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Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, Mississippi 39406-5018
SUMMARY INTRODUCTION NATURE OF THE SIGNAL Signaling without Extracellular Sensing, Transport, or Phosphorylation of Glucose Redundant but Unified Glucose Signaling Pathways Early Steps in Signal Transduction The Ras/cAMP/PKA pathway. The Gpr1/Gpa2 pathway. Activation of Ras and Gpa2 by glucose. (i) Extracellular sensing. (ii) Intracellular sensing. Extracellular sensing by Snf3/Rgt2. SIGNAL RECEPTION: GLUCOSE REPRESSION Involvement of the Hexokinase Hxk2 in the Glucose Response Snf1-Mediated Signal Transduction in Glucose Repression and Derepression Dueling Snf1 kinase and Glc7 phosphatase mediate glucose regulation. Transmission of the Ras/cAMP-dependent glucose signal to Reg1/Glc7 and Snf1/Snf4. DNA-bound repressors controlled by Snf1. DNA-bound activators controlled by Snf1. SIGNAL RECEPTION: GLUCOSE INDUCTION Snf3/Rgt2 Signaling of the Rgt1 Repressor Complex The Cell Cycle Connection Transcriptional Induction of Glycolytic and Ribosomal Protein Genes The Repressor/Activator Theme in Glucose Regulation Reverse Recruitment: Rap1/Gcr1 Activation at the Nuclear Periphery GENE REGULATION VIA REVERSE RECRUITMENT A New Model for Eukaryotic Transcriptional Activation and Repression Glucose May Signal a Highly Networked and Structurally Stable Perinuclear Machine ACKNOWLEDGMENTS REFERENCES
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This review will focus on the cytoplasmic regulators that transmit the glucose signal from the plasma membrane into the yeast nucleus, as well as the nuclear apparatus that mediates the global transcriptomic response to the sugar (both glucose repression and glucose induction). The response to glucose also involves direct modification of metabolic pathways (termed glucose or catabolite inactivation) and other processes such as mRNA turnover (7, 76, 103, 121, 153, 154, 156, 161, 166, 171-173, 232, 294, 307, 329, 334, 335, 337, 391). Although the latter phenomena are outside the scope of this review, they appear to be mediated by the same two central signal transduction pathways, Ras/cyclic AMP (cAMP)/protein kinase A (PKA) and Snf3/Rgt2/Yck, which are described below in detail.
The recent application of yeast genomics and proteomics is a first step towards comprehensive characterization of the glucose response. For example, we now know that within 20 min of glucose addition, 
20% of the roughly 6,200 genes in S. cerevisiae exhibit a greater-than-threefold change in expression (either induction or repression), and 40% show at least a twofold change (408). Glucose repression largely affects genes encoding enzymes involved in respiration or alternative carbon source metabolism, and glucose induction stimulates transcription of glycolytic genes and ribosomal protein (RP) genes (see below). Genomic and proteomic analyses are particularly useful when supplemented with high-throughput genetic techniques such as synthetic genetic array analysis, which allows the identification of synthetic phenotypes by simultaneous screening of the entire collection of mutants in which each of the 4,700 nonessential yeast genes has been deleted (373).
The continued pursuit of such large data sets, in combination with the now-facile elimination, overexpression, or point mutation of relevant yeast regulators, should eventually permit the delineation of a global wiring diagram that mirrors its biological template when virtually challenged with a wide range of concentrations of the sugar. For such a computational model to be valid, it must at a minimum be informed by (i) an accurate molecular map of cytoplasmic and nuclear substructure, (ii) a detailed diagram of the physical and functional connections that link the relevant signal transduction components to each other and to adjacent networks, and (iii) the temporal progression of the response following the appearance of glucose. Here I review our progress toward that ambitious goal and attempt to identify the most important issues that require further clarification.
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The Snf1 serine-threonine kinase represents another example of downstream regulatory overlap; Snf1 activates genes that are repressed in glucose-containing media but are derepressed and needed for growth in the presence of nonfermentable carbon sources (46). Recent transcriptomic and proteomic analyses of snf1 cells revealed a previously unnoticed upshift in expression of genes (e.g., ADH1) involved in glucose catabolism (hereafter the term "glycolytic genes" will be used for this group) (432). Thus, in addition to its well-established role in derepressing glucose-repressed genes, Snf1 also somehow represses transcription of glucose-induced genes (including HXT1) (372). Snf1 repression of glycolytic gene expression has been further substantiated by proteomic analysis of cells lacking Snf4, the 
subunit of the Snf1 kinase complex (140).
A final example of downstream regulatory overlap is the phosphoprotein Gcr1, which was first identified as an activator of glycolytic genes (13, 63, 64, 152). Transcriptomics and other analyses have confirmed that expression of numerous glucose-repressed genes is derepressed in the absence of Gcr1 at high glucose concentrations, suggesting a new role for this regulator in repression of transcription (327, 383; K. Barbara and G. M. Santangelo, unpublished data).
The roles of Rgt1, Hxk2, Snf1/Snf4, and Gcr1, as well as numerous other repressor/activator polypeptides that participate in the glucose response, are presented in greater detail in later sections of this review. In addition to these specific regulators, at least eight components of the general transcription machinery that interact with the carboxy-terminal domain of RNA polymerase II (Gal11, Sin4, Rgr1, Rox3, Srb8, Ssn2, Ssn3, and Ssn8) have been found to play both positive and negative regulatory roles; the function of many of these factors is known to be influenced by glucose signaling (43, 195). Thus, the overlapping positive and negative circuitry in the glucose response may extend from the plasma membrane through to the ultimate choice of induction or shutoff of transcription initiation of each glucose-regulated gene by RNA polymerase II.
The induction/repression duality of signal recipients such as Rgt1, Hxk2, Snf1/Snf4, Gcr1, and other transcription factors clearly requires further characterization of the precise mechanisms involved. Nonetheless, together with the transcriptomic analysis of activated Ras, it suggests the existence of a more unified and interdigitated response to glucose than had previously been appreciated. Although it now seems certain that transmission of the glucose signal is redundant (408), those signaling pathways may converge on only a relatively small group of transcription factors in the yeast nucleus (see below). Coordinated remodeling of the nuclear regulatory apparatus in response to glucose might therefore involve a parsimonious series of alterations in key regulators. Although for the sake of clarity and historical perspective the discussion of nuclear regulators is divided into separate sections of this review, in reality the mechanisms of "glucose repression" and "glucose induction" may be no more segregated than the two faces of a coin.
The Ras/cAMP/PKA pathway. Ras proteins are monomeric GTPases that function as switches; these so-called G proteins are inactive in the GDP-bound state and active when GTP is bound. The switch from the active to the inactive form involves hydrolysis of bound GTP by the intrinsic GTPase and is stimulated by GTPase-activating proteins (GAPs). The reverse (inactive-to-active) switch requires replacement of bound GDP with GTP, which is normally accomplished with the aid of guanine nucleotide exchange factors (GEFs). Activating mutations in Ras polypeptides (e.g., human rasVal12 or yeast Ras2Val19) increase GTP association in a GEF-independent fashion and are responsible for many human cancers (78, 419). Ras mutations are found in approximately 30% of human tumors; the frequency of oncogenic forms of Ras is as high as 50% or 90% in colorectal or pancreatic tumors, respectively (345). Although it remains dimly understood, it seems likely that there is an important connection between these recent data and a very old observation that altered glucose metabolism is a hallmark of cancerous cells in solid tumors (71, 179).
The polypeptides encoded by the two RAS genes in S. cerevisiae, Ras1 (309 residues) and Ras2 (322 residues), are >70% identical overall and approximately 90% identical over the N-terminal 180 residues (291). Growth on glucose is unaffected by the absence of either Ras1 or Ras2, whereas loss of both causes arrest in the G1 phase of the cell cycle (362, 367). These early findings were suggestive of a connection to nutrient sensing, since nutrient-deprived cells also arrest in G1 phase. Overexpression of RAS1 was also found to suppress the failure of ras2
cells to grow on nonfermentable carbon sources (363), suggesting that the latter defect is due to the fact that Ras1 is more weakly expressed than Ras2 (249, 361), rather than a specific function that maps to the divergent and slightly extended C terminus of Ras2.
Important effector molecules modulate Ras activity during the glucose response; posttranslational modification and membrane localization of Ras are two important features of these regulatory inputs. For example, Ras farnesylation contributes to nucleotide exchange by the GEF Cdc25 (34, 73, 134); CDC25 is an essential gene and was originally identified by a screen for mutations that cause G1 arrest (139). A second yeast RasGEF that can substitute for Cdc25 when overexpressed is encoded by the dispensable SDC25 gene (31) (note that this gene was originally named SCD25 [79]).
Ras stimulation by the RasGEFs in turn stimulates production of cAMP by the essential product of the adenylate cyclase gene (CYR1) (229). Inactivation of Ras, resulting in lower cAMP levels, is facilitated by the GAPs Ira1 and Ira2 (358, 360) (Fig. 2). Presumably due to their competing effects on Ras activation, IRA1 deletion rescues the lethality caused by deletion of the RasGEF CDC25 (359). Low-affinity and high-affinity cAMP phosphodiesterases in S. cerevisiae (Pde1 and Pde2, respectively) also antagonize the Ras signal through enzymatic removal of cAMP; in the presence of exogenous cAMP, deletion of PDE2 restores growth to ras1 ras2 strains (266, 420).
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The palmitoyl moiety anchors Ras to the cytoplasmic face of the yeast plasma membrane (199); blocking addition of palmitate via the single point mutation C318S causes mislocalization of the mutated Ras2 protein to the cytoplasm (176). Ras palmitoylation is reversible and therefore may be a regulatory step. This is of particular interest because mutation of palmitoylation sites abrogates both the glucose response in yeast cells (see below) and transformation of mammalian cells by oncogenic forms of Ras (418).
The appearance of glucose thus generates one or more signals that converge on membrane-bound Ras and its modifiers (in particular the RasGEFs and RasGAPs). This glucose signaling results in a rapid but transient spike in the level of intracellular cAMP, which increases 5- to 50-fold within 1 to 2 min of glucose addition and returns to near-basal levels within 20 min. When cells express the nonpalmitoylated cytoplasmic Ras2C318S form as their only Ras protein, they fail to exhibit the glucose-induced cAMP spike and are defective in transitioning to rapid growth upon addition of glucose (160). Thus, glucose-regulated cAMP production appears to require attachment of Ras to the yeast plasma membrane. Interestingly, membrane localization of yeast Ras2 was recently shown to occur despite disruption of the classical secretory pathway that normally mediates translocation from the endoplasmic reticulum to the plasma membrane (92).
Additional signal transduction components appear to contribute to glucose signaling by contacting adenylate cyclase (Cyr1); such Cyr1 protein-protein interactions identified thus far include those with (i) an N-terminal SH3 domain in the RasGEF Cdc25 (237), (ii) the farnesylated Ras2 C terminus (73, 74, 184, 199, 338, 355), (iii) the C terminus of the putative cochaperonin Sgt1 (95), (iv) the N terminus of the ancillary factor Srv2 (CAP) via coiled-coil formation (267), and (v) the RasGAP Ira1, which mediates peripheral association of adenylate cyclase with the yeast plasma membrane (241).
It seems likely that the sole purpose of the glucose-induced spike in cAMP levels is to block the inhibitory effect of the BCY1-encoded regulatory subunit on the catalytic subunits of PKA, encoded redundantly by TPK1, TPK2, and TPK3; overexpression of any of these three genes, or deletion of BCY1, restores growth to ras1 ras2 strains (369). PKA is a heterotetramer consisting of two catalytic (Tpk) subunits and two regulatory (Bcy1) subunits (Fig. 2); addition of regulatory subunits to catalytic subunits in vitro converts protein kinase activity into a completely cAMP-dependent form (151, 177, 370).
Interestingly, the subcellular localization of PKA catalytic and regulatory subunits appears to respond to glucose signaling; Bcy1 is present in both the cytoplasm and nucleus in the absence of glucose but is exclusively nuclear in its presence (129, 130). Cross talk from the TOR (target of rapamycin) pathway, another major set of nutrient-responsive signaling components, may help to maintain PKA activity in part by regulating nuclear translocation of Tpk1 (330). Although it has yet to be demonstrated that the observed translocation of PKA subunits between compartments plays an important role in glucose signaling in yeast cells, targeting of PKA regulatory substrates would appear to undergo a qualitative and/or quantitative shift in response to the appearance or disappearance of the sugar. Further progress in this area is complicated by the fact that signaling via each of the three different catalytic subunits of PKA (Tpk1, Tpk2, and Tpk3) results in a distinct pattern of downstream gene expression readouts (309).
Although active PKA is thought to phosphorylate proteins involved in transcription, energy metabolism, and cell cycle progression (129), further study is required to identify the exact targets and sequence of protein translocation and phosphorylation events that transmit the glucose regulatory signal to the yeast nucleus. Significantly, mutations impairing specific components of the RNA polymerase II transcriptional machinery are synthetically lethal with the hyperactive Ras2Val19 allele (157), and PKA can phosphorylate a component of the Srb complex (Ssn2) both in vitro and in vivo (55). The latter may be one of but a few Ras/cAMP/PKA-mediated modifications in nuclear factors needed to accomplish the broad sweep of alterations to the yeast transcriptome upon glucose addition or depletion.
The Ras/cAMP/PKA pathway has also been implicated in aging (212, 216), thermotolerance (439), bud site selection, actin repolarization (333), glycogen accumulation (342), stress resistance (39, 407), and sporulation (39, 41); it may also regulate pseudohyphal differentiation (growth as chain-like multicellular filaments) in response to nutrient limitation. Each of these interesting topics (see reference 411 for a recent review), as well as the involvement in these processes of other nutrient-stimulated kinases (e.g., Snf1 and TOR [45, 75, 196, 197, 401, 441]), GTPases (e.g., Gpa2 [277]), and transcription factors (e.g., Msn2/Msn4 [30, 96, 112]), is outside the scope of this review.
The Gpr1/Gpa2 pathway.
Heterotrimeric G proteins are signaling molecules composed of 
, ß, and subunits. They represent a second class of factors that, like monomeric Ras polypeptides, are capable of binding guanine nucleotides (Fig. 2). The G subunit Gpa2 and its negatively acting regulator (GAP) Rgs2 have been shown to participate in glucose signaling in S. cerevisiae (190, 394). Gpa2 was originally identified by screening a yeast genomic library with cDNA probes encoding mammalian G subunits (252), and the putative ß subunits Gpb1 and Gpb2 were identified as interaction partners of Gpa2 (135); no corresponding G subunits involved in glucose signaling have yet been identified (but see reference 135). Whereas deletion of either GPA2 or RAS2 causes little or no impairment of growth on glucose media, deletion of both causes a severe synthetic growth defect (428). Removal of the major cAMP phosphodiesterase in the cell (Pde2) restores normal growth to gpa2 ras2 strains, suggesting that an increase in cAMP levels bypasses the loss of both Gpa2 and Ras2 (428). Most importantly, as mentioned above, the transcriptomic response to glucose is successfully mimicked in the absence of the sugar by turning on expression of either activated Gpa2 or activated Ras2; the fact that this signaling is PKA dependent confirms their redundant participation in the cAMP-dependent branch of glucose signaling (408).
A two-hybrid protein interaction screen with GPA2 as the bait led to the identification of the GPR1 gene, which encodes a member of the G protein-coupled seven-transmembrane receptor superfamily (Fig. 2). Gpr1 is located on the yeast cell surface (428). Genetic analysis strongly suggests that Gpa2 acts downstream from Gpr1 in the same signaling pathway: the low growth rates of gpr1 ras2, gpa2 ras2, and gpa2 gpr1 ras2 strains are essentially identical, and introduction of either multicopy or activated GPA2 suppresses the defect of the gpr1 ras2 strain (428). Deletion of GPR1 diminishes (although does not eliminate) genome-wide transcriptional induction and repression in response to glucose (408). The simplest explanation for these data is that Gpr1 is the only receptor coupled to Gpa2 and that it too contributes to activation of the cAMP pathway in response to glucose (190).
Although Gpa2 was originally thought to function upstream of the Sch9 kinase (368, 428), it was subsequently found that SCH9 deletion is synthetically lethal with gpr1, gpa2, or ras2, suggesting that Sch9 acts instead in the pseudohyphal differentiation pathway that is parallel to both Gpr1/Gpa2 and Ras (217). Sch9 may nevertheless participate somehow in glucose signaling, since (i) it is the yeast ortholog of a component of the mammalian insulin response pathway (Aft/protein kinase B), (ii) it shares similarity with PKA catalytic subunits, (iii) its overexpression compensates for the loss of Ras function, and (iv) PKA activity is elevated in a sch9 background (72, 109).
Activation of Ras and Gpa2 by glucose. Since artificial activation of Ras and Gpa2 is sufficient to generate the glucose response, and it seems unlikely that glucose interacts directly with the G proteins themselves, how do Ras and Gpa2 become activated in wild-type yeast cells exposed to the sugar? It is convenient to separate potential answers to this question into two categories: recognition of glucose by receptors on the outside surface of the cell versus direct signaling of Gpa2 and Ras (or their regulators) by intracellular glucose or its metabolites.
(i) Extracellular sensing. There are 18 known hexose transporter genes in S. cerevisiae (HXT1 to HXT17 and GAL2). Deletion of seven of these genes results in the failure to transport or grow on glucose (308, 413). In this hxt1 to hxt7 null background, constitutive expression of the GAL2-encoded galactose permease restores glucose-induced cAMP synthesis (313). This suggests that the yeast hexose transporters do not participate in glucose signaling but are required only for transport.
The seven-transmembrane receptor Gpr1 may detect extracellular glucose and thereby activate Gpa2, but it is not required to signal Ras; Gpr1 or Gpa2 deletion does not prevent the glucose-induced increase in Ras2-GTP levels (66). In contrast, the Ras2-GTP increase is absent in a glucose phosphorylation-deficient (hxk1 hxk2 glk1) strain (66). Thus, Ras appears to be activated by intracellular phosphorylated glucose but not by extracellular glucose. A clear demonstration of extracellular sensing by Gpr1 will require direct evidence, for example, as suggested by recent mutagenic analysis (205), that it interacts with glucose.
(ii) Intracellular sensing. If extracellular signaling does not participate in Ras activation (66, 313), it will be particularly important to understand how intracellular glucose triggers the glucose response. The longstanding idea that glucose phosphorylation (in particular by the glucose-induced hexokinase Hxk2; see below) and/or subsequent glycolytic reactions are required to alter yeast gene expression in response to glucose has been controversial (see references 20, 121, 244, and 314 for reviews of this topic). Since repression occurs even when the metabolic steps immediately following glucose phosphorylation are reduced to less than 2% of wild-type levels, it seems likely that nothing beyond the production of glucose-6-phosphate is required (317). Further, since cells can be tricked into the full-blown glucose response by expressing activated Ras or Gpa2, glucose phosphorylation must generate the signal through the G proteins and/or through a redundant and as-yet-obscure alternative pathway. A recent interesting suggestion is that glucose phosphorylation increases Ras2-GTP levels by inhibiting the Ira (RasGAP) proteins (66).
Although progress has been made, much more work is needed to establish the precise mechanism(s) through which Ras, Gpa2, and/or their regulatory GEFs and GAPs is signaled by glucose. Intracellular acidification (316, 365) and energy charge (the AMP/ATP ratio) (134, 244) are additional potential signaling mechanisms that require further testing. The events that immediately follow activation of either G protein are much clearer (Fig. 2). Activated Ras or Gpa2 can each generate a spike in cAMP production by adenylate cyclase; cAMP in turn releases PKA catalytic (Tpk) subunits from inhibition by its regulatory (Bcy1) subunits.
Extracellular sensing by Snf3/Rgt2. The putative glucose sensors Snf3 and Rgt2 are 60% identical to each other and, like hexose transporters found in bacteria, plants, and mammals (228, 259, 332), contain 12 predicted transmembrane-spanning domains (272). Snf3 and Rgt2 do not appear to act as glucose transporters but instead signal the presence of the sugar (118, 178, 182, 247, 273, 314). Since the transcriptional response of Snf3/Rgt2 target genes (e.g., HXT genes encoding the glucose transporters) is generated in the absence of glucose by expression of hyperactive Ras or Gpa2, the Snf3 and Rgt2 sensors appear either to act upstream of Ras/cAMP/PKA or to play an ancillary or redundant role in the initial detection of glucose. This view is consistent with the possibility that glucose sensing by Snf3/Rgt2 is required for maintenance of glucose induction or repression for at least a subset of the transcriptome (182, 272). Further, it seems clear that the Snf3/Rgt2 regulatory pathway helps to ensure that changes in gene expression are commensurate with the glucose concentration in the environment, a mechanism that is likely to be particularly important in nature.
As mentioned above for Gpr1, a clear demonstration of extracellular sensing by Snf3 and Rgt2 will require direct evidence that they interact with glucose. Intriguing hints along these lines have already been uncovered. The V404I mutation in Rgt2 appears to abolish glucose signaling; this residue is orthologous to one in Hxt1 (F371) that is necessary for glucose transport (247). V404 in Rgt2 may therefore be required for glucose sensing because it helps to form the glucose binding domain in the receptor. Similarly, dominant mutations in both Snf3 (R229K) and Rgt2 (R231K) that cause constitutive signaling, even in the absence of glucose, map to the putative glucose binding pocket (272, 273). Progress has also been made with respect to the elaboration of a mechanism for subsequent signaling by Snf3 and Rgt2 across the plasma membrane (see below).
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Most of the 1,000 genes in S. cerevisiae that are transcriptionally downshifted during growth on glucose can be sorted into one of three groups: gluconeogenic genes, Krebs cycle/respiratory genes, and genes involved in alternate carbon source metabolism. This section will review known mechanisms through which the glucose signal is received in the yeast nucleus and modulates expression of glucose-repressed target genes.
Interestingly, approximately 14% of Hxk2 is nuclear in glucose-grown cells (146). This discovery suggests an alternative explanation for the regulatory involvement of this hexokinase in the glucose response (244, 304). Hxk2 has been detected in the nucleus by enzymatic assay, immunoblotting, and fluorescent visualization of a green fluorescent protein (GFP)-tagged chimera, confirming controversial reports of nuclear hexokinase that first appeared in the early 1960s (234, 340, 390). The negatively acting DNA-bound glucose regulator Mig1 (see below) appears to mediate nuclear localization of Hxk2; Mig1 and Hxk2 have been shown to interact via two-hybrid, immunoprecipitation, and glutathione S-transferase pull-down assays (2, 245). An interaction between Hxk2 and the Mediator component Med8 has also been described, and the latter appears to bind directly to both positively and negatively acting glucose regulatory elements in DNA (57, 80, 246). A previously reported example of a metabolic enzyme that doubles as a transcriptional regulator is the galactokinase Gal1, which both phosphorylates galactose and appears to activate the transcription factor Gal4 by binding to the Gal4 inhibitor Gal80 (436).
Despite these intriguing hints, contradictory results prevent an unambiguous assignment of the Hxk2 function in glucose regulation to the nuclear form of the protein. For example, removal of a small N-terminal segment of Hxk2 (residues 7 through 16) in a putative nuclear localization sequence (Lys8-Pro-Gln-Ala-Arg12) has been reported to abolish both Hxk2 nuclear localization and glucose repression of SUC2, HXK1, and GLK1 without impairing hexokinase catalytic activity (146, 311). The exact opposite result (intact glucose signaling and low catalytic activity), however, was obtained by others upon deletion of an overlapping and slightly larger Hxk2 segment (residues 1 through 15) (231). Likewise, the phosphorylatable Ser14 residue in Hxk2 (193) is reported either to mediate (305) or not to mediate (146, 231) glucose repression. Further analysis is needed to resolve these discrepancies. Most importantly, proof of a regulatory role for nuclear Hxk2 awaits an unambiguous demonstration that its role in the glucose response relies upon the relatively small fraction of the protein that resides in the nucleus.
Dueling Snf1 kinase and Glc7 phosphatase mediate glucose regulation. SNF1 was first identified in screens for regulatory factors in the glucose response (it was then named either CAT1 or CCR1) (62, 102, 440). SNF1 was also identified in a screen for mutants that could ferment glucose but not sucrose (46). Since inactivation of SNF1 also impairs utilization of galactose, maltose, and nonfermentable carbon sources, soon after its identification it was recognized as an important regulatory gene mediating glucose derepression. The SNF1 gene was cloned and found to encode a serine-threonine protein kinase (51); the existing evidence also suggested a physical and functional link to SNF4, another gene that had been identified in the "sucrose nonfermenting" screen (49, 50, 259, 261). The snf4 and snf1 phenotypes are similar, including defective utilization of the many carbon sources (e.g., sucrose, galactose, maltose, raffinose, and lactate) that require expression of glucose-repressed genes. Increased SNF1 dosage and SNF1 mutations (e.g., the SNF1-G53R allele, which produces a hyperactive kinase) can partially restore glucose derepression in the absence of SNF4 (53, 108, 204). Since Snf4 also coimmunoprecipitates with Snf1 and is required for maximal Snf1 protein kinase activity (in cells grown on a nonfermentable carbon source) (52), it is a physically associated activator of the Snf1 kinase (108, 113). Like Glc7 (and perhaps Reg1; see below), Snf1 appears to have a regulatory role in glycogen metabolism that is somehow influenced by PKA signaling (see reference 119 for a review). While this apparent connection between the signal transduction pathways governing glucose catabolism and glycogen storage is intriguing, it remains poorly defined and will not be considered further here.
Identification of a role in glucose repression for Glc7, the yeast homolog of mammalian type I protein phosphatase (PP1) (111), began with a selection for mutants defective in glucose repression of a target gene (SUC2, encoding invertase) (260). All of the recovered mutations were recessive; two of the three complementation groups found in this screen proved to be allelic with HXK2 and REG1, respectively. The third group identified a new locus (designated cid1, for constitutive invertase derepression). Complementation of one of these mutations (cid1-226, now designated glc7-T152K) (378) led to the isolation of GLC7, a locus that was also identified independently in a screen for glycogen-deficient mutants. Interestingly, in addition to SNF1 (GLC2), other glc lesions were allelic with IRA1 (GLC1), IRA2 (GLC4), and RAS2 (GLC5) (42), consistent with numerous studies connecting Ras and Gpa2 signaling with accumulation of the storage carbohydrates trehalose and glycogen (40, 41, 67, 125, 190, 284, 330, 363). Further investigation is needed to pursue this implicit regulatory linkage between glucose catabolic pathways and carbohydrate storage.
Complicating matters further, in addition to glucose repression and carbohydrate accumulation, GLC7 phosphatase is needed for a variety of other cellular functions in S. cerevisiae (14, 350, 437), including normal progression through the G2/M phase of the cell cycle (23, 24, 150, 159, 223, 288), actin organization (6, 54), translation (412), and sporulation (12, 42, 302, 357, 378, 380). Recent work has further expanded this list of functions assigned to yeast PP1; it is also required for the glucose-induced vacuolar degradation of fructose-1,6-bisphosphatase (76) and for normal Mex67-mediated export of mRNA from the nucleus to the cytoplasm (127; see also references 381, 382, and 404).
Interestingly, isolated PP1 molecules exhibit little substrate specificity and instead require regulatory subunits that alter the conformation of the phosphatase and/or target it to its substrates (65, 97). This targeting hypothesis is supported by the Glc7 localization pattern, which suggests dynamic changes throughout the yeast cell cycle (24). Glc7 binding proteins specific to the following functions have been reported: glucose repression (Reg1) (380), glycogen metabolism (Gac1 and Pig1) (59, 353, 426), cell cycle progression (Reg2 and Sds22) (120, 149, 288), actin organization (Scd5) (54), and sporulation (Gip1) (357, 380).
A short motif present in most known regulatory subunits ([RK]-X0-1-[VI]-X-[FW]) plays a critical role in the interaction with a hydrophobic groove at the interface of two ß-sheets in PP1 (97, 403). Thus, multiple regulatory subunits appear to compete for Glc7 binding in vivo; for example, Gac1 overexpression affects glucose repression (426). In addition to binding regulatory subunits, the hydrophobic groove may mediate changes in the structure and/or activity of the phosphatase. It also now seems likely that many if not all targeting subunits interact with more than one site on the PP1 surface (425). In the two-hybrid assay the F468R mutation in Reg1 dramatically reduces its interaction with Glc7; the PP1 binding motif that contains this residue (RHIHF468) therefore appears to be one of the moieties required for formation of the Reg1/Glc7 complex (325). Importantly, Reg1 is the only regulatory subunit known to participate in glucose repression.
The idea that Reg1 (first identified in reference 230 and in an earlier screen as Hex2 [101]) mediates the Glc7 role in glucose repression arose because (i) reg1 and glc7 alleles were both isolated in the screen for mutants with impaired repression of SUC2 (260, 378) (see above), (b) a combination of those defective alleles exhibited no synergy in relief from glucose repression of SUC2, (iii) SNF1 deletion is epistatic to both alleles, and (iv) Reg1 overexpression suppresses the repression defect caused by glc7-T152K (379). Genetic and biochemical experiments confirmed that Reg1 and Glc7 are linked both physically and functionally (325, 379). The results of alanine-scanning mutagenesis suggested that Reg1 interacts with at least two distinct areas of the resolved PP1 structure (14). Satisfyingly, one of the Glc7 surfaces specific to its role in glucose repression is adjacent to Thr152 (14); the glc7-T152K lesion had earlier been shown to impair Reg1-Glc7 interaction (379).
A functional link between Reg1 and Snf1 was suspected long ago, primarily because REG1 deletion results in derepression of glucose-repressed genes and SNF1 deletion results in a failure to derepress many (if not all) of those same genes. The demonstration that Reg1 and Snf1 interact (218) left little doubt that the Reg1-targeted Glc7 phosphatase and Snf4-activated Snf1 kinase are indeed interconnected components of the glucose signaling pathway (106, 120, 162, 174, 378,379; see references 45, 121, and 178 for reviews) (Fig. 3). Sincethennumerous additional participants in this regulatory duel have been discovered. For example, three ancillary components of the Snf1/Snf4 kinase complex (Sip1, Sip2, and Gal83) strengthen Snf1/Snf4 association (175, 429, 430). Each of these three proteins contains an Snf1 binding internal region and an Snf4 binding C-terminal region (their N-terminal regions are divergent); they therefore appear to bridge the interaction between Snf1 and Snf4. By analogy with the heterotrimeric mammalian Snf1 homolog, the AMP-activated protein kinase, SNF1 encodes the yeast subunit, SNF4 encodes the subunit, and SIP1, SIP2, and GAL83 redundantly encode the ß subunit (136).
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sip2 gal83 triple mutant does indeed display an snf1 phenotype (331). Despite the observed redundancy, the three ß subunits play distinct roles in vivo, in particular with respect to Snf1 substrate specificity (143, 144, 175, 213, 254, 331, 396, 397, 430). Besides its role in the response to glucose limitation, Snf1 has also been implicated in several other processes, including aging, thermotolerance, peroxisome biogenesis, filamentation, invasive growth, biofilm formation, meiosis, and (like Glc7) sporulation (8, 45, 137, 196, 197, 297, 401). Clearly more work is needed before we will fully understand the respective relationships between the distinct forms of the Snf1 kinase complex and each of these regulatory mechanisms. However, Gal83 is likely to be a primary contributor to glucose regulation by the Snf1 complex, since Gal83 is the most abundant ß subunit, it is the only ß subunit in the nucleus when cells are grown on a nonfermentable carbon source, and Snf1/Gal83 is the form responsible for most Snf1 kinase activity (144, 397).
Another trio of factors that mediate Snf1 function are the redundantly acting Snf1 kinase kinases Sak1 (YER129W, formerly known as Pak1; the new name was chosen to avoid confusion with Prk1 or p21-activated kinases), Tos1, and Elm1, each of which shares sequence similarity with the human tumor suppressor and AMP-activated protein kinase LKB1 (155, 255, 354, 422). Removal of all three yeast activities confers an snf1 phenotype. A one-to-one correspondence between these three upstream kinases and the distinct forms that contain each of the three ß subunits has been ruled out (143). Since Sak1 is the most important of the trio of Snf1 kinase kinases in activating the Snf1/Gal83 form, it is likely also to be the most important regulator of Snf1-dependent glucose regulation (143). This conclusion is further supported by the observation that deletion of SAK1 suppresses many of the Snf1-dependent phenotypes observed in reg1 cells (255).
Like Glc7 and its regulatory subunits, the Snf1 kinase appears to be localized by its ß subunits to distinct cellular subcompartments, thereby targeting it to different substrates (331, 397). In glucose-grown cells Snf1 is found exclusively in the cytoplasm; following a shift to a nonfermentable carbon source, Snf1 is targeted by Gal83 to the nucleus. The N terminus of Gal83, which is not shared by the other two ß subunits Sip1 and Sip2, is required for this function; the upstream kinase Sak1 also controls Snf1 localization to the nucleus in the absence of glucose (144, 397). Upstream events in glucose signaling may also be involved, since it has been reported that PKA regulates nucleocytoplasmic shuttling of Snf1/Sip1 (144). If PKA also regulates shuttling of Snf1/Gal83, it appears to do so redundantly with another signaling pathway.
With respect to subnuclear localization, three as-yet-unconnected observations suggest the possibility that the Snf1 kinase complex operates at least in part at the nuclear periphery: Snf1 is concentrated in nuclear envelope fractions prepared from pyruvate-grown (but not glucose-grown) cells (B. Menon and G. M. Santangelo, unpublished data), Glc7 has been implicated in mRNA processing as a component of the cleavage/polyadenylation complex (127, 256, 404), and Reg1 deletion causes an RNA processing defect (381, 382). Further elaboration of this potential connection with perinuclear phenomena is needed and may provide critical information about the function and organization of the regulatory apparatus in the yeast nucleus that responds to glucose (see below).
An overview of the regulatory duel between the Reg1/Glc7 phosphatase and the Snf1 kinase complex is presented in Fig. 3. Snf1 has two domains, an N-terminal kinase domain and a C-terminal regulatory domain. In high concentrations of glucose the regulatory domain remains bound to the catalytic domain, maintaining Snf1 in an autoinhibited conformation (labeled "inactive" in Fig. 3) (325). Upon depletion of glucose, Snf4 counteracts autoinhibition of Snf1 by interacting with its regulatory domain; deletion of the latter bypasses the requirement for SNF4 in derepression of glucose-repressed genes (52, 174, 204). Snf4 binding to Snf1 therefore produces an active, open conformation of the complex (labeled "active" in Fig. 3) (174). Phosphorylation of a conserved threonine (T210) in the activation loop of Snf1 is essential for activation of the kinase; the upstream kinase Sak1 has been shown to phosphorylate this residue and is required for full Snf1 kinase activity (255). Blocking phosphorylation at this position via a T210A mutation impairs Snf4 binding to the regulatory domain and prevents the release of Snf1 autoinhibition (174, 198, 218). The T210A lesion also interferes with normal nuclear enrichment of Snf1 after a shift from high to low glucose (144). Since Snf4 localization to the nucleus is essentially carbon source independent, in glucose-grown cells nuclear Snf4 is apparently not associated with other subunits of the Snf1 kinase complex (397). Thus, Snf4 apparently does not contribute to the cytoplasmic roles of Snf1 that are distinct from its mediation of glucose derepression.
Glucose repression involves Reg1/Glc7-facilitated conversion of the kinase complex from the active to the autoinhibited state, presumably via dephosphorylation of Snf1 (325). Reg1/Glc7 appears to associate only with active kinase complexes, since the catalytic domain of Snf1 (phosphorylated at T210) is required for the interaction with Reg1 (218). Upon activation in a reg1 or glc7-T152K background, the Snf1 complex becomes trapped in the active conformation. Overexpression of Reg1, but not overexpression of the mutated form that fails to bind Glc7 (ReglF468R; see above), interferes with the Snf1/Snf4 interaction in low glucose (325). Sip5, the first protein shown to bind to both Reg1/Glc7 and the Snf1 kinase complex, may stabilize the interaction between them and thereby facilitate glucose repression, since the two-hybrid interaction between Reg1 and Snf1 is reduced threefold in a sip5 mutant (326).
The critical regulatory interaction between Reg1 and the Snf1 complex was presumed to occur in the nucleus, especially since that is where active Snf1 is predominantly located prior to being switched off and exported following the appearance of glucose, and Glc7 is known to be largely nuclear in growing cells (24, 437). However, in contrast with a previous report that found Reg1 in the nucleus (265), fluorescently tagged Reg1 appears to be exclusively cytoplasmic (90, 91). To resolve this discrepancy it was proposed that the activated form of Snf1 cycles rapidly between the nuclear and cytoplasmic compartments, allowing inactivation by Reg1/Glc7 to occur in the cytoplasm following the appearance of glucose (91). This hypothesis has not yet been tested, but it may explain the apparent role of Bmh1 and Bmh2 in glucose-regulated gene expression (168). Bmh1 and Bmh2 are the yeast homologs of the somewhat mysterious 14-3-3 proteins (33, 388), which may serve as mediators of Reg1 function (90) and are known to respond to Ras/PKA signaling (124, 232).
Additional information concerning the connection between upstream glucose signaling events and the Glc7/Snf1 regulatory duel will be the next topic of discussion, followed by a review of the downstream DNA-bound phosphoproteins that are known Snf1 regulatory targets and that account for much of the transcriptomic response to glucose in yeast cells.
Transmission of the Ras/cAMP-dependent glucose signal to Reg1/Glc7 and Snf1/Snf4. Although the regulatory duel between Snf1 kinase and Glc7 phosphatase has now largely been explained at a molecular level, the input by glucose signaling that shifts the balance in favor of the closed, inactive form of the kinase remains poorly characterized. A proposal that Snf1 responds to the AMP/ATP ratio (421), which drops dramatically upon removal of glucose (7, 421), has been difficult to substantiate, since Snf1 kinase activity does not respond to these nucleotides in vitro (136). In contrast, the ample evidence that PKA and Snf1 act antagonistically in affecting a similar set of cellular functions (7, 164, 366) is reinforced by the similarity of the transcriptome-wide response of Snf1 target genes following either the addition of glucose or the expression of activated G proteins in the absence of glucose (408).
Sak1, the recently identified upstream activator of Snf1 that regulates both Snf1 kinase activity and its nuclear localization (144, 255), is an obvious potential intermediary between PKA and Snf1 (question mark in Fig. 3). If the gap between the earliest events in glucose signaling and Sak1 could be bridged, it would represent a complete chain of causality between glucose sensing at the cellular periphery and the response of DNA-bound regulators in the nucleus. Not surprisingly, however, given that its role as an Snf1 activator was only recently uncovered, little is yet known about regulation of Sak1 and whether or not the PKA pathway might be involved.
Equally mysterious is the apparent capacity of the glucose signal to change the target of Glc7 phosphatase from Reg1 to Snf1 (no. 7 and 1 in Fig. 3, respectively), which in the absence of Snf1 phosphorylation (no. 3) traps Snf1 in the autoinhibited inactive state. Interestingly, PKA signal transduction might alter Glc7 targeting through the negatively acting regulator Hxk2. As mentioned above, PKA signaling appears to cause reduced phosphorylation of Hxk2, which favors its dimeric form; the phosphatase that dephosphorylates Hxk2 in response to glucose is known to be Reg1/Glc7 (4, 305, 398). Hxk2 (presumably its underphosphorylated dimeric form) in turn interferes with dephosphorylation of Reg1 by Glc7 (325). Since only the phosphorylated form of Reg1 promotes dissociation of Snf4 from Snf1 and inactivation of the kinase, the PKA-mediated transition from monomeric to dimeric Hxk2 is potentially a direct glucose trigger that stabilizes the repressing form of Reg1/Glc7 and thereby inactivates Snf1. Reg1 and Hxk2 were reported to interact as predicted by this model (325). This is further supported by a wealth of additional data: Reg1 overexpression suppresses the glucose repression defect of an hxk2 mutant (325), the association between Reg1 and Glc7 is unaffected by removal of Hxk2, and the Snf1-Snf4 interaction is glucose insensitive in the absence of Hxk2 (174).
A near-complete chain of causality describing the glucose response can thus be stated as follows (Fig. 3): early events in glucose signaling stimulate PKA to inhibit phosphorylation of Hxk2, thereby targeting it to Reg1/Glc7, where Hxk2 is dephosphorylated, dimerizes, and blocks dephosphorylation of Reg1 by Glc7; Glc7 then switches targets and inactivates the Snf1 kinase, which can no longer phosphorylate its DNA-bound substrates, resulting in altered expression of the relevant target genes. Ambiguity unfortunately accompanies even this parsimonious view of the glucose regulatory cascade, since the relationship between subcellular distribution and function awaits clarification for several of the factors involved (most notably Hxk2 and Reg1).
DNA-bound repressors controlled by Snf1. Several downstream repressors are known to be phosphorylated and inactivated by the Snf1 kinase in the absence of glucose. The first of these to be discovered was Mig1, originally identified as a multicopy inhibitor of GAL gene expression (257). Deletion of the MIG1 gene relieves glucose repression of numerous target genes (115, 131, 180, 189, 257, 258, 335, 336, 406), and its product is thought to interact with the intergenic regions of at least 90 different genes in vivo (221, 251). Mig1 is presumed to function downstream of Snf1, because deletion of MIG1 suppresses snf1 defects in glucose derepression (180, 386).
Mig1 is a Cys2His2 zinc finger protein that binds to a GC-rich core sequence (219). The consensus Mig1 binding site determined by in vitro selection is 5'-ATAAAATGCGGGGAA-3' (221). At the time of its discovery, other zinc finger proteins were known to be capable of functioning as either activators or repressors, depending upon the chromosomal and cellular context (207). This was therefore also considered a possibility for Mig1 (257), a suggestion that later proved to be correct: both Mig1 and the related Cys2His2 zinc finger-containing repressor Mig3 (Yer028) can also function as activators (220, 221, 303, 376, 423). Although this has complicated the interpretation of Mig1 function (37, 160, 189, 376, 384, 386, 423), clearly the primary physiological role of Mig1 seems to be that of a negative regulator of glucose-repressed genes (e.g., SUC2 and GAL1).
Interestingly, the Mig1 N-terminal zinc fingers are very similar to those in the C terminus of WT1 (257), a human tumor suppressor that collaborates with p53 to repress transcription and (like p53) also activates transcription (133, 224, 235, 248, 264, 268, 306, 348). Mig1 and WT1 are both phosphoproteins, and PKA phosphorylation of WT1 appears to make an important contribution to its repressor function (323); the significance of a potential PKA target site found in the internal regulatory region of Mig1 is not yet known (257, 270).
Glucose-dependent repression by a LexA-Mig1 fusion is readily detected by a heterologous reporter gene with upstream LexA operator sites, suggesting that Mig1 can repress transcription without the assistance of other promoter-bound factors (376, 377). Analysis of LexA-Mig1 repression also led to the discovery that the Mig1 polypeptide, which has a predicted molecular mass of 55 kDa (504 residues), is phosphorylated to different extents in glucose-repressed and derepressed cells and might be a downstream target of Snf1 kinase (87, 376). Indeed, three consensus Snf1 kinase sites (Ser278, Ser311, and Ser381) map within a region of Mig1 required for glucose regulation (77, 87, 269). All three of these sites, and a fourth one that matches the Snf1 consensus imperfectly (Ser222), are phosphorylated by Snf1 in vitro (343). Mutation of these serine residues to alanine, including a Mig1 mutant in which all four residues were mutated (S222A+S278A+S311A+S381A), impaired but did not eliminate Mig1 phosphorylation and glucose repression (377). Nevertheless, in vivo phosphorylation of Mig1 is dramatically reduced in an snf1 mutant, and immunoprecipitated Snf1 (but not the kinase-dead Snf1-K84R) phosphorylates coprecipitated Mig1. Also, as expected based upon their capacity to mediate glucose repression by inhibiting Snf1 kinase activity, deletion of either REG1 or HXK2 results in Mig1 hyperphosphorylation under repressing conditions (377). Taken together, the simplest interpretation of these data is that phosphorylation of Mig1 by Snf1 is at least partly responsible for inactivation of Mig1 repressor function.
Deletions that map within an internal region containing most of the Snf1 phosphorylation sites (residues 261 to 400) converts Mig1 into a constitutive repressor that is not inactivated when glucose is depleted (270). Interestingly, deletions in Mig1 that remove all or part of the internal region from residue 261 to 400 cause it to persist in the nucleus in the absence of glucose, whereas under the same conditions wild-type Mig1 is largely exported to the cytoplasm (87). Fusion of Mig1 residues 261 to 400 to a GFP-ß-galactosidase chimera confers glucose-regulated nuclear import and export upon the resulting fusion protein, and its import in the presence of glucose requires both REG1 and HXK2. In glycerol-grown cells Mig1 becomes increasingly nuclear as more Snf1 kinase sites are removed (86). Msn5, an importin ß homolog originally identified due to its suppression of an snf1 mutation when overexpressed (107), interacts with Mig1 and mediates its nuclear export in response to glucose removal (86). These and other data suggest that Snf1 phosphorylation of Mig1 inactivates it by changing its subcellular localization.
According to this Mig1 nucleocytoplasmic shuttling hypothesis, Hxk2- and Reg1/Glc7-dependent inactivation of Snf1 in the presence of glucose results in hypophosphorylation of Mig1, which causes it to relocate rapidly to the nucleus; subsequent binding of Mig1 to its recognition sites upstream from glucose-regulated genes mediates transcriptional repression (86, 87). Although this model is attractive, it is difficult to reconcile with two strikingly contrasting results. First, the Mig1 target gene GAL1 is derepressed normally in an msn5 mutant grown on glycerol, despite the constitutive presence of Mig1 in the nucleus (86). Second, Mig1 remains bound to the GAL1 promoter in vivo under these same derepressing conditions (278). Both of these results suggest that Mig1 transcriptional repression of target genes is regulated via a mechanism other than nuclear export and that the key function of Snf1 phosphorylation in relieving Mig1-dependent glucose repression is to inactivate the relatively small amount of Mig1 that remains in the nucleus and is bound upstream of target genes in derepressed cells (87, 270). How this is accomplished remains obscure.
Mig3, Nrg1, and Nrg2 are three additional Snf1-regulated repressors in S. cerevisiae. Mig3 is somewhat mysterious; it was identified because it contains Cys2His2 zinc fingers that are very similar to those of Mig1 and Mig2 (26, 220). Several additional results seem to suggest that Mig3, like Mig1 but not Mig2, is an Snf1-controlled, DNA-bound glucose regulator (182, 221). It binds to the Mig1 binding sites upstream from SUC2, and a LexA-Mig3 chimera functions as a glucose-dependent repressor of reporter gene expression when bound to upstream LexA operators. When cells are shifted from glucose to galactose, Mig3 is extensively phosphorylated (at least in part by Snf1) and rapidly subjected to Snf1-dependent proteolysis (94). Nevertheless, physiologically significant regulation of target genes by Mig3 is yet to be demonstrated; in wild-type cells under standard conditions, it seems unlikely to be more than a minor regulator of glucose-responsive gene expression (220, 221). Recently the MIG3 gene was also isolated in a screen for multicopy suppressors of Rad53-GFP toxicity, but the relationship between Mig3 function and the DNA damage response awaits further clarification (94). It is hoped that transcriptomic analysis of the effects of an as-yet-untested environmental condition or genetic background will eventually reveal the Mig3 role in glucose regulation and explain the results obtained to date.
Nrg1 and Nrg2 contain Cys2His2 zinc fingers that are very similar both to each other and to those in Mig1, Mig2, and Mig3 (400). Nrg1 was first identified in two separate screens for factors mediating repression of glucose-regulated genes (1, 282), and Nrg2 was discovered because of its two-hybrid interaction with Snf1 (400). Outside of their DNA binding domains Nrg1 and Nrg2 share only 27% identity, and this is thought to account for their somewhat distinct regulatory roles in glucose repression, filamentous invasive growth, and biofilm formation (18, 196, 282, 400, 438). In the absence of glucose, neither factor is degraded, nor is their capacity to bind target sites in vivo impaired. Both Nrg1 and Nrg2 exhibit glucose-dependent repression of a heterologous reporter gene; they are also both capable of interacting with Snf1 kinase, but (unlike Mig1 and Mig3) neither appears to be phosphorylated by it (18, 400). Although Snf1 appears to modulate Nrg2 levels and is clearly required for normal Nrg1 function, the regulatory relationship between these factors is complex and in need of further study. Interestingly, like Mig1 and Mig3 (see above), Nrg1 can function as an activator in some circumstances (18).
DNA-bound activators controlled by Snf1. The Snf1 protein kinase also regulates a number of positively acting transcription factors required for the utilization of nonfermentable carbon sources. Two of these factors, Cat8 and Sip4, both contain zinc finger DNA binding domains similar to that in Gal4 (a C6 zinc cluster) (145, 206) and bind specifically to a set of carbon source responsive elements (CSRE) under derepressing conditions (300, 395). Cat8 and Sip4 are closely related structurally and appear to have slightly different affinities for variants of the CSRE sequence (319). Although Sip4-dependent transcriptional activation has been shown to require phosphorylation of Sip4 by Snf1 kinase, removal of Sip4 has little or no effect on CSRE-regulated genes (141, 145, 206, 299). Also, unlike deletion of CAT8, which causes defective growth on nonfermentable carbon sources, deletion of SIP4 results in no obvious phenotype (206).
Cat8 regulates the expression of a wide array of genes involved in the metabolism of nonfermentable carbon sources, including genes necessary for ethanol utilization (e.g., ACS1 and ACR1/YJR095W) (28, 192), gluconeogenesis (141, 145), the glyoxylate cycle (141), lactate utilization (JEN1) (27), and isocitrate metabolism (IDP2) (27); Cat8 also regulates expression of SIP4 (141, 395). Cat8 activity is regulated on two levels: its expression is Mig1 repressed (145, 257), and its capacity to activate transcription requires a functional Snf1 kinase (303). Snf1 phosphorylates Cat8 in ethanol-grown cells (56, 303); following the addition of glucose, it is dephosphorylated in a Glc7-independent manner (303). Interestingly, constitutively expressed Cat8 fused to the Ino2 activation domain bypasses the need for a functional Snf1 in cells growing on ethanol (300).
Cat8 works together with another Snf1-regulated transcriptional activator, Adr1, for maximal expression of a small group of shared target genes (including ADH2, ACS1, and ALD6) under derepressing conditions (25, 98, 192, 356, 405, 432). Like the Mig1, Mig3, Nrg1, and Nrg2 repressors, Adr1 has a Cys2His2-type DNA binding domain (283), and its function requires Snf1 kinase (84), although the mechanism of Adr1 regulation remains unclear. It has been suggested that Snf1 and Reg1/Glc7 contribute to regulation of Adr1 by affecting its binding to promoters of target genes. Snf1 promotes Adr1 binding under derepressing conditions, while Reg1/Glc7 inhibits Adr1 binding to promoters in the presence of glucose (91, 433); it is currently unknown whether Adr1 is a direct target of Snf1 and Reg1/Glc7 or whether this effect occurs through intermediary factors. Adr1 activity has also been reported to be negatively regulated by PKA (60) in a manner independent of its DNA binding (364).
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1,000 glucose-induced genes is even more mysterious than the mechanisms discussed above that control the 1,000 genes whose expression is repressed in the presence of glucose. Our clearest picture is of glucose induction by the Snf3/Rgt2 sensing mechanism, a pathway that appears to operate in a largely PKA-independent fashion and (at minimum) contributes the critical regulatory trim that allows S. cerevisiae to grow well on a broad range of glucose concentrations (from micromolar to molar concentrations) (272). The integral plasma membrane proteins Snf3 and Rgt2 mediate 
