by GPCRs in living cells following changes in environmental conditions, such as changes in concentrations. The method capitalizes on the intrinsic capability of FRET spectrometry to extract oligomer geometrical information from distributions of FRET efficiencies (or FRET spectrograms) determined from pixel-level imaging of cells, combined with the ability of the statistical ensemble approaches to FRET to probe the proportion of different quaternary structures (such as dimers, rhombus or parallelogram shaped tetramers, etc.) from averages over entire cells. Our approach revealed that the yeast pheromone receptor Ste2 forms predominantly tetramers at average expression levels of 2 to 25 molecules per pixel (2.8·10 to 3.5·10molecules/nm), and a mixture of tetramers and octamers at expression levels of 25-100 molecules per pixel (3.5·10 to 1.4·10molecules/nm). Ste2 is a class D GPCR found in the yeast Saccharomyces cerevisiae of the mating type a, and binds the pheromone α-factor secreted by cells of the mating type α. Such investigations may inform development of antifungal therapies targeting oligomers of pheromone receptors. The proposed FRET imaging platform may be used to determine the quaternary structure sub-states and stoichiometry of any GPCR and, indeed, any membrane protein in living cells. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.