glucose signaling in saccharomyces cerevisiae · glucose induction and are also essentially ras...

MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Mar. 2006, p. 253–282 Vol. 70, No. 11092-2172/06/$08.00�0 doi:10.1128/MMBR.70.1.253–282.2006Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Glucose Signaling in Saccharomyces cerevisiaeGeorge M. Santangelo*

Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, Mississippi 39406-5018

INTRODUCTION .......................................................................................................................................................253NATURE OF THE SIGNAL......................................................................................................................................254

Signaling without Extracellular Sensing, Transport, or Phosphorylation of Glucose..................................254Redundant but Unified Glucose Signaling Pathways ........................................................................................255Early Steps in Signal Transduction .....................................................................................................................256

The Ras/cAMP/PKA pathway............................................................................................................................256The Gpr1/Gpa2 pathway ....................................................................................................................................258Activation of Ras and Gpa2 by glucose ...........................................................................................................258

(i) Extracellular sensing ................................................................................................................................258(ii) Intracellular sensing................................................................................................................................258

Extracellular sensing by Snf3/Rgt2 ..................................................................................................................259SIGNAL RECEPTION: GLUCOSE REPRESSION ..............................................................................................259

Involvement of the Hexokinase Hxk2 in the Glucose Response ......................................................................259Snf1-Mediated Signal Transduction in Glucose Repression and Derepression ............................................260

Dueling Snf1 kinase and Glc7 phosphatase mediate glucose regulation....................................................260Transmission of the Ras/cAMP-dependent glucose signal to Reg1/Glc7 and Snf1/Snf4..........................263DNA-bound repressors controlled by Snf1......................................................................................................263DNA-bound activators controlled by Snf1.......................................................................................................265

SIGNAL RECEPTION: GLUCOSE INDUCTION.................................................................................................265Snf3/Rgt2 Signaling of the Rgt1 Repressor Complex ........................................................................................265The Cell Cycle Connection ....................................................................................................................................267Transcriptional Induction of Glycolytic and Ribosomal Protein Genes .........................................................268The Repressor/Activator Theme in Glucose Regulation....................................................................................269Reverse Recruitment: Rap1/Gcr1 Activation at the Nuclear Periphery..........................................................269

GENE REGULATION VIA REVERSE RECRUITMENT.....................................................................................272A New Model for Eukaryotic Transcriptional Activation and Repression .....................................................272Glucose May Signal a Highly Networked and Structurally Stable Perinuclear Machine............................273

ACKNOWLEDGMENTS ...........................................................................................................................................274REFERENCES ............................................................................................................................................................274

INTRODUCTION

Jacques Monod’s seminal discovery of the diauxic growth ofbacterial cultures (243) demonstrated that microbes are par-ticularly adept at targeting and responding to glucose, the mostabundant monosaccharide in nature. A wealth of subsequentwork has shown that virtually all cells possess a sophisticatedgenetic program that responds to this rich source of carbonand energy. Study of the glucose response in eukaryotes hasbeen greatly aided by the development of the malleable bud-ding yeast Saccharomyces cerevisiae as a model system (see,e.g., references 361 and 417). S. cerevisiae shares with complexmulticellular eukaryotes many of the signal transduction com-ponents that detect glucose, transduce the corresponding sig-nals to the interior of the cell, and make the needed adjust-ments to cellular metabolism and gene expression profiles.

This review will focus on the cytoplasmic regulators thattransmit the glucose signal from the plasma membrane into theyeast nucleus, as well as the nuclear apparatus that mediatesthe global transcriptomic response to the sugar (both glucose

repression and glucose induction). The response to glucosealso involves direct modification of metabolic pathways (termedglucose or catabolite inactivation) and other processes such asmRNA turnover (7, 76, 103, 121, 153, 154, 156, 161, 166,171–173, 232, 294, 307, 329, 334, 335, 337, 391). Although thelatter phenomena are outside the scope of this review, theyappear to be mediated by the same two central signal trans-duction pathways, Ras/cyclic AMP (cAMP)/protein kinase A(PKA) and Snf3/Rgt2/Yck, which are described below indetail.

The recent application of yeast genomics and proteomics isa first step towards comprehensive characterization of the glu-cose response. For example, we now know that within 20 minof 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 atwofold change (408). Glucose repression largely affects genesencoding enzymes involved in respiration or alternative carbonsource metabolism, and glucose induction stimulates transcrip-tion of glycolytic genes and ribosomal protein (RP) genes (seebelow). Genomic and proteomic analyses are particularly use-ful when supplemented with high-throughput genetic tech-niques such as synthetic genetic array analysis, which allows theidentification of synthetic phenotypes by simultaneous screen-

* Mailing address: Department of Biological Sciences, University ofSouthern Mississippi, Hattiesburg, MS 39406-5018. Phone: (601) 266-6080. Fax: (601) 266-5068. E-mail: [email protected].

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ing 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 combina-tion with the now-facile elimination, overexpression, or pointmutation of relevant yeast regulators, should eventually permitthe delineation of a global wiring diagram that mirrors itsbiological template when virtually challenged with a widerange of concentrations of the sugar. For such a computationalmodel to be valid, it must at a minimum be informed by (i) anaccurate molecular map of cytoplasmic and nuclear substruc-ture, (ii) a detailed diagram of the physical and functionalconnections that link the relevant signal transduction compo-nents to each other and to adjacent networks, and (iii) thetemporal progression of the response following the appearanceof glucose. Here I review our progress toward that ambitiousgoal and attempt to identify the most important issues thatrequire further clarification.

NATURE OF THE SIGNAL

Two recent surprises uncovered by global analyses of Saccha-romyces cerevisiae are the extent to which the glucose inductionand repression mechanisms appear to overlap and the fact thatyeast cells can be tricked into generating the full-blown response(both induction and repression) in the complete absence of glu-cose. These and other recent developments place G-protein sig-naling, in particular the Ras/cAMP/PKA pathway, front and cen-ter among the cellular components that participate in early eventsin the glucose response.

Signaling without Extracellular Sensing, Transport,or Phosphorylation of Glucose

A recent microarray experiment from the Broach laboratoryhas clearly demonstrated the universality of induction/repres-sion overlap in glucose signal transduction. A unified transcrip-tome-wide response (both upshifted and downshifted gene ex-pression) was observed in yeast cells by stimulating early stepsin the glucose signaling pathway; a clever experimental schemeallowed the generation and detection of this comprehensiveglucose response without addition of the sugar (408). The yeasttranscriptome was analyzed after heterologous induction ofactivated alleles of Ras2 or Gpa2, signal transmitters that act inparallel early in the cAMP-dependent protein kinase (PKA)pathway (see below). Astonishingly, virtually all of the changesin transcript levels after addition of glucose (both up-regula-tion and down-regulation) were also observed in the absence ofglucose following expression of the activated form of Ras2. Ofthe genes that showed at least a threefold change in expressionwithin an hour following glucose addition, almost all (92%)changed at least twofold in the same direction after Ras acti-vation (Fig. 1; Table 1). The result was similar with activatedGpa2, though the intensity of the transcriptional response wasreduced relative to that seen with activated Ras. Those authorsalso demonstrated that all of the changes in transcript levelsresulting from Ras2 and Gpa2 activation were mediated byPKA (408) (Fig. 1). Importantly, since much of this transcrip-tional reprogramming can also be accomplished in the absenceof signaling through cAMP, redundant overlapping pathways(Ras2/Gpa2/PKA dependent and Ras2/Gpa2/PKA indepen-

dent) appear to be fully capable of transducing the glucosesignal. Little is known about the PKA-independent mecha-nism, although it presumably targets a similar set of down-stream factors in the absence of normal PKA catalytic activity.

The observed transcriptome-wide up-regulation and down-regulation of gene expression in response to activated Ras wasparticularly surprising, since it had long been assumed (despiteearly hints to the contrary [see, e.g., reference 233]) that theprimary glucose repression pathways in yeast are distinct from

FIG. 1. Wild-type versus Ras2 mimicry of the glucose response.Each curve represents the expression profile of genes exhibiting atwofold or greater change, both within 20 min of glucose addition andwithin 20 min of the appearance of activated Ras2. The average valuesfor 250 such induced genes (upper graph) and repressed genes (lowergraph) are shown (see supplemental data associated with reference208); error bars represent standard errors of the means. Squares,expression profile of wild-type cells following glucose addition; filledtriangles, expression profile of cells containing Gal-regulated Ras2*(activated Ras) following addition of galactose; open triangles, expres-sion profile of cells containing Gal-regulated Ras2* (activated Ras) ina tpkw (weak PKA) background following addition of galactose. It isimportant to note that the �100-fold induction and repression ob-served for a small subset of glucose regulated genes (e.g., HXT genes;see reference 272) is not seen within the 60-minute time frame of thisdata set.

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glucose induction and are also essentially Ras independent (44,45, 118, 121, 123, 178, 315, 335, 365). Further, stimulation ofthe glucose response in the absence of either transport orphosphorylation of the sugar (408) is particularly significant.Transport of glucose and its conversion to glucose-6-phosphateby hexokinases had been explored as a potentially essentialfeature of the response (121, 314, 316) and now appears to atmost participate in redundant signaling that operates throughor in parallel with the cAMP-PKA pathway. Whatever extra-cellular or intracellular glucose sensing is needed to activateRas or Gpa2 is therefore sufficient for both glucose inductionand repression. This interpretation has the added advantage ofbringing our assessment of glucose-stimulated signal transduc-tion in yeast cells closer to what is known about the analogousresponse in bacterial and mammalian cells (for reviews, seereferences 35, 129, and 322).

Redundant but Unified Glucose Signaling Pathways

The first hint of overlap between glucose induction andrepression resulted from analysis of regulators that are “down-stream” in the signaling cascade, i.e., factors that for the mostpart function within the yeast nucleus. For example, the Rgt1repressor blocks transcription of HXT1 through HXT4 (4 ofthe 18 glucose transporter genes in S. cerevisiae) (see refer-ences 118 and 272 for reviews). At high glucose concentrations,although its contact with the HXT1 promoter can no longer bedetected, Rgt1 is converted into an activator through hyper-phosphorylation (142, 186, 250). The hexokinase Hxk2, longknown to participate in glucose repression, is also required forglucose induction of HXT1 (231, 271).

The Snf1 serine-threonine kinase represents another exam-ple of downstream regulatory overlap; Snf1 activates genesthat are repressed in glucose-containing media but are dere-pressed and needed for growth in the presence of nonferment-able carbon sources (46). Recent transcriptomic and proteomicanalyses of snf1 cells revealed a previously unnoticed upshift inexpression of genes (e.g., ADH1) involved in glucose catabo-lism (hereafter the term “glycolytic genes” will be used for thisgroup) (432). Thus, in addition to its well-established role inderepressing glucose-repressed genes, Snf1 also somehow re-presses transcription of glucose-induced genes (including HXT1)(372). Snf1 repression of glycolytic gene expression has beenfurther substantiated by proteomic analysis of cells lacking Snf4,the � subunit of the Snf1 kinase complex (140).

A final example of downstream regulatory overlap is thephosphoprotein Gcr1, which was first identified as an activatorof glycolytic genes (13, 63, 64, 152). Transcriptomics and otheranalyses have confirmed that expression of numerous glucose-repressed genes is derepressed in the absence of Gcr1 at highglucose concentrations, suggesting a new role for this regulatorin repression of transcription (327, 383; K. Barbara and G. M.Santangelo, unpublished data).

The roles of Rgt1, Hxk2, Snf1/Snf4, and Gcr1, as well asnumerous other repressor/activator polypeptides that partici-pate in the glucose response, are presented in greater detail inlater sections of this review. In addition to these specific reg-ulators, at least eight components of the general transcriptionmachinery that interact with the carboxy-terminal domain ofRNA polymerase II (Gal11, Sin4, Rgr1, Rox3, Srb8, Ssn2,Ssn3, and Ssn8) have been found to play both positive andnegative regulatory roles; the function of many of these factors

TABLE 1. Genes that respond similarly to both glucose addition and Ras2 activationa

Induced genes Repressed genes

Proteinbiosynthesisb Transcription Transportc Energy reserve

metabolismdProtein

turnoverStress

response

RPS9A RPA43 NUP1 BCY1 PEP4 HSP30RPS22B RPA49 NUP100 SDC25 RPN2 SSE2RPS27A RPB5 NUP133 GAL83 RPN3 SSA3RPL7B RPB9 NUP170 PHO85 RPN5 SSA4RPL14B RPC40 MEX67 TPS1 RPN6 SSD1RPL17B RPC82 RNA1 CIT3 RPN7 STO1TIF11 RPO26 GSP1 GPD1 RPT1 CDC55TIF3 RPO31 PSE1 MDH3 RPT3 UBC5TIF34 RRP43 SXM1 PGM2 RPT4 PRE1TEF4 RRP45 ALR21 HXT10 RPT5 PRE3CDC60 RRP5 FET3 MRPL23 DOA1 GTS1ARG8 ARG80 FET4 MRPL32 DOA4 CRS5ARO7 BAS1 FKS1 PET112 APG13 CTA1ARO8 DBP2 HNM1 PET117 APG14 GRX2ASP1 DBP3 HXT2 PET191 APC9 TRX2HIS1 GCR2 HXT4 PET54 HRD1 MDR1HIS3 IFH1 LYP1 COQ1 HRD3 PAU1ILS1 HMALPHA1 NPL3 COQ3 PEX14 PAU2ILV1 IMP4 PHO84 COX10 MPD2 PAU3LYS4 LEU3 ZRT1 COX11 SBA1 PPZ2

a See supplemental data associated with reference 408; a grand total of 758 genes exhibited a twofold or greater change in expression both within 20 min of glucoseaddition and within 20 min of the appearance of activated Ras2. Gene categories are based on Munich Information Center for Protein Sequences functionalclassification; a representative list from each category is shown. A total of 329 and 429 genes were in the induced and repressed categories, respectively.

b Includes genes encoding translational components (including ribosomal proteins), rRNA-processing factors, and amino acid biosynthetic enzymes.c Includes genes encoding nucleocytoplasmic transport factors and plasma membrane transporters.d Includes genes encoding factors involved in carbon storage and respiration (including mitochondrial factors).

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is known to be influenced by glucose signaling (43, 195). Thus,the overlapping positive and negative circuitry in the glucoseresponse may extend from the plasma membrane through tothe ultimate choice of induction or shutoff of transcription initi-ation of each glucose-regulated gene by RNA polymerase II.

The induction/repression duality of signal recipients such asRgt1, Hxk2, Snf1/Snf4, Gcr1, and other transcription factorsclearly requires further characterization of the precise mecha-nisms involved. Nonetheless, together with the transcriptomicanalysis of activated Ras, it suggests the existence of a moreunified and interdigitated response to glucose than had previ-ously been appreciated. Although it now seems certain thattransmission of the glucose signal is redundant (408), thosesignaling pathways may converge on only a relatively smallgroup of transcription factors in the yeast nucleus (see below).Coordinated remodeling of the nuclear regulatory apparatus inresponse to glucose might therefore involve a parsimoniousseries of alterations in key regulators. Although for the sake ofclarity and historical perspective the discussion of nuclear reg-ulators is divided into separate sections of this review, in realitythe mechanisms of “glucose repression” and “glucose induc-tion” may be no more segregated than the two faces of a coin.

Early Steps in Signal Transduction

Given the sufficiency of Ras or Gpa2 activation to producethe transcriptome-wide spectrum of changes in response toglucose, it is important first to review what is known aboutthese factors, their respective signaling pathways, and how theyare activated immediately following appearance of the sugar.

The Ras/cAMP/PKA pathway. Ras proteins are monomericGTPases that function as switches; these so-called G proteinsare inactive in the GDP-bound state and active when GTP isbound. The switch from the active to the inactive form involveshydrolysis of bound GTP by the intrinsic GTPase and is stim-ulated by GTPase-activating proteins (GAPs). The reverse (in-active-to-active) switch requires replacement of bound GDPwith GTP, which is normally accomplished with the aid ofguanine nucleotide exchange factors (GEFs). Activating mu-tations in Ras polypeptides (e.g., human rasVal12 or yeastRas2Val19) increase GTP association in a GEF-independentfashion and are responsible for many human cancers (78, 419).Ras mutations are found in approximately 30% of humantumors; the frequency of oncogenic forms of Ras is as high as50% or 90% in colorectal or pancreatic tumors, respectively(345). Although it remains dimly understood, it seems likelythat there is an important connection between these recentdata and a very old observation that altered glucose metabo-lism 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 overthe N-terminal 180 residues (291). Growth on glucose is unaf-fected by the absence of either Ras1 or Ras2, whereas loss ofboth causes arrest in the G1 phase of the cell cycle (362, 367).These early findings were suggestive of a connection to nutri-ent 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 carbonsources (363), suggesting that the latter defect is due to the factthat Ras1 is more weakly expressed than Ras2 (249, 361),rather than a specific function that maps to the divergent andslightly extended C terminus of Ras2.

Important effector molecules modulate Ras activity duringthe glucose response; posttranslational modification and mem-brane localization of Ras are two important features of theseregulatory inputs. For example, Ras farnesylation contributesto nucleotide exchange by the GEF Cdc25 (34, 73, 134);CDC25 is an essential gene and was originally identified by ascreen for mutations that cause G1 arrest (139). A second yeastRasGEF that can substitute for Cdc25 when overexpressed isencoded by the dispensable SDC25 gene (31) (note that thisgene was originally named SCD25 [79]).

Ras stimulation by the RasGEFs in turn stimulates produc-tion of cAMP by the essential product of the adenylate cyclasegene (CYR1) (229). Inactivation of Ras, resulting in lowercAMP levels, is facilitated by the GAPs Ira1 and Ira2 (358,360) (Fig. 2). Presumably due to their competing effects on Rasactivation, IRA1 deletion rescues the lethality caused by dele-tion of the RasGEF CDC25 (359). Low-affinity and high-affin-ity cAMP phosphodiesterases in S. cerevisiae (Pde1 and Pde2,respectively) also antagonize the Ras signal through enzymaticremoval of cAMP; in the presence of exogenous cAMP, dele-tion of PDE2 restores growth to ras1� ras2� strains (266, 420).

The Ras C terminus also undergoes functionally significantposttranslational modification. Yeast Ras2 is normally pal-mitoylated via a thioester linkage at cysteine 318 and farnesy-lated via a thioether linkage at cysteine 319. Blocking bothmodifications with the double point mutation C318S C319Sresults in a nonfunctional molecule (92). Farnesylation is re-quired for palmitoylation and all subsequent modifications,which include removal of the three C-terminal residues byproteolytic cleavage and methyl esterification of the revealedC-terminal cysteine 319 (19).

The palmitoyl moiety anchors Ras to the cytoplasmic face ofthe yeast plasma membrane (199); blocking addition of palmi-tate via the single point mutation C318S causes mislocalizationof the mutated Ras2 protein to the cytoplasm (176). Ras pal-mitoylation is reversible and therefore may be a regulatorystep. This is of particular interest because mutation of pal-mitoylation sites abrogates both the glucose response in yeastcells (see below) and transformation of mammalian cells byoncogenic forms of Ras (418).

The appearance of glucose thus generates one or more sig-nals that converge on membrane-bound Ras and its modifiers(in particular the RasGEFs and RasGAPs). This glucose sig-naling results in a rapid but transient spike in the level ofintracellular cAMP, which increases 5- to 50-fold within 1 to 2min of glucose addition and returns to near-basal levels within20 min. When cells express the nonpalmitoylated cytoplasmicRas2C318S form as their only Ras protein, they fail to exhibitthe glucose-induced cAMP spike and are defective in transi-tioning to rapid growth upon addition of glucose (160). Thus,glucose-regulated cAMP production appears to require attach-ment of Ras to the yeast plasma membrane. Interestingly,membrane localization of yeast Ras2 was recently shown to



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occur despite disruption of the classical secretory pathway thatnormally mediates translocation from the endoplasmic reticu-lum to the plasma membrane (92).

Additional signal transduction components appear to con-tribute to glucose signaling by contacting adenylate cyclase(Cyr1); such Cyr1 protein-protein interactions identified thusfar include those with (i) an N-terminal SH3 domain in theRasGEF Cdc25 (237), (ii) the farnesylated Ras2 C terminus(73, 74, 184, 199, 338, 355), (iii) the C terminus of the putativecochaperonin Sgt1 (95), (iv) the N terminus of the ancillaryfactor Srv2 (CAP) via coiled-coil formation (267), and (v) theRasGAP Ira1, which mediates peripheral association of ade-nylate cyclase with the yeast plasma membrane (241).

It seems likely that the sole purpose of the glucose-inducedspike in cAMP levels is to block the inhibitory effect of theBCY1-encoded regulatory subunit on the catalytic subunits ofPKA, encoded redundantly by TPK1, TPK2, and TPK3; over-expression of any of these three genes, or deletion of BCY1,

restores growth to ras1� ras2� strains (369). PKA is a het-erotetramer consisting of two catalytic (Tpk) subunits and tworegulatory (Bcy1) subunits (Fig. 2); addition of regulatory sub-units to catalytic subunits in vitro converts protein kinase ac-tivity into a completely cAMP-dependent form (151, 177, 370).

Interestingly, the subcellular localization of PKA catalyticand regulatory subunits appears to respond to glucose signal-ing; Bcy1 is present in both the cytoplasm and nucleus in theabsence 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 signalingcomponents, may help to maintain PKA activity in part byregulating nuclear translocation of Tpk1 (330). Although it hasyet to be demonstrated that the observed translocation of PKAsubunits between compartments plays an important role inglucose signaling in yeast cells, targeting of PKA regulatorysubstrates would appear to undergo a qualitative and/or quan-titative shift in response to the appearance or disappearance of

FIG. 2. Cytoplasmic events in PKA signaling. Glucose regulation relies upon the intracellular signaling molecule (“second messenger”) cAMP.GTP-bound G proteins (Ras and Gpa2) bind independently to adenylate cyclase (Cyr1) and stimulate its production of cAMP. The monomericRas G proteins are anchored in the plasma membrane via a posttranslationally added palmitoyl moiety (red squiggle). RasGEFs (Cdc25 and Sdc25)and RasGAPs (Ira1 and Ira2) are also present in the Ras/Cyr1 complex and regulate adenylate cyclase by controlling the Ras switch (see text).The seven-transmembrane polypeptide Gpr1 acts upstream of Gpa2 in glucose signaling and is a member of the G protein-coupled receptor(GPCR) superfamily. Gpa2 was identified as a result of its similarity to the mammalian G� subunits of heterotrimeric G proteins, and the putativeG� subunits Gpb1 and Gpb2 were found via their interaction with Gpa2; a corresponding � subunit has yet to be identified (question mark). AGpa2GEF also awaits identification; Rgs2 is a known Gpa2GAP. Phosphodiesterases (Pde1 and Pde2) antagonize glucose signaling via enzymaticinactivation of cAMP (conversion to AMP). The PKA tetramer is the regulatory target of cAMP. While bound to the kinase subunits (TPK), theregulatory Bcy1 subunits keep PKA in an inactive state; cAMP activates the catalytic subunits by binding to Bcy1 subunits and promotingdissociation of the complex (dashed arrow).



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the sugar. Further progress in this area is complicated by thefact that signaling via each of the three different catalytic sub-units of PKA (Tpk1, Tpk2, and Tpk3) results in a distinctpattern of downstream gene expression readouts (309).

Although active PKA is thought to phosphorylate proteinsinvolved in transcription, energy metabolism, and cell cycleprogression (129), further study is required to identify theexact targets and sequence of protein translocation and phos-phorylation events that transmit the glucose regulatory signalto the yeast nucleus. Significantly, mutations impairing specificcomponents of the RNA polymerase II transcriptional machin-ery are synthetically lethal with the hyperactive Ras2Val19 allele(157), and PKA can phosphorylate a component of the Srbcomplex (Ssn2) both in vitro and in vivo (55). The latter may beone of but a few Ras/cAMP/PKA-mediated modifications innuclear factors needed to accomplish the broad sweep of al-terations to the yeast transcriptome upon glucose addition ordepletion.

The Ras/cAMP/PKA pathway has also been implicated inaging (212, 216), thermotolerance (439), bud site selection,actin repolarization (333), glycogen accumulation (342), stressresistance (39, 407), and sporulation (39, 41); it may also reg-ulate pseudohyphal differentiation (growth as chain-like mul-ticellular filaments) in response to nutrient limitation. Each ofthese 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 thisreview.

The Gpr1/Gpa2 pathway. Heterotrimeric G proteins are sig-naling molecules composed of �, �, and � subunits. Theyrepresent a second class of factors that, like monomeric Raspolypeptides, 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 signal-ing in S. cerevisiae (190, 394). Gpa2 was originally identified byscreening a yeast genomic library with cDNA probes encodingmammalian G� subunits (252), and the putative � subunitsGpb1 and Gpb2 were identified as interaction partners ofGpa2 (135); no corresponding G� subunits involved in glucosesignaling have yet been identified (but see reference 135).Whereas deletion of either GPA2 or RAS2 causes little or noimpairment of growth on glucose media, deletion of bothcauses a severe synthetic growth defect (428). Removal of themajor cAMP phosphodiesterase in the cell (Pde2) restoresnormal growth to gpa2� ras2� strains, suggesting that an in-crease in cAMP levels bypasses the loss of both Gpa2 and Ras2(428). Most importantly, as mentioned above, the transcrip-tomic response to glucose is successfully mimicked in the ab-sence of the sugar by turning on expression of either activatedGpa2 or activated Ras2; the fact that this signaling is PKAdependent confirms their redundant participation in thecAMP-dependent branch of glucose signaling (408).

A two-hybrid protein interaction screen with GPA2 as thebait led to the identification of the GPR1 gene, which encodesa member of the G protein-coupled seven-transmembrane re-ceptor superfamily (Fig. 2). Gpr1 is located on the yeast cellsurface (428). Genetic analysis strongly suggests that Gpa2 actsdownstream 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 ei-ther multicopy or activated GPA2 suppresses the defect of thegpr1� ras2� strain (428). Deletion of GPR1 diminishes (al-though does not eliminate) genome-wide transcriptional in-duction and repression in response to glucose (408). The sim-plest explanation for these data is that Gpr1 is the onlyreceptor coupled to Gpa2 and that it too contributes to acti-vation of the cAMP pathway in response to glucose (190).

Although Gpa2 was originally thought to function upstreamof the Sch9 kinase (368, 428), it was subsequently found thatSCH9 deletion is synthetically lethal with gpr1�, gpa2�, orras2�, suggesting that Sch9 acts instead in the pseudohyphaldifferentiation pathway that is parallel to both Gpr1/Gpa2 andRas (217). Sch9 may nevertheless participate somehow in glu-cose signaling, since (i) it is the yeast ortholog of a componentof the mammalian insulin response pathway (Aft/protein ki-nase B), (ii) it shares similarity with PKA catalytic subunits,(iii) its overexpression compensates for the loss of Ras func-tion, and (iv) PKA activity is elevated in a sch9� background(72, 109).

Activation of Ras and Gpa2 by glucose. Since artificial acti-vation of Ras and Gpa2 is sufficient to generate the glucoseresponse, and it seems unlikely that glucose interacts directlywith the G proteins themselves, how do Ras and Gpa2 becomeactivated in wild-type yeast cells exposed to the sugar? It isconvenient to separate potential answers to this question intotwo categories: recognition of glucose by receptors on the outsidesurface of the cell versus direct signaling of Gpa2 and Ras (ortheir regulators) by intracellular glucose or its metabolites.

(i) Extracellular sensing. There are 18 known hexose trans-porter genes in S. cerevisiae (HXT1 to HXT17 and GAL2).Deletion of seven of these genes results in the failure to trans-port or grow on glucose (308, 413). In this hxt1 to hxt7 nullbackground, constitutive expression of the GAL2-encoded ga-lactose permease restores glucose-induced cAMP synthesis(313). This suggests that the yeast hexose transporters do notparticipate in glucose signaling but are required only fortransport.

The seven-transmembrane receptor Gpr1 may detect extra-cellular glucose and thereby activate Gpa2, but it is not re-quired to signal Ras; Gpr1 or Gpa2 deletion does not preventthe glucose-induced increase in Ras2-GTP levels (66). In con-trast, the Ras2-GTP increase is absent in a glucose phosphor-ylation-deficient (hxk1 hxk2 glk1) strain (66). Thus, Ras ap-pears to be activated by intracellular phosphorylated glucosebut not by extracellular glucose. A clear demonstration ofextracellular sensing by Gpr1 will require direct evidence, forexample, as suggested by recent mutagenic analysis (205), thatit interacts with glucose.

(ii) Intracellular sensing. If extracellular signaling does notparticipate in Ras activation (66, 313), it will be particularlyimportant to understand how intracellular glucose triggers theglucose response. The longstanding idea that glucose phos-phorylation (in particular by the glucose-induced hexokinaseHxk2; see below) and/or subsequent glycolytic reactions arerequired to alter yeast gene expression in response to glucosehas been controversial (see references 20, 121, 244, and 314 forreviews of this topic). Since repression occurs even when themetabolic steps immediately following glucose phosphoryla-



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tion are reduced to less than 2% of wild-type levels, it seemslikely that nothing beyond the production of glucose-6-phos-phate is required (317). Further, since cells can be tricked intothe full-blown glucose response by expressing activated Ras orGpa2, glucose phosphorylation must generate the signalthrough the G proteins and/or through a redundant and as-yet-obscure alternative pathway. A recent interesting sugges-tion is that glucose phosphorylation increases Ras2-GTP levelsby inhibiting the Ira (RasGAP) proteins (66).

Although progress has been made, much more work isneeded to establish the precise mechanism(s) through whichRas, Gpa2, and/or their regulatory GEFs and GAPs is signaledby glucose. Intracellular acidification (316, 365) and energycharge (the AMP/ATP ratio) (134, 244) are additional poten-tial signaling mechanisms that require further testing. Theevents that immediately follow activation of either G proteinare much clearer (Fig. 2). Activated Ras or Gpa2 can eachgenerate a spike in cAMP production by adenylate cyclase;cAMP in turn releases PKA catalytic (Tpk) subunits frominhibition by its regulatory (Bcy1) subunits.

Extracellular sensing by Snf3/Rgt2. The putative glucosesensors Snf3 and Rgt2 are 60% identical to each other and, likehexose transporters found in bacteria, plants, and mammals(228, 259, 332), contain 12 predicted transmembrane-spanningdomains (272). Snf3 and Rgt2 do not appear to act as glucosetransporters but instead signal the presence of the sugar (118,178, 182, 247, 273, 314). Since the transcriptional response ofSnf3/Rgt2 target genes (e.g., HXT genes encoding the glucosetransporters) is generated in the absence of glucose by expres-sion of hyperactive Ras or Gpa2, the Snf3 and Rgt2 sensorsappear either to act upstream of Ras/cAMP/PKA or to play anancillary or redundant role in the initial detection of glucose.This view is consistent with the possibility that glucose sensingby Snf3/Rgt2 is required for maintenance of glucose inductionor repression for at least a subset of the transcriptome (182,272). Further, it seems clear that the Snf3/Rgt2 regulatorypathway helps to ensure that changes in gene expression arecommensurate with the glucose concentration in the environ-ment, a mechanism that is likely to be particularly important innature.

As mentioned above for Gpr1, a clear demonstration ofextracellular sensing by Snf3 and Rgt2 will require direct evi-dence that they interact with glucose. Intriguing hints alongthese lines have already been uncovered. The V404I mutationin Rgt2 appears to abolish glucose signaling; this residue isorthologous to one in Hxt1 (F371) that is necessary for glucosetransport (247). V404 in Rgt2 may therefore be required forglucose sensing because it helps to form the glucose bindingdomain in the receptor. Similarly, dominant mutations in bothSnf3 (R229K) and Rgt2 (R231K) that cause constitutive sig-naling, even in the absence of glucose, map to the putativeglucose binding pocket (272, 273). Progress has also beenmade with respect to the elaboration of a mechanism for sub-sequent signaling by Snf3 and Rgt2 across the plasma mem-brane (see below).

SIGNAL RECEPTION: GLUCOSE REPRESSION

Direct sensing of extracellular glucose by yeast cells, ratherthan intracellular sensing of its metabolic products, is a rela-

tively new concept. Further study of receptor-mediated signal-ing in yeast may prove critical in understanding similar nutrientsensors in mammalian cells (179). How PKA, once unleashed,remodels the architecture of the yeast nucleus to produce theultimate transcriptomic consequences of glucose induction andrepression will be a central consideration of the remainder ofthis review.

Most of the �1,000 genes in S. cerevisiae that are transcrip-tionally downshifted during growth on glucose can be sortedinto one of three groups: gluconeogenic genes, Krebs cycle/respiratory genes, and genes involved in alternate carbon sourcemetabolism. This section will review known mechanisms throughwhich the glucose signal is received in the yeast nucleus andmodulates expression of glucose-repressed target genes.

Involvement of the Hexokinase Hxk2in the Glucose Response

The first, irreversible step in the intracellular metabolism ofglucose is C6 phosphorylation. Three enzymes (encoded byHXK1, HXK2, and GLK1) can catalyze this reaction; of these,only HXK2 is highly expressed in the presence of glucose (147).HXK2 lesions (both point and null mutations) were long agoshown to cause failures in glucose repression (99, 100, 104,222). After much controversy, it is now deemed unlikely thatHxk2 catalytic activity plays an important role in the intracel-lular glucose signaling pathway (244). The surviving versionof this model postulates that, following the onset of thephosphoryl transfer reaction, a stable transition intermediatealters Hxk2 conformation and mediates its regulatory functionin altering target gene expression (20, 191). However, thismodel fails to explain the fact that sugars metabolized in anHxk2-independent fashion can cause genome-wide transcrip-tional repression comparable to that observed after the addi-tion of glucose to wild-type cells or upon expression of acti-vated Ras or Gpa2 (17, 214, 312, 397, 408).

Interestingly, approximately 14% of Hxk2 is nuclear in glu-cose-grown cells (146). This discovery suggests an alternativeexplanation for the regulatory involvement of this hexokinasein the glucose response (244, 304). Hxk2 has been detected inthe nucleus by enzymatic assay, immunoblotting, and fluores-cent visualization of a green fluorescent protein (GFP)-taggedchimera, confirming controversial reports of nuclear hexoki-nase that first appeared in the early 1960s (234, 340, 390). Thenegatively acting DNA-bound glucose regulator Mig1 (see be-low) appears to mediate nuclear localization of Hxk2; Mig1and Hxk2 have been shown to interact via two-hybrid, immu-noprecipitation, and glutathione S-transferase pull-down as-says (2, 245). An interaction between Hxk2 and the Mediatorcomponent Med8 has also been described, and the latter ap-pears to bind directly to both positively and negatively actingglucose regulatory elements in DNA (57, 80, 246). A previouslyreported example of a metabolic enzyme that doubles as atranscriptional regulator is the galactokinase Gal1, which bothphosphorylates galactose and appears to activate the transcrip-tion factor Gal4 by binding to the Gal4 inhibitor Gal80 (436).

Despite these intriguing hints, contradictory results preventan unambiguous assignment of the Hxk2 function in glucoseregulation to the nuclear form of the protein. For example,removal of a small N-terminal segment of Hxk2 (residues 7



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through 16) in a putative nuclear localization sequence (Lys8-Pro-Gln-Ala-Arg12) has been reported to abolish both Hxk2nuclear localization and glucose repression of SUC2, HXK1,and GLK1 without impairing hexokinase catalytic activity (146,311). The exact opposite result (intact glucose signaling andlow catalytic activity), however, was obtained by others upondeletion of an overlapping and slightly larger Hxk2 segment(residues 1 through 15) (231). Likewise, the phosphorylatableSer14 residue in Hxk2 (193) is reported either to mediate (305)or not to mediate (146, 231) glucose repression. Further anal-ysis is needed to resolve these discrepancies. Most importantly,proof of a regulatory role for nuclear Hxk2 awaits an unam-biguous demonstration that its role in the glucose responserelies upon the relatively small fraction of the protein thatresides in the nucleus.

Snf1-Mediated Signal Transduction in GlucoseRepression and Derepression

The uncertainty surrounding the role of Hxk2 subcellularlocalization and phosphorylation state in the glucose responseis particularly important to resolve because it represents one ofseveral potential connections between Ras/cAMP/PKA-depen-dent events that occur early in the signal transduction cascadeand downstream modifications that have a direct impact ontarget gene expression. Importantly, increasing the extracellu-lar glucose concentration, or hyperactivation of PKA via bcy1deletion, results in reduced Hxk2 phosphorylation, whereasattenuation of PKA activity increases 32P labeling of Hxk2(398). These data are consistent with a model in which Hxk2dephosphorylation, which converts it from a monomer to adimer (305), somehow transduces the glucose signal to down-stream effectors, eventually resulting in repression (and per-haps also induction) of glucose-regulated genes. The nuclearform of Hxk2 might accomplish this via its direct interactionwith Med8 and/or Mig1 (see above). Alternatively or addition-ally, such an Hxk2 signal might be received by two additionalphosphoproteins that are well established as downstream com-ponents in the glucose signal transduction pathway: the Reg1-targeting subunit of the Glc7 phosphatase and the Snf1 kinase(325).

Dueling Snf1 kinase and Glc7 phosphatase mediate glucoseregulation. SNF1 was first identified in screens for regulatoryfactors in the glucose response (it was then named either CAT1or CCR1) (62, 102, 440). SNF1 was also identified in a screenfor 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 itsidentification it was recognized as an important regulatorygene mediating glucose derepression. The SNF1 gene wascloned and found to encode a serine-threonine protein kinase(51); the existing evidence also suggested a physical and func-tional link to SNF4, another gene that had been identified inthe “sucrose nonfermenting” screen (49, 50, 259, 261). The snf4and snf1 phenotypes are similar, including defective utilizationof the many carbon sources (e.g., sucrose, galactose, maltose,raffinose, and lactate) that require expression of glucose-re-pressed genes. Increased SNF1 dosage and SNF1 mutations(e.g., the SNF1-G53R allele, which produces a hyperactivekinase) can partially restore glucose derepression in the ab-

sence of SNF4 (53, 108, 204). Since Snf4 also coimmunopre-cipitates with Snf1 and is required for maximal Snf1 proteinkinase activity (in cells grown on a nonfermentable carbonsource) (52), it is a physically associated activator of the Snf1kinase (108, 113). Like Glc7 (and perhaps Reg1; see below),Snf1 appears to have a regulatory role in glycogen metabolismthat is somehow influenced by PKA signaling (see reference119 for a review). While this apparent connection between thesignal transduction pathways governing glucose catabolism andglycogen storage is intriguing, it remains poorly defined andwill not be considered further here.

Identification of a role in glucose repression for Glc7, theyeast homolog of mammalian type I protein phosphatase (PP1)(111), began with a selection for mutants defective in glucoserepression of a target gene (SUC2, encoding invertase) (260).All of the recovered mutations were recessive; two of the threecomplementation groups found in this screen proved to beallelic with HXK2 and REG1, respectively. The third groupidentified a new locus (designated cid1, for constitutive inver-tase derepression). Complementation of one of these muta-tions (cid1-226, now designated glc7-T152K) (378) led to theisolation of GLC7, a locus that was also identified indepen-dently in a screen for glycogen-deficient mutants. Interestingly,in addition to SNF1 (GLC2), other glc lesions were allelic withIRA1 (GLC1), IRA2 (GLC4), and RAS2 (GLC5) (42), consis-tent with numerous studies connecting Ras and Gpa2 signalingwith accumulation of the storage carbohydrates trehalose andglycogen (40, 41, 67, 125, 190, 284, 330, 363). Further investi-gation is needed to pursue this implicit regulatory linkagebetween glucose catabolic pathways and carbohydrate storage.

Complicating matters further, in addition to glucose repres-sion and carbohydrate accumulation, GLC7 phosphatase isneeded for a variety of other cellular functions in S. cerevisiae(14, 350, 437), including normal progression through the G2/Mphase of the cell cycle (23, 24, 150, 159, 223, 288), actin orga-nization (6, 54), translation (412), and sporulation (12, 42, 302,357, 378, 380). Recent work has further expanded this list offunctions assigned to yeast PP1; it is also required for theglucose-induced vacuolar degradation of fructose-1,6-bisphos-phatase (76) and for normal Mex67-mediated export of mRNAfrom the nucleus to the cytoplasm (127; see also references381, 382, and 404).

Interestingly, isolated PP1 molecules exhibit little substratespecificity and instead require regulatory subunits that alter theconformation of the phosphatase and/or target it to its sub-strates (65, 97). This targeting hypothesis is supported by theGlc7 localization pattern, which suggests dynamic changesthroughout the yeast cell cycle (24). Glc7 binding proteinsspecific to the following functions have been reported: glucoserepression (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 interactionwith a hydrophobic groove at the interface of two �-sheets inPP1 (97, 403). Thus, multiple regulatory subunits appear tocompete for Glc7 binding in vivo; for example, Gac1 overex-pression affects glucose repression (426). In addition to bind-ing regulatory subunits, the hydrophobic groove may mediate



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changes in the structure and/or activity of the phosphatase. Italso now seems likely that many if not all targeting subunitsinteract with more than one site on the PP1 surface (425). Inthe two-hybrid assay the F468R mutation in Reg1 dramaticallyreduces its interaction with Glc7; the PP1 binding motif thatcontains this residue (RHIHF468) therefore appears to be oneof the moieties required for formation of the Reg1/Glc7 com-plex (325). Importantly, Reg1 is the only regulatory subunitknown to participate in glucose repression.

The idea that Reg1 (first identified in reference 230 and inan earlier screen as Hex2 [101]) mediates the Glc7 role inglucose repression arose because (i) reg1 and glc7 alleles wereboth isolated in the screen for mutants with impaired repres-sion of SUC2 (260, 378) (see above), (b) a combination ofthose defective alleles exhibited no synergy in relief from glu-cose repression of SUC2, (iii) SNF1 deletion is epistatic to bothalleles, and (iv) Reg1 overexpression suppresses the repressiondefect caused by glc7-T152K (379). Genetic and biochemicalexperiments confirmed that Reg1 and Glc7 are linked bothphysically and functionally (325, 379). The results of alanine-scanning mutagenesis suggested that Reg1 interacts with atleast two distinct areas of the resolved PP1 structure (14).Satisfyingly, one of the Glc7 surfaces specific to its role inglucose repression is adjacent to Thr152 (14); the glc7-T152Klesion had earlier been shown to impair Reg1-Glc7 interaction(379).

A functional link between Reg1 and Snf1 was suspected longago, primarily because REG1 deletion results in derepressionof glucose-repressed genes and SNF1 deletion results in afailure to derepress many (if not all) of those same genes. Thedemonstration that Reg1 and Snf1 interact (218) left littledoubt that the Reg1-targeted Glc7 phosphatase and Snf4-activated Snf1 kinase are indeed interconnected compo-nents of the glucose signaling pathway (106, 120, 162, 174,378, 379; see references 45, 121, and 178 for reviews) (Fig. 3).Since then numerous additional participants in this regulatoryduel have been discovered. For example, three ancillary com-ponents of the Snf1/Snf4 kinase complex (Sip1, Sip2, and Gal83)strengthen Snf1/Snf4 association (175, 429, 430). Each of thesethree proteins contains an Snf1 binding internal region and anSnf4 binding C-terminal region (their N-terminal regions aredivergent); they therefore appear to bridge the interactionbetween Snf1 and Snf4. By analogy with the heterotrimericmammalian Snf1 homolog, the AMP-activated protein kinase,SNF1 encodes the yeast � subunit, SNF4 encodes the � subunit,and SIP1, SIP2, and GAL83 redundantly encode the � subunit(136).

It was initially puzzling that little or no detectable phenotyperesulted from mutation of all three genes that encode the �subunits of the Snf1 kinase complex (105, 106, 121, 175). How-ever, the construction of true null alleles revealed that, asexpected, the sip1� sip2� gal83� triple mutant does indeeddisplay an snf1 phenotype (331). Despite the observed redun-dancy, the three � subunits play distinct roles in vivo, in par-ticular with respect to Snf1 substrate specificity (143, 144, 175,213, 254, 331, 396, 397, 430).

Besides its role in the response to glucose limitation, Snf1has also been implicated in several other processes, includingaging, thermotolerance, peroxisome biogenesis, filamentation,invasive growth, biofilm formation, meiosis, and (like Glc7)

sporulation (8, 45, 137, 196, 197, 297, 401). Clearly more workis needed before we will fully understand the respective rela-tionships between the distinct forms of the Snf1 kinase com-plex and each of these regulatory mechanisms. However,Gal83 is likely to be a primary contributor to glucose regula-tion by the Snf1 complex, since Gal83 is the most abundant �subunit, it is the only � subunit in the nucleus when cells aregrown on a nonfermentable carbon source, and Snf1/Gal83 isthe form responsible for most Snf1 kinase activity (144, 397).

Another trio of factors that mediate Snf1 function are theredundantly acting Snf1 kinase kinases Sak1 (YER129W, for-merly known as Pak1; the new name was chosen to avoidconfusion with Prk1 or p21-activated kinases), Tos1, and Elm1,each of which shares sequence similarity with the human tumorsuppressor and AMP-activated protein kinase LKB1 (155, 255,354, 422). Removal of all three yeast activities confers an snf1phenotype. A one-to-one correspondence between these threeupstream kinases and the distinct forms that contain each ofthe three � subunits has been ruled out (143). Since Sak1 is themost important of the trio of Snf1 kinase kinases in activatingthe Snf1/Gal83 form, it is likely also to be the most importantregulator of Snf1-dependent glucose regulation (143). Thisconclusion is further supported by the observation that dele-tion of SAK1 suppresses many of the Snf1-dependent pheno-types observed in reg1� cells (255).

Like Glc7 and its regulatory subunits, the Snf1 kinase ap-pears to be localized by its � subunits to distinct cellular sub-compartments, thereby targeting it to different substrates (331,397). In glucose-grown cells Snf1 is found exclusively in thecytoplasm; following a shift to a nonfermentable carbonsource, Snf1 is targeted by Gal83 to the nucleus. The N termi-nus of Gal83, which is not shared by the other two � subunitsSip1 and Sip2, is required for this function; the upstream ki-nase Sak1 also controls Snf1 localization to the nucleus in theabsence of glucose (144, 397). Upstream events in glucosesignaling may also be involved, since it has been reported thatPKA regulates nucleocytoplasmic shuttling of Snf1/Sip1 (144).If PKA also regulates shuttling of Snf1/Gal83, it appears to doso redundantly with another signaling pathway.

With respect to subnuclear localization, three as-yet-uncon-nected observations suggest the possibility that the Snf1 kinasecomplex operates at least in part at the nuclear periphery: Snf1is concentrated in nuclear envelope fractions prepared frompyruvate-grown (but not glucose-grown) cells (B. Menon andG. M. Santangelo, unpublished data), Glc7 has been impli-cated in mRNA processing as a component of the cleavage/polyadenylation complex (127, 256, 404), and Reg1 deletioncauses an RNA processing defect (381, 382). Further elabora-tion of this potential connection with perinuclear phenomenais needed and may provide critical information about the func-tion and organization of the regulatory apparatus in the yeastnucleus that responds to glucose (see below).

An overview of the regulatory duel between the Reg1/Glc7phosphatase and the Snf1 kinase complex is presented in Fig.3. Snf1 has two domains, an N-terminal kinase domain and aC-terminal regulatory domain. In high concentrations of glu-cose the regulatory domain remains bound to the catalyticdomain, 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



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regulatory domain; deletion of the latter bypasses the require-ment 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 theactivation loop of Snf1 is essential for activation of the kinase;the upstream kinase Sak1 has been shown to phosphorylatethis residue and is required for full Snf1 kinase activity (255).Blocking phosphorylation at this position via a T210A muta-tion impairs Snf4 binding to the regulatory domain and pre-vents the release of Snf1 autoinhibition (174, 198, 218). TheT210A lesion also interferes with normal nuclear enrichmentof Snf1 after a shift from high to low glucose (144). Since Snf4

localization to the nucleus is essentially carbon source inde-pendent, in glucose-grown cells nuclear Snf4 is apparently notassociated with other subunits of the Snf1 kinase complex(397). Thus, Snf4 apparently does not contribute to the cyto-plasmic roles of Snf1 that are distinct from its mediation ofglucose derepression.

Glucose repression involves Reg1/Glc7-facilitated conver-sion of the kinase complex from the active to the autoinhibitedstate, 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) isrequired for the interaction with Reg1 (218). Upon activationin a reg1 or glc7-T152K background, the Snf1 complex becomes

FIG. 3. The opposing roles of Reg1/Glc7 phosphatase and the Snf1 kinase complex. The duel between Reg1/Glc7 and Snf1/Snf4/Gal83 turnsthe Snf1 kinase on (ACTIVE) and off (INACTIVE) by determining whether the Snf1 regulatory domain (RD) binds to and autoinhibits its kinasedomain (KD). This switch plays a central role in determining the transcriptomic response to the presence or absence of glucose. The � subunitGal83 acts as a scaffolding factor for the nuclear form of the Snf1 (�) and Snf4 (�) subunits of the kinase. Addition of glucose to yeast cells growingon an alternative carbon source results in the dephosphorylation of active Snf1 kinase by Reg1/Glc7 (no. 1); this returns Snf1 to the autoinhibitedstate (no. 2) and results in export of Snf1/Gal83 to the cytoplasm (not shown). Once glucose is depleted, Sak1 phosphorylates Snf1 (no. 3),Snf1/Gal83 enters the nucleus (not shown), and Snf4 binds to the Snf1 regulatory domain, thereby releasing the catalytic domain (no. 4); Reg1 thenundergoes rapid Snf1-dependent phosphorylation (no. 6), which stabilizes the interaction between Snf1 and the Reg1/Glc7 phosphatase (no. 7) andprimes the latter to repress Snf1 anew should glucose reappear. Std1 interacts with the Snf1 catalytic domain and stimulates kinase activity (no.5), although it is stoichiometrically underrepresented in Snf1 complexes. A dynamic equilibrium (purple arrows between no. 6 and 7), in which Glc7appears to counteract Snf1 phosphorylation of Reg1 and promote its own dissociation from active kinase complexes, is at the heart of thisregulatory duel. Sip5 may stabilize the Reg1/Snf1 interaction in no. 6 (not shown). Hxk2 interacts with Reg1 (not shown), and Hxk2 may respondto PKA signaling by dimerizing and mediating the switch in Glc7 phosphatase substrate selection (from Reg1 [no. 7] to Snf1 [no. 1]; see the text).



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trapped in the active conformation. Overexpression of Reg1,but not overexpression of the mutated form that fails to bindGlc7 (ReglF468R; see above), interferes with the Snf1/Snf4 in-teraction in low glucose (325). Sip5, the first protein shown tobind to both Reg1/Glc7 and the Snf1 kinase complex, maystabilize the interaction between them and thereby facilitateglucose repression, since the two-hybrid interaction betweenReg1 and Snf1 is reduced threefold in a sip5� mutant (326).

The critical regulatory interaction between Reg1 and theSnf1 complex was presumed to occur in the nucleus, especiallysince that is where active Snf1 is predominantly located priorto being switched off and exported following the appearance ofglucose, and Glc7 is known to be largely nuclear in growingcells (24, 437). However, in contrast with a previous report thatfound Reg1 in the nucleus (265), fluorescently tagged Reg1appears to be exclusively cytoplasmic (90, 91). To resolve thisdiscrepancy it was proposed that the activated form of Snf1cycles rapidly between the nuclear and cytoplasmic compart-ments, allowing inactivation by Reg1/Glc7 to occur in the cy-toplasm following the appearance of glucose (91). This hypoth-esis has not yet been tested, but it may explain the apparentrole of Bmh1 and Bmh2 in glucose-regulated gene expression(168). Bmh1 and Bmh2 are the yeast homologs of the some-what mysterious 14-3-3 proteins (33, 388), which may serve asmediators of Reg1 function (90) and are known to respond toRas/PKA signaling (124, 232).

Additional information concerning the connection betweenupstream glucose signaling events and the Glc7/Snf1 regula-tory duel will be the next topic of discussion, followed by areview of the downstream DNA-bound phosphoproteins thatare known Snf1 regulatory targets and that account for muchof the transcriptomic response to glucose in yeast cells.

Transmission of the Ras/cAMP-dependent glucose signal toReg1/Glc7 and Snf1/Snf4. Although the regulatory duel be-tween Snf1 kinase and Glc7 phosphatase has now largely beenexplained at a molecular level, the input by glucose signalingthat shifts the balance in favor of the closed, inactive form ofthe kinase remains poorly characterized. A proposal that Snf1responds to the AMP/ATP ratio (421), which drops dramati-cally upon removal of glucose (7, 421), has been difficult tosubstantiate, since Snf1 kinase activity does not respond tothese nucleotides in vitro (136). In contrast, the ample evi-dence that PKA and Snf1 act antagonistically in affecting asimilar set of cellular functions (7, 164, 366) is reinforced bythe similarity of the transcriptome-wide response of Snf1 targetgenes following either the addition of glucose or the expressionof activated G proteins in the absence of glucose (408).

Sak1, the recently identified upstream activator of Snf1 thatregulates both Snf1 kinase activity and its nuclear localization(144, 255), is an obvious potential intermediary between PKAand Snf1 (question mark in Fig. 3). If the gap between theearliest events in glucose signaling and Sak1 could be bridged,it would represent a complete chain of causality between glu-cose sensing at the cellular periphery and the response ofDNA-bound regulators in the nucleus. Not surprisingly, how-ever, given that its role as an Snf1 activator was only recentlyuncovered, little is yet known about regulation of Sak1 andwhether or not the PKA pathway might be involved.

Equally mysterious is the apparent capacity of the glucosesignal to change the target of Glc7 phosphatase from Reg1to Snf1 (no. 7 and 1 in Fig. 3, respectively), which in theabsence of Snf1 phosphorylation (no. 3) traps Snf1 in theautoinhibited inactive state. Interestingly, PKA signal trans-duction might alter Glc7 targeting through the negativelyacting regulator Hxk2. As mentioned above, PKA signalingappears to cause reduced phosphorylation of Hxk2, whichfavors its dimeric form; the phosphatase that dephosphory-lates Hxk2 in response to glucose is known to be Reg1/Glc7(4, 305, 398). Hxk2 (presumably its underphosphorylateddimeric form) in turn interferes with dephosphorylation ofReg1 by Glc7 (325). Since only the phosphorylated form ofReg1 promotes dissociation of Snf4 from Snf1 and inactiva-tion of the kinase, the PKA-mediated transition from mo-nomeric to dimeric Hxk2 is potentially a direct glucose trig-ger that stabilizes the repressing form of Reg1/Glc7 andthereby inactivates Snf1. Reg1 and Hxk2 were reported tointeract as predicted by this model (325). This is furthersupported by a wealth of additional data: Reg1 overexpres-sion suppresses the glucose repression defect of an hxk2�mutant (325), the association between Reg1 and Glc7 isunaffected by removal of Hxk2, and the Snf1-Snf4 interac-tion is glucose insensitive in the absence of Hxk2 (174).

A near-complete chain of causality describing the glucoseresponse can thus be stated as follows (Fig. 3): early eventsin glucose signaling stimulate PKA to inhibit phosphoryla-tion of Hxk2, thereby targeting it to Reg1/Glc7, where Hxk2is dephosphorylated, dimerizes, and blocks dephosphoryla-tion of Reg1 by Glc7; Glc7 then switches targets and inac-tivates the Snf1 kinase, which can no longer phosphorylateits DNA-bound substrates, resulting in altered expression ofthe relevant target genes. Ambiguity unfortunately accom-panies even this parsimonious view of the glucose regulatorycascade, since the relationship between subcellular distribu-tion and function awaits clarification for several of the fac-tors involved (most notably Hxk2 and Reg1).

DNA-bound repressors controlled by Snf1. Several down-stream repressors are known to be phosphorylated and inacti-vated by the Snf1 kinase in the absence of glucose. The first ofthese to be discovered was Mig1, originally identified as amulticopy inhibitor of GAL gene expression (257). Deletion ofthe MIG1 gene relieves glucose repression of numerous targetgenes (115, 131, 180, 189, 257, 258, 335, 336, 406), and itsproduct is thought to interact with the intergenic regions of atleast 90 different genes in vivo (221, 251). Mig1 is presumed tofunction downstream of Snf1, because deletion of MIG1 sup-presses snf1 defects in glucose derepression (180, 386).

Mig1 is a Cys2His2 zinc finger protein that binds to a GC-richcore sequence (219). The consensus Mig1 binding site deter-mined by in vitro selection is 5�-ATAAAATGCGGGGAA-3�(221). At the time of its discovery, other zinc finger proteinswere known to be capable of functioning as either activators orrepressors, depending upon the chromosomal and cellularcontext (207). This was therefore also considered a possibilityfor Mig1 (257), a suggestion that later proved to be correct:both Mig1 and the related Cys2His2 zinc finger-containingrepressor Mig3 (Yer028) can also function as activators (220,



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221, 303, 376, 423). Although this has complicated theinterpretation of Mig1 function (37, 160, 189, 376, 384, 386,423), clearly the primary physiological role of Mig1 seems to bethat of a negative regulator of glucose-repressed genes (e.g.,SUC2 and GAL1).

Interestingly, the Mig1 N-terminal zinc fingers are very sim-ilar to those in the C terminus of WT1 (257), a human tumorsuppressor that collaborates with p53 to repress transcriptionand (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 impor-tant contribution to its repressor function (323); the signifi-cance of a potential PKA target site found in the internalregulatory region of Mig1 is not yet known (257, 270).

Glucose-dependent repression by a LexA-Mig1 fusion isreadily detected by a heterologous reporter gene with up-stream LexA operator sites, suggesting that Mig1 can represstranscription without the assistance of other promoter-boundfactors (376, 377). Analysis of LexA-Mig1 repression also ledto the discovery that the Mig1 polypeptide, which has a pre-dicted molecular mass of 55 kDa (504 residues), is phosphor-ylated to different extents in glucose-repressed and dere-pressed 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 forglucose regulation (77, 87, 269). All three of these sites, and afourth one that matches the Snf1 consensus imperfectly(Ser222), are phosphorylated by Snf1 in vitro (343). Mutationof these serine residues to alanine, including a Mig1 mutantin which all four residues were mutated (S222A�S278A�S311A�S381A), impaired but did not eliminate Mig1 phos-phorylation and glucose repression (377). Nevertheless, in vivophosphorylation of Mig1 is dramatically reduced in an snf1mutant, and immunoprecipitated Snf1 (but not the kinase-dead Snf1-K84R) phosphorylates coprecipitated Mig1. Also,as expected based upon their capacity to mediate glucose re-pression by inhibiting Snf1 kinase activity, deletion of eitherREG1 or HXK2 results in Mig1 hyperphosphorylation underrepressing conditions (377). Taken together, the simplest in-terpretation of these data is that phosphorylation of Mig1 bySnf1 is at least partly responsible for inactivation of Mig1repressor function.

Deletions that map within an internal region containingmost of the Snf1 phosphorylation sites (residues 261 to 400)converts Mig1 into a constitutive repressor that is not inacti-vated when glucose is depleted (270). Interestingly, deletionsin Mig1 that remove all or part of the internal region fromresidue 261 to 400 cause it to persist in the nucleus in theabsence of glucose, whereas under the same conditions wild-type Mig1 is largely exported to the cytoplasm (87). Fusion ofMig1 residues 261 to 400 to a GFP–�-galactosidase chimeraconfers glucose-regulated nuclear import and export upon theresulting fusion protein, and its import in the presence ofglucose requires both REG1 and HXK2. In glycerol-grown cellsMig1 becomes increasingly nuclear as more Snf1 kinase sitesare removed (86). Msn5, an importin � homolog originallyidentified due to its suppression of an snf1 mutation whenoverexpressed (107), interacts with Mig1 and mediates its nu-

clear export in response to glucose removal (86). These andother data suggest that Snf1 phosphorylation of Mig1 inacti-vates it by changing its subcellular localization.

According to this Mig1 nucleocytoplasmic shuttling hypoth-esis, Hxk2- and Reg1/Glc7-dependent inactivation of Snf1 inthe presence of glucose results in hypophosphorylation of Mig1,which causes it to relocate rapidly to the nucleus; subsequentbinding 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 withtwo strikingly contrasting results. First, the Mig1 target geneGAL1 is derepressed normally in an msn5 mutant grown onglycerol, despite the constitutive presence of Mig1 in the nu-cleus (86). Second, Mig1 remains bound to the GAL1 pro-moter in vivo under these same derepressing conditions (278).Both of these results suggest that Mig1 transcriptional repres-sion of target genes is regulated via a mechanism other thannuclear export and that the key function of Snf1 phosphoryla-tion in relieving Mig1-dependent glucose repression is to in-activate the relatively small amount of Mig1 that remains in thenucleus and is bound upstream of target genes in derepressedcells (87, 270). How this is accomplished remains obscure.

Mig3, Nrg1, and Nrg2 are three additional Snf1-regulatedrepressors in S. cerevisiae. Mig3 is somewhat mysterious; it wasidentified because it contains Cys2His2 zinc fingers that arevery similar to those of Mig1 and Mig2 (26, 220). Severaladditional results seem to suggest that Mig3, like Mig1 but notMig2, is an Snf1-controlled, DNA-bound glucose regulator(182, 221). It binds to the Mig1 binding sites upstream fromSUC2, and a LexA-Mig3 chimera functions as a glucose-de-pendent repressor of reporter gene expression when bound toupstream LexA operators. When cells are shifted from glucoseto galactose, Mig3 is extensively phosphorylated (at least inpart by Snf1) and rapidly subjected to Snf1-dependent prote-olysis (94). Nevertheless, physiologically significant regulationof target genes by Mig3 is yet to be demonstrated; in wild-typecells under standard conditions, it seems unlikely to be morethan a minor regulator of glucose-responsive gene expression(220, 221). Recently the MIG3 gene was also isolated in ascreen for multicopy suppressors of Rad53-GFP toxicity, butthe relationship between Mig3 function and the DNA damageresponse awaits further clarification (94). It is hoped that tran-scriptomic analysis of the effects of an as-yet-untested environ-mental condition or genetic background will eventually revealthe Mig3 role in glucose regulation and explain the resultsobtained to date.

Nrg1 and Nrg2 contain Cys2His2 zinc fingers that are verysimilar both to each other and to those in Mig1, Mig2, andMig3 (400). Nrg1 was first identified in two separate screensfor factors mediating repression of glucose-regulated genes (1,282), and Nrg2 was discovered because of its two-hybrid inter-action with Snf1 (400). Outside of their DNA binding domainsNrg1 and Nrg2 share only 27% identity, and this is thought toaccount for their somewhat distinct regulatory roles in glucoserepression, filamentous invasive growth, and biofilm formation(18, 196, 282, 400, 438). In the absence of glucose, neitherfactor is degraded, nor is their capacity to bind target sites invivo impaired. Both Nrg1 and Nrg2 exhibit glucose-dependentrepression of a heterologous reporter gene; they are also bothcapable of interacting with Snf1 kinase, but (unlike Mig1 and



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Mig3) neither appears to be phosphorylated by it (18, 400).Although Snf1 appears to modulate Nrg2 levels and is clearlyrequired for normal Nrg1 function, the regulatory relationshipbetween these factors is complex and in need of further study.Interestingly, like Mig1 and Mig3 (see above), Nrg1 can func-tion as an activator in some circumstances (18).

DNA-bound activators controlled by Snf1. The Snf1 proteinkinase also regulates a number of positively acting transcrip-tion factors required for the utilization of nonfermentable car-bon sources. Two of these factors, Cat8 and Sip4, both containzinc finger DNA binding domains similar to that in Gal4 (a C6zinc cluster) (145, 206) and bind specifically to a set of carbonsource responsive elements (CSRE) under derepressing con-ditions (300, 395). Cat8 and Sip4 are closely related structurallyand appear to have slightly different affinities for variants of theCSRE sequence (319). Although Sip4-dependent transcrip-tional activation has been shown to require phosphorylation ofSip4 by Snf1 kinase, removal of Sip4 has little or no effect onCSRE-regulated genes (141, 145, 206, 299). Also, unlike dele-tion of CAT8, which causes defective growth on nonferment-able carbon sources, deletion of SIP4 results in no obviousphenotype (206).

Cat8 regulates the expression of a wide array of genes in-volved in the metabolism of nonfermentable carbon sources,including genes necessary for ethanol utilization (e.g., ACS1and ACR1/YJR095W) (28, 192), gluconeogenesis (141, 145),the glyoxylate cycle (141), lactate utilization (JEN1) (27), andisocitrate metabolism (IDP2) (27); Cat8 also regulates expres-sion of SIP4 (141, 395). Cat8 activity is regulated on two levels:its expression is Mig1 repressed (145, 257), and its capacity toactivate 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 aGlc7-independent manner (303). Interestingly, constitutivelyexpressed Cat8 fused to the Ino2 activation domain bypassesthe need for a functional Snf1 in cells growing on ethanol(300).

Cat8 works together with another Snf1-regulated transcrip-tional activator, Adr1, for maximal expression of a small groupof shared target genes (including ADH2, ACS1, and ALD6)under derepressing conditions (25, 98, 192, 356, 405, 432). Likethe Mig1, Mig3, Nrg1, and Nrg2 repressors, Adr1 has aCys2His2-type DNA binding domain (283), and its functionrequires Snf1 kinase (84), although the mechanism of Adr1regulation remains unclear. It has been suggested that Snf1and Reg1/Glc7 contribute to regulation of Adr1 by affecting itsbinding to promoters of target genes. Snf1 promotes Adr1binding under derepressing conditions, while Reg1/Glc7 inhib-its Adr1 binding to promoters in the presence of glucose (91,433); it is currently unknown whether Adr1 is a direct target ofSnf1 and Reg1/Glc7 or whether this effect occurs throughintermediary factors. Adr1 activity has also been reported to benegatively regulated by PKA (60) in a manner independent ofits DNA binding (364).

SIGNAL RECEPTION: GLUCOSE INDUCTION

The connection between PKA function and up-regulation ofthe �1,000 glucose-induced genes is even more mysteriousthan 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/Rgt2sensing mechanism, a pathway that appears to operate in alargely PKA-independent fashion and (at minimum) contrib-utes the critical regulatory trim that allows S. cerevisiae to growwell on a broad range of glucose concentrations (from micro-molar to molar concentrations) (272). The integral plasmamembrane proteins Snf3 and Rgt2 mediate both transcrip-tional repression and derepression of four of the genes thatencode glucose transporters (HXT1 to HXT4). This pathwayestablishes a regulatory link between glucose concentrationand expression of the transporters, and it operates in a largelySnf1-independent fashion (for reviews, see references 179 and272). Snf3 and Rgt2 are putative glucose sensors that appear toact redundantly with the Ras2/Gpa2/PKA pathway, since thelatter seems sufficient for glucose signaling through both theSnf1-dependent and Snf1-independent branches (408) (seeabove). Interestingly, however, like activating mutations inRAS2 and GPA2, at least part of the glucose signal can be atleast partially mimicked in the absence of glucose via dominantmutations in SNF3 or RGT2 (228, 272, 273). This may meanthat the Snf3/Rgt2 regulatory pathway, in addition to modu-lating expression of key glucose transporters, signals PKA or aPKA-independent glucose response pathway and thus aug-ments or tailors the transcriptomic response beyond its well-established effect on transporter genes.

Snf3/Rgt2 Signaling of the Rgt1 Repressor Complex

Upon transmission to the cytoplasmic side of the plasmamembrane, the Snf3/Rgt2-mediated glucose signal appears tooperate through YCK1 and its 77% identical paralog, YCK2, anessential gene pair encoding casein kinase (Fig. 4) (310). LikeRas (see above), Yck1 and Yck2 are tethered to the membranevia palmitate moieties attached to C-terminal Cys-Cys se-quences (9, 318). Membrane anchoring of the Yck proteins isthought to facilitate an interaction with Snf3 and Rgt2 thatactivates the kinases following the glucose-induced conforma-tion change in the sensors; Yck1 has been shown to interactwith Rgt2 in vivo (179, 247). The targets of activated Yckkinases in this pathway are Std1 and Mth1 (247), the corepres-sors that interact with the Rgt1 repressor; the latter is a C6 zinccluster protein that binds directly to DNA sites upstream fromtarget genes (122, 163, 202, 272, 289, 332). Mutations in Rgt1restore HXT gene expression and were originally isolated viasuppression of defective glucose regulation in the snf3� andgrr1� backgrounds (106, 227). Note that the Rgt1 repressorblocks transcription of glucose-induced genes, unlike the Migand Nrg repressors, which block transcription of glucose-re-pressed genes (see above). Thus, the Mig and Nrg proteins actas repressors only in the presence of glucose, and Rgt1 acts asa repressor only when glucose is absent (Fig. 4).

The large (�200-residue) cytoplasmic C-terminal tails ofSnf3 and Rgt2 are an important feature of this signaling path-way. Deletion of the tail domains impairs Snf3- and Rgt2-dependent gene regulation in response to glucose, and fusingthe tail to Hxt1 or Hxt2 complements those defects, as doesexpression of the Snf3 tail domain by itself (69, 89, 228, 247,272, 274). Although they were originally thought to play animportant role in receiving the glucose signal, recent evidence



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suggests instead that a dynamic interaction with Mth1 and Std1(201, 332) is the important function of the Snf3 and Rgt2C-terminal tails in glucose signaling (247).

The membrane anchoring of Mth1 and Std1 by the cytoplas-mic tails is thought to mediate phosphorylation of these Rgt1corepressors by Snf3/Rgt2-associated Yck kinases (Fig. 4)(247). Both Std1 and Mth1 can be phosphorylated by affinity-purified Yck1 in vitro, and membrane fractions of crude ex-tracts yield maximal Mth1 phosphorylation; mutant forms ofYck kinase were used to demonstrate that the production ofphosphoforms of these regulators is Yck dependent (247). Thelikely site of Yck phosphorylation is a region conserved in bothStd1 (residues 129 to 147) and Mth1 (residues 118 to 136),each of which contains several matches with the consensustarget sequence of casein kinase I (SXXS) (247).

Once Std1 and Mth1 are phosphorylated by the Yck kinases,they are degraded via a Grr1-dependent mechanism (Fig. 4)

(117, 179, 247). Mutations in GRR1 were shown long ago toimpair the regulatory response to glucose (11) and have sincebeen associated with a broad array of phenotypes, includingcell elongation, decreased divalent cation transport, defects insporulation, and slow growth or lethality in combination withdefective amino acid biosynthetic pathways (21, 114, 158, 215,427, 431).

Interestingly, the F-box motif of Grr1, which mediates itsinteraction with Skp1 (187), may explain the pleiotropic phe-notype of grr1� cells (10, 209). Skp1 is a component of severalstructurally distinct but functionally related ubiquitin ligasecomplexes that target a wide variety of regulators to the 26Sproteasome for degradation (for a review, see reference 414).These complexes are termed SCF to indicate their two com-mon components, Skp1 and the cullin Cdc53, and a third ex-changeable component, the F-box protein. In S. cerevisiae atotal of 21 F-box proteins have been identified by sequence

FIG. 4. The Snf3/Rgt2 signaling pathway. Transcription of hexose transporter genes (HXT) is repressed by Rgt1 in the absence of glucose; incontrast, Mig1 represses transcription of its target genes in the presence of glucose and cross-regulates the Snf3/Rgt2 pathway as shown. Relieffrom the repressor function of Rgt1 occurs via glucose signaling of the Snf3/Rgt2 sensors, which stimulate phosphorylation of Mth1 and Std1 byYck kinases; the C-terminal tails of Snf3 and Rgt2 also appear to facilitate this event by interacting with both the kinases and their substrates. TheYck polypeptides are tethered to the plasma membrane via palmitate (red squiggles). Yck-phosphorylated Mth1 and Std1 are subjected toSCFGrr1-mediated ubiquitination and degradation by the proteasome. This releases Rgt1 from its upstream DNA binding sites and results inderepression of downstream HXT target genes. Removal of Mth1 and Std1 converts Rgt1 into a transcriptional activator that may stimulate HXTexpression in the absence of specific DNA binding activity.



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alignment (414). Further analysis is urgently needed, sincemost of these factors have neither been verified as SCF com-ponents nor characterized as to their function, and each bonafide SCF subunit is expected to confer a distinct substratespecificity upon its respective complex. Like Grr1, any one ofthese F-box proteins could contribute to nutrient signaling bytargeting kinase substrates in response to the appearance ordisappearance of glucose.

Two F-box proteins that target cell cycle regulators for deg-radation were first identified as a result of their important rolesin cell cycle progression: Cdc4, which targets the cyclin-depen-dent kinase inhibitor Sic1, and Grr1, which (long after itsdiscovery as a regulator of the glucose response) was found totarget the G1 cyclins Cln1 and Cln2 (16, 110, 158, 209, 341, 393,414). Grr1 was shown subsequently to also target Gic2, aCdc42 effector that mediates actin polarization at bud emer-gence (170, 414). A breakthrough in understanding the initiallypuzzling GRR1 contribution to the glucose response (11, 209,385, 387) came with the discovery that SCFGrr1, in addition totargeting the aforementioned cell cycle regulators, mediatesthe ubiquitination and degradation of the Rgt1 corepressorsStd1 and Mth1 (117, 209, 347).

Grr1-mediated ubiquitination and degradation of Mth1 andStd1 appear to require, in addition to the Skp1 interaction withthe Grr1 F box, an LRR domain comprised of 12 leucine-richrepeats that form the substrate-targeting domain in Grr1 (158,187). This is consistent with our knowledge of structure-func-tion relationships in other F-box proteins, which invariablypossess a second motif that mediates substrate targeting (414).For example, in Cdc4 a WD40 repeat domain is sufficient forhigh-affinity interaction with its substrates (253).

Degradation of Mth1 in glucose-grown cells has been shownto require both phosphorylation by Yck kinase (247) and theLRR domain of Grr1, although Grr1 residues identified asessential for Cln2 interaction appear to be distinct from thoserequired for interaction with Mth1 (117, 158, 347). No infor-mation is yet available regarding the structural requirements ofthe Grr1-Std1 interaction; this will likely be more difficult todetermine, since Std1 apparently does not disappear as com-pletely as Mth1 in the presence of glucose (182).

In the absence of glucose Rgt1 binds to a consensus sequence(5�-CGGANNA-3�) (186) found in multiple copies upstream ofthe HXT target genes (250, 275). Transcriptional repression byRgt1 requires Mth1, which causes a conformation change thatallows Rgt1 to bind to its recognition sites in DNA (117, 202,289). Std1 also interacts with Rgt1 in the absence of glucose(289, 372), although it does not regulate the Rgt1 DNA bind-ing activity (179). More work is needed to determine thecontribution of Std1 to Rgt1-mediated repression in the pres-ence of glucose; an interesting possibility is that its interactionwith Rgt1 shifts the activator/repressor equilibrium (see below)in favor of repressor function (289). Elimination of both Mth1and Std1 appears to be required for maximal derepression ofRgt1 target genes (202).

The central events in the Snf3/Rgt2 pathway, from glucosesignal generation at the plasma membrane to signal receptionby a DNA-bound repressor (179, 247), are depicted in Fig. 4.As identified by global profiling (182), Snf3/Rgt2 signaling alsoinitiates feedback and feedforward regulatory loops that areintertwined with the Snf1-dependent pathway. The Snf3/Rgt2

contribution to these regulatory loops involve three zinc finger-containing repressors, Mig2, Mig3, and Rgt1, as well as theRgt1 corepressors Std1 and Mth1. For example, glucose sig-naling through Snf3/Rgt2 induces expression of the MIG2-encoded repressor, and in the absence of glucose Std1 binds toand activates Snf1 kinase (Fig. 3) (see above). The Snf1-Mig1pathway in turn represses expression of Snf3 and Mth1. Im-portantly, despite these links to Snf1 kinase activity, signalingby Snf3/Rgt2 does appear to operate as an independent glu-cose response pathway (Fig. 4) (179, 182, 228, 272, 273). Thus,Snf3 and Rgt2 initiate a component of the glucose responsethat overlaps at least partially with both the PKA pathway andits Snf1 kinase branch but that can also operate independentlyof either.

An important remaining aspect of this glucose regulatorypathway that awaits clarification is the apparent capacity ofRgt1, under conditions (high glucose concentration) in which itno longer interacts with its recognition sites in DNA, to reverseroles and function as an activator of HXT transcription and ofa heterologous reporter gene (186, 250, 271, 275) (however,see also reference 117). Although its functional significanceremains unclear, transcriptional activation by Rgt1 requiresthe glucose sensors Snf3 and Rgt2 and the F-box protein Grr1(275).

The Cell Cycle Connection

Eukaryotic cells grow and divide at maximal rates only whenglucose is freely available. This requires sophisticated regula-tory coordination between the metabolic engine of growth andthe wide variety of structural events required for cellular re-duplication. With respect to glucose signaling it has longseemed likely that there is a connection with the essentialcyclin-dependent kinase Cdc28; the latter acts in concert withthe G1 cyclins Cln1 to -3 at the primary gating event (Start) inthe cell division cycle (3, 116, 132, 165, 262, 263, 276, 285, 286,321, 371, 416, 424; see references 83, 365, and 389 for reviews).Unfortunately many of the critical relationships between thegrowth and cell cycles have resisted elucidation. The slowprogress on this front is evident when one considers that Hart-well first codified the basic questions over 30 years ago (138):how does macromolecular synthesis communicate with theCDC28 gene product, and how are all of these diverse inputs(e.g., the pathways for carbon, nitrogen, sulfur, phosphorus,and potassium assimilation) integrated at Start?

Two mechanisms of apparent coordination between glucosesignaling and cell cycle progression seem particularly worthy offurther attention. The first involves Grr1, the aforementionedF-box protein that both relieves glucose repression as a com-ponent of the Snf3/Rgt2 pathway and regulates Cln-dependentCdc28 function (209). Little is yet known about how Grr1might integrate glucose signaling with cell cycle regulation, butit now seems clear that it responds directly to glucose sensingby Snf3 and Rgt2 at the plasma membrane. Second is the PKApathway, which has long been considered a general regulatorof cell growth in any carbon source and as such is thought toparticipate in the dramatic increase in the rate of cell divisionupon the appearance of fermentable sugars, including glucose.As discussed above, many components of the PKA pathway



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that were originally identified in connection with their role inglucose signaling have now been thoroughly characterized.

Progress has also been made in understanding the ultimaterecipients of the glucose response in the yeast nucleus; PKAsignaling increases transcription of Rap1 target genes and ap-pears to operate through the newly identified Rap1 coregula-tors Fhl1/Ifh1 as well as the Rap1/Gcr1/Gcr2 transcriptionalactivation assemblage (81, 83, 188, 320, 328, 402, 416). Targetgenes activated by Rap1 fall into two main groups, glycolyticand ribosomal protein (RP) genes (211). Not surprisingly,these are the genes most abundantly transcribed by RNA poly-merase II in S. cerevisiae cultures growing rapidly on glucose;26 of the 30 most highly expressed transcription units are inone of these two groups (392). Overall the Rap1 activationmechanism(s) accounts for 60 to 75% of RNA polymerase IItranscription in rapidly growing cells (324, 410).

Transcriptional Induction of Glycolytic andRibosomal Protein Genes

Rap1 is an essential DNA binding protein required for bothtranscriptional repression and activation of target genes (128,339); interestingly, it also binds to telomeres and negativelyregulates telomere length (for a review, see reference 344).The promoters of most genes encoding glycolytic enzymesor ribosomal proteins contain sites occupied by Rap1 in vivo(211). The Rap1 site appears to mediate the response ofthese genes to the appearance or disappearance of glucose;for example, their expression is coordinately down-regu-lated upon depletion of glucose at the diauxic shift (85). ThePKA and TOR signaling pathways are thought to regulatethis response of Rap1-activated genes to nutritional status(328, 402, 410, 441).

Recent reports suggest that the PKA and TOR pathwaysintersect with respect to their shared role in stimulating RPexpression. The transcription factors Sfp1, Fhl1, Ifh1, and Crf1appear to be key downstream targets of this signaling in theyeast nucleus, and various unified models suggest how PKAand TOR signaling might intersect to regulate cell cycle pro-gression by controlling the rate of ribosome production (181,226, 292). Although additional work is needed to clarify theroles of these newly identified transcriptional regulators, to-gether they appear to comprise the downstream targets ofnutrient sensing through the TOR pathway and thus are themost likely recipients of signaling by the anticancer agent rapa-mycin. In contrast, the Rap1 coactivator Gcr1 appears to re-spond only to PKA; gcr1� cells exhibit no alteration in rapa-mycin sensitivity or rapamycin-induced down-regulation of RPgene expression (S. J. Deminoff, K. A. Willis, and G. M. San-tangelo, unpublished data). Like Fhl1/Ifh1, Gcr1 makes animportant contribution to nutrient sensing, since its removalcauses a long delay in the glucose response (as measured byupshift of RP gene transcription, polysome formation, trans-lation rate, and growth rate [416; see below]).

Transcriptional activation by Rap1 was shown long ago toexhibit sensitivity to disruption of GCR1 (81, 324, 435). Ge-nome-wide analysis has now demonstrated for the entire ge-nome that genes with a Rap1 binding site in their promoter arealso Gcr1 target genes (81, 211, 324, 375, 435). A wealth ofadditional evidence suggests that the Rap1/Gcr1 assemblage

participates directly in an activation mechanism shared by thiscommon set of target genes. First, Gcr1 activation of glycolyticand RP genes is eliminated by point mutations or small dele-tions that eliminate the Rap1 binding site(s) in the correspond-ing promoters (374, 375). Thus, the capacity of Gcr1 to stim-ulate transcription of most if not all of its target genes isentirely dependent upon Rap1 binding upstream of thosegenes. Second, mutational inactivation of leucine zipper-me-diated Gcr1 homodimerization eliminates both the GCR1 con-tribution to activation of Rap1 target genes and complemen-tation of the gcr1� phenotype (81, 82). Indeed, loss of Gcr1function and reduced transcriptional activation of Rap1 targetgenes exhibit a tight correlation across an entire spectrum oflesions that cause mild to severe impairment. Finally, fluores-cently tagged Rap1 and Gcr1 colocalize to foci in the yeastnucleus in live cells (236), and epitope-tagged Rap1 and Gcr1coimmunoprecipitate (375) and can be detected in a large(�1-MDa) complex by size affinity chromatography; this Rap1complex exhibits faster migration in the absence of Gcr1, in-dicating that it is not an aggregation artifact (H. Zhou andG. M. Santangelo, unpublished data).

Despite these data and other experimental support for thehypothesis that Rap1 and Gcr1 collaborate directly to stimu-late transcription of both glycolytic and RP genes, it has beendifficult to rule out a competing idea, that the effect of GCR1deletion on RP gene expression is indirect. This alternativehypothesis is based on the observation of coordinate regulationof the 138 RP genes in S. cerevisiae. Coordinate regulationlinks transcription of RP and other translational componentgenes to the growth rate of yeast cells (93, 185, 409). The ideathat removal of Gcr1 indirectly causes a drop in RP transcrip-tion involves the following reasoning: the reduction in glyco-lytic gene transcription, which is a direct result of GCR1 dele-tion, causes a severe defect (slow growth relative to wild-typecells); this low growth rate in turn causes reduced RP genetranscription via a Gcr1-independent mechanism of coordinateregulation. According to this hypothesis, the rationale for as-signing the “sick phenotype” label to the gcr1� mutant is thesupposition that a bottleneck in energy generation takes place:reduced glycolytic flux in glucose-grown gcr1� cells impairsanaerobic metabolism, while glucose repression of aerobic me-tabolism remains intact. This unfortunate combination wouldleave cells with no good way to generate energy, thereby ex-plaining the slow growth and indirect reduction in RP tran-scription.

Three independent lines of evidence demonstrate that thisalternative hypothesis is inviable. First, it is now clear thatglucose repression is not intact in gcr1� cells (327, 383). Sec-ond, analysis of strains that harbor partially functional GCR1alleles identified several that grow and ferment glucose almostas well as the wild type but nevertheless exhibit RP transcrip-tion as defective as that observed in gcr1� cells (81). Third, thegcr1� defect in RP transcription is equivalent in all ferment-able carbon sources tested thus far (glucose, sucrose, raffinose,and galactose), which, significantly (see above), includes non-repressing sugars and pyruvate, a nonfermentable carbonsource (Fig. 5). Together these data eliminate the key under-lying assumption of the alternative hypothesis, i.e., that in theabsence of Gcr1, glucose repression and/or slow growth causesan indirect effect on RP expression.



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Therefore, especially when combined with new insights con-cerning the shared participation of Rap1, Gcr1, and Gcr2 inperinuclear gene regulation (see below), it is now apparentthat these global regulators work together to stimulate tran-scription of both glycolytic and RP genes, the two largest andmost abundantly transcribed classes of glucose-induced targetgenes in yeast cells. Much work remains in deciphering thephysical organization and relative contributions of the Rap1/Fhl1/Ifh1 and Rap1/Gcr1/Gcr2 assemblages and how they aresignaled by the PKA and TOR pathways in response to theappearance of glucose. Significantly in this regard, the conse-quences of removing Gcr1 include a dramatic drop in transla-tion rate, a decrease in CLN transcription, a cln3�-like in-crease in cell size, and conditional cell cycle arrest andelongated morphology in the cln3� and cln1� cln2� back-grounds (83, 416). An important topic for future investigation

is whether (and to what extent) the respective Sfp1/Fhl1/Ifh1and Gcr1/Gcr2 contributions to Rap1 transcriptional activa-tion and cell cycle control are coordinated.

The Repressor/Activator Theme in Glucose Regulation

Although some information has been gleaned regarding theall-important events downstream of the transcriptional regula-tory factors that mediate repression and derepression of glu-cose-responsive target genes, a more than rudimentary under-standing of the central mechanism(s) of gene regulation in S.cerevisiae and other eukaryotes has thus far eluded our grasp.Like Mig1, Mig3, Nrg1, and Rgt1, many of the factors that bindDNA either directly or indirectly are capable of both activatingand repressing transcription. This puzzling property of glucoserepressors is also true of positively acting regulators such asRap1 and Gcr1, as well as RNA polymerase II holoenzymecomponents such as Med1, Ssn8, Sin4, and Rgr1 (15, 58, 210,242, 346).

How a distinction is made between these diametrically op-posite activities of activation and repression based on chromo-somal and/or environmental context, as well as the molecularmechanisms that execute this decision, awaits clarification. Forexample, an unambiguous finding regarding the mechanism ofglucose repression in yeast cells is that the Ssn6/Tup1 core-pressor complex plays an important role downstream of mostof the factors that bind DNA directly, such as the MIG-, NRG-,and RGT1-encoded repressors. However, upon derepressionboth Mig1 and Ssn6/Tup1 remain associated with glucose-repressed promoters (29, 278, 279). Further, even Ssn6 appearsto be capable of stimulating transcription under some circum-stances (68). Thus, most if not all of the relevant negativelyacting regulators remain associated with target promoters butare somehow inactivated (or their activities are reversed) un-der derepressing conditions. This is a much more subtle regu-latory mechanism, and it operates on a much more static pro-tein-DNA complex, than was previously believed to exist. Thefinal sections of this review will describe a new paradigm foreukaryotic gene activation and regulation that may provide asuperior context in which to understand these and other inter-esting new observations about glucose regulation in the yeastnucleus.

Reverse Recruitment: Rap1/Gcr1 Activationat the Nuclear Periphery

Beyond its interesting regulatory role in yeast cell physiol-ogy, a second mystery has shrouded the Rap1/Gcr1 mechanismof transcriptional activation: it conforms poorly with the re-cruitment paradigm of gene regulation (36, 70, 295, 296, 352,434). For example, when a Rap1 binding site is placed up-stream from heterologous reporter genes, or when the activityof Rap1-Gal4 or Rap1-LexA fusion proteins is measured,Rap1 activation is surprisingly weak (in some contexts it isundetectable) (38, 88, 349, 415). In contrast, mutational inac-tivation of Rap1 binding sites in the very strong glycolyticpromoters typically results in a 10- to 20-fold decrease in tran-scription, suggesting that Rap1 is a powerful activator (see,e.g., reference 374). This discrepancy, with identical Rap1binding elements yielding very different results depending on

FIG. 5. Transcriptomic analysis of defective gene expression ingcr1� cells. Microarray hybridization was done with RNA from iso-genic gcr1� versus wild-type cultures grown to mid-logarithmic phase(a density of 1 106 cells/ml) on each of the carbon sources indicated(2% glucose, 3% sucrose, or 3% pyruvate). Total RNA was extracted(408) and labeled with either Cy3-CTP or Cy5-CTP by using a low-RNA-input fluorescent linear amplification kit (Agilent Technologies,Palo Alto, CA). Labeled RNA was purified with the RNeasy MinElutekit (QIAGEN) and hybridized to yeast 60-mer oligonucleotide arrays(Agilent Technologies, Palo Alto, CA) according to the manufactur-er’s recommendations. Array slides were scanned at 10-m resolutionwith two-line averaging by using an Axon GenePix 4200A scanner andGenePix 6.0 software. Hierarchical clustering was done with Acuity4.0, with a centered Pearson similarity metric. The data shown hererepresent an average of two replicate arrays, including a dye-swapcontrol. The scale shows color intensity proportional to the log ratio ofexpression in gcr1� cells (relative to the corresponding wild-type val-ues). The cluster diagram shows the results for the 192 genes (11glycolytic, 110 ribosomal protein, 4 translational component, and 67other genes) that, in all three carbon sources, exhibit at least a twofolddecrease in expression in gcr1� cells.



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whether they are in their native location or upstream from areporter, is paradoxical because no yeast protein other thanRap1 binds to its recognition motif in DNA. Moreover, weakRap1 activation of reporter genes is not due to a structuralperturbation resulting from fusion to a heterologous DNAbinding domain; Rap1-LexA fusion proteins that can fullycomplement a deletion of endogenous RAP1 show little or noheterologous activity as transcriptional activators. This wasoriginally thought to indicate a requirement for additionalDNA-bound proteins (such as chromatin-remodeling factors)for full activation by Rap1 and its coactivators, but to date noarrangement of DNA motifs has been described that allows anisolated Rap1 site to promote transcription as efficiently as itsnative counterparts in glycolytic promoters.

An alternative explanation, which remains untested, is thatcooperativity resulting from coupling of the various steps intranscription (i.e., activation, elongation, termination, process-ing, and export of the final products to the cytoplasm [225])explains the impressive strength of Rap1 activation when theprotein is bound at its native sites. Inclusion of mRNA exportin such a holistic mechanism of gene expression implies afunctional and/or physical connection with nuclear pores. This“gene expression machine” (GEM) explanation for the dis-crepancy in Rap1 activation efficiency is plausible because apas de deux between chromatin and GEMs, an arrangementthat could have evolved to optimize expression of naturallyoccurring promoter-open reading frame combinations whoseproducts are in high demand, would be difficult to reproducewith a heterologous gene. (A similar discrepancy has beenobserved for transcriptional repression by Ssn6/Tup1, which ismuch stronger in natural promoters than in artificial constructs[194, 203]). Since most GEMs in glucose-grown cells appearto be occupied with Rap1-regulated transcription units (seeabove) (392, 416), this would explain why Rap1 activation (andperhaps all glucose-regulated transcription) is unusually diffi-cult to reproduce via the ubiquitous heterologous reporterassay. An important corollary of this idea is that an accurateworking model for gene regulation will require detailed map-ping of nuclear architecture (169, 200, 238–240, 280, 281).Interestingly, the “gene gating” hypothesis proposed byGunther Blobel in 1985 (22) may prove to have been a partic-ularly perceptive early suggestion for how three-dimensionalstructure in the nucleus is used to mediate (and regulate) geneexpression.

A second prominent aspect of the Rap1 activation mecha-nism that conforms poorly with the recruitment model of geneactivation is the nature of the Gcr1 activation domain. Thisdomain is irreducibly large (280 residues) and can be subdi-vided into at least four distinct hypomutable regions (82, 83);two of these hypomutable regions coincide with predictedtransmembrane domains (236), and transmembrane-disrupt-ing lesions in these domains are known to inactivate bothGCR1 function and Gcr1-LexA activation of a heterologousreporter gene (82). If, as predicted by the recruitment model,these transmembrane motifs are required for incorporation ofGcr1 into its activation complex, it should have been possibleto make compensatory extragenic lesions that would suppressat least one of the inactivating mutations. Exhaustive screensfor such suppressing lesions by using either irradiation or

chemical mutagens have failed. Further, every mutation in thisregion that inactivates GCR1 function also eliminates Gcr1-LexA activation of a LexA-driven reporter gene. Since Gcr1-dependent transcriptional activation of target genes is elimi-nated by removal of the Rap1 binding site in the correspondingpromoters, this was originally interpreted to mean that contactbetween Rap1 and Gcr1 is a requirement for the latter torecruit the transcriptional machinery. However, targeted mu-tagenesis of RAP1 has been equally unsuccessful in generatingdetectable suppression of lesions in Gcr1 that impair activa-tion. Furthermore, activation of a heterologous reporter byGcr1 persists long after the disappearance of Rap1 in RAP1ts

strains. Together these data suggest that when Gcr1 is boundupstream from a reporter gene, its Rap1 interaction domain isrequired but (paradoxically) Rap1 itself is not. The large bodyof existing data regarding the Gcr2 contribution to the Rap1/Gcr1 activation mechanism has likewise been difficult to rec-oncile with the recruitment paradigm (81, 435). Thus, all at-tempts to resist the straightforward interpretation of anactivation domain that overlaps with transmembrane domains,i.e., that Gcr1 association with the nuclear membrane is animportant feature of its transcriptional activation function,have failed.

A recent genome-wide genetic screen (synthetic genetic ar-ray analysis; see above) of gcr1� and gcr2� query strainsyielded a surprising result that has finally provided an im-proved context for resolving these disparities and elucidatingthe mechanism of Rap1/Gcr1 transcriptional activation. BothGCR1 and GCR2 were found to be members of a large geneticnetwork of nucleoporin genes (236). More specifically, dele-tion of each nonessential gene encoding a component of theNup84 subcomplex exhibits a synthetic genetic defect in com-bination with GCR1 deletion. Follow-up biochemical, cell bi-ological, and gene expression analyses confirmed that Rap1/Gcr1 activation occurs at the nuclear periphery and is impairedby disruption of genes encoding components of the Nup84subcomplex. For example, Rap1 and Gcr1 are 100% and 71%as enriched in nuclear envelope fractions as the integral nu-clear membrane protein Pom152, unlike control proteins suchas the nucleolar factor Nop1 (�1% enriched) (236). Impor-tantly, perinuclear localization of Rap1/Gcr1 target genes hasbeen observed via chromatin immunoprecipitation of nuclearpore-associated genes (48), which provides independent sup-port for the conclusion that there is a physical and functionallink between the Rap1/Gcr1 assemblage and the nuclear rim.

Most surprisingly, not only do Rap1 and its coactivatorsGcr1 and Gcr2 activate transcription at the nuclear periphery,but many yeast nucleoporins function as transcriptional acti-vators; two of these were shown to do so at their normalperinuclear location (236). Transcriptional activation by nu-cleoporins is severely impaired by GCR1 deletion, suggest-ing that disruption of the Rap1 activation assemblage inter-feres with the normal organization of GEMs at the yeastnuclear periphery. This result may recapitulate a phenomenonrelevant to human cancer. Perinuclear gene activation appearsto be oncogenic in acute myeloid leukemia and related syn-dromes in humans, in which chromosomal rearrangementsfuse Hox DNA binding domains to the nucleoporin hNup98



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(126, 183). Interestingly, human Nup98 also activates tran-scription in yeast cells, and its ability to do so is impaired in theabsence of Gcr1 (236). Integral GEM components, includingseveral Mediator subunits (Ssn8, Sin4, and Med1) and Rpo21,also associate with the nuclear periphery and participate inperinuclear activation.

These data indicate the existence of an unusually intimaterelationship between the Rap1/Gcr1 assemblage and GEMcomponents, a conclusion that is further suggested by proteinsequence similarity searches. For example, PSI-BLAST analy-sis of Gcr1 identified homology with the RNA polymerase IIlarge subunit in the free-living protist Mastigamoeba invertens(Fig. 6) (351). The highly significant E value of this pairing is2.0 10�30 (marginal significance is attained at an E value of0.05 [5]). M. invertens and other primitively amitochondriatemembers of the order Pelobiontida may represent the earliesteukaryotic lineages (351). Thus, the RNA polymerase II large-subunit gene in M. invertens could represent a composite pre-cursor of both Rpo21 and Gcr1 (Fig. 6). This potential primor-dial connection between nuclear pores and Rap1/Gcr1/Gcr2function may help to explain the lack of evolutionary conser-vation of the latter; divergence of the nucleoporin Nup96 hasbeen shown to cause hybrid inviability in Drosophila and maytherefore contribute to speciation (293).

These new data begin to place the Rap1/Gcr1 activationassemblage within its proper architectural context in the yeastnucleus and (at least where this unusual regulatory complex isconcerned) suggest an interesting alternative to the recruit-ment model of transcriptional activation. We have termed thisalternative paradigm “reverse recruitment” to highlight its cen-tral feature, that Rap1/Gcr1 target genes are recruited to pe-rinuclear GEMs; the term references the opposite presump-tion of the recruitment model, that the requisite factors aredrawn to (or assembled on) the relevant promoters. Impor-tantly, although this mechanism was uncovered through the

discovery of a functional connection between the Rap1/Gcr1/Gcr2 activation assemblage and GEMs at the nuclear rim, thereverse recruitment model does not imply that all activationoccurs at the nuclear periphery. Indeed, promyelocytic leuke-mia protein bodies in the nuclear lumina of mammalian cells(61, 290, 399) could represent nodes of GEM organization thatparallel what we have observed to occur at nuclear pores inyeast cells. In this regard, the reverse recruitment idea is con-sistent with an earlier proposal by Hutchison and Weintraubthat active eukaryotic genes are localized along interchromatinchannels that communicate with the nuclear periphery (167).Thus, the central feature of the reverse recruitment idea is thatgenes become transcriptionally active by docking with com-partmentalized GEMs. Although they are relatively abundantat the periphery, it seems likely that at least some GEMs areluminal; these internal GEMs may nevertheless maintain func-tional and/or physical communication with their perinuclearcounterparts.

The reverse recruitment idea explains much or all of thedata that seemed paradoxical when attempting to fit themwithin the recruitment model. It presumes that Rap1 and Gcr1are separate cogs in a large and multifaceted GEM and thatthe site of Gcr1 attachment to this megalith is an extensivehydrophobic interaction with either the Nup84 subcomplex, anadjacent site on the nuclear envelope itself, or both (Fig. 7).Thus, there may be two types of reverse recruitment in S.cerevisiae, i.e., transient occupation of GEMs by tightly regu-lated (and often weakly expressed) genes and constitutive oc-cupation by strongly expressed genes that bind directly to aGEM component; the Rap1/Gcr1 mechanism is in the lattercategory. According to this idea, regulation of Rap1/Gcr1 tar-get genes, which clearly does occur (e.g., upshift upon theappearance of glucose), might involve a novel type of regula-tory control: alteration of the functional or physical relation-ship between perinuclear GEMs and the Rap1/Gcr1 assem-blage.

FIG. 6. Both Gcr1 and Rpo21 are related to the RNA polymerase II large-subunit gene from M. invertens. The N terminus and carboxy-terminaldomain (CTD) of Rpo21 exhibit strong homology with their M. invertens counterparts, whereas the internal region of the M. invertens proteinexhibits weaker (although highly significant) homology with both Rpo21 and Gcr1. The transmembrane domains of Gcr1 (TM1 and TM2), as wellas its primary homodimerization domain (1LZ), all of which make a critical contribution to Gcr1 function (see the text), exhibit �80% similaritywith the M. invertens RNA polymerase large subunit, as indicated.



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GENE REGULATION VIA REVERSE RECRUITMENT

A New Model for Eukaryotic Transcriptional Activationand Repression

As described above, the existing data suggest that reverserecruitment accounts for the bulk of transcriptional activationin growing yeast cells. What about more conventional regula-tory systems: does reverse recruitment explain all types oftranscriptional activation and regulation in eukaryotic nuclei?Data favoring this conclusion are already available for twoconventional regulatory mechanisms, INO and GAL (32, 48)(Fig. 8). With respect to the well-studied GAL system, tran-scriptional activation in galactose-grown cells is accompaniedby association between GAL genes and the nuclear periphery,which is dramatically reduced in glucose-grown cells (48). Thisresult is particularly significant because analyses of galactose-regulated gene expression and the Gal4 activator were usedinitially to establish the recruitment paradigm. The link be-tween transcriptional activation and perinuclear associationhas also been reported for INO1 (32). Thus, a growing body ofevidence supports the ideas that a link exists between thenuclear periphery and transcriptional activation and that re-versible association with the nuclear rim may be an integralfeature of all eukaryotic gene regulation (see also a recentanalysis of var genes in Plasmodium falciparum [301]).

Universal application of the reverse recruitment paradigmmay provide a context in which to understand numerous per-plexing phenomena that have heretofore defied explanation,most importantly the capacity of regulators to exert influenceon gene transcription under circumstances where either director indirect contact with the DNA is thought to be unlikely. Thebest-characterized examples of such “ghost regulation” are

Rap1 repression of target genes upon disruption of the secre-tory pathway (208) and Rgt1 activation of HXT1 (see above).Ghost regulation cannot be reconciled with a competitor of thereverse recruitment idea that postulates an alternative expla-nation for the perinuclear location of activated GAL and INOgenes, i.e., that transcription initiation complexes form as pre-

FIG. 7. Perinuclear transcriptional activation of Rap1/Gcr1 target genes via reverse recruitment. Known components of the Rap1 activation(Gcr1/Gcr2) or silencing (Sir complex) assemblages are shown. Essential nucleoporins are indicated with an asterisk. Dashed lines highlightpresumptive perinuclear tethering interactions; nuclear pore complex-associated factors shown to interact with Gcr1 are underlined. Therepresentation shown here is not intended to rule out the existence of a unified complex that can switch between activation and repression oftranscription. (Reprinted from reference 236 with permission of the publisher. Copyright 2005 National Academy of Sciences, U.S.A.)

FIG. 8. Perinuclear transcriptional activation of GAL genes via re-verse recruitment. The glucose-regulated transcriptional activatorGal4 associates with its DNA binding sites whether the downstreamtarget genes are inactive or active, as shown. The GAL1, GAL7, andGAL10 genes are not detectably associated with the nuclear peripheryunder conditions (in glucose-grown cells) in which their transcription isrepressed (UNINDUCED, left side). When cells are grown in thepresence of galactose, these same GAL genes associate with the nu-clear periphery (INDUCED, right side).



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dicted by the recruitment model and move to the peripheryonly during the final stages of gene expression (i.e., duringmRNA processing and export).

Glucose May Signal a Highly Networked and StructurallyStable Perinuclear Machine

Another recent development favoring the reverse recruit-ment idea is that proteins capable of both induction and re-pression of transcription, including many involved in glucoseregulation (e.g., Rap1, Gcr1, Snf1, Mig1, Mig3, Nrg1, Rgt1,Ssn6, Med1, Ssn8, Sin4, Rgr1, and Fhl1), appear to be the rulerather than the exception. We have now shown that many ofthese factors are concentrated at the periphery of the yeastnucleus (236; B. B. Menon and G. M. Santangelo, unpublisheddata). It is increasingly clear that subtle allosteric alteration ofbifunctional activator/repressor-type regulators is a commonmechanism responsible for major regulatory shifts in the tran-scriptome (e.g., at Snf1/Mig1/Ssn6 targets). Reverse recruit-ment has the advantage of allowing a straightforward and par-simonious depiction of this type of subtle gene regulationmechanism (Fig. 9), a task that has become more and moredifficult and convoluted when one adheres to the recruitmentparadigm. This is in part because the latter requires so manyfactors to assemble in the correct configuration at each pro-moter. It is especially so when also considering that the re-

cruitment model requires them to assemble in the propersequence (i.e., in a temporally flawless fashion) at each pro-moter, or to preassemble and then bind as a complex or seriesof complexes, in order to obtain the proper transcriptionalreadout for each corresponding gene. Indeed, a recent ge-nome-wide analysis of quiescence revealed that, in contrastwith the recruitment model, RNA polymerase II is alreadypositioned upstream of many inactive genes prior to their in-duction upon exit from stationary phase (298).

With respect to glucose regulation in particular, it is alsonow apparent that DNA-bound transcriptional activators,readily identified in some systems (e.g., Gal4), may be curi-ously missing from others. Given that SUC2 has been a modeltarget gene in the study of glucose repression, it is reasonableto have expected by now the identification of a DNA-boundSUC2 activator. (Although it seems unlikely, it remains a for-mal possible that the gene encoding the SUC2 activator proteinhas simply eluded detection.) The very successful screens fornon-sucrose-fermenting mutants (46, 259, 385) have insteadidentified the critically important kinase subunits Snf1 andSnf4, a putative glucose sensor localized to the plasma mem-brane (Snf3), endosomal factors (Snf7 and Snf8 [148, 287]),and components of the Swi/Snf chromatin remodeling complex(Snf2, Snf5, Snf6, Snf11, and Snf12). None of these proteinsare thought to bind DNA directly. Subsequent screens forsuppressors of Snf1 (47, 386) identified two critical down-

FIG. 9. A model for glucose regulation at the nuclear periphery of S. cerevisiae. Numerous regulators involved in the transcriptomic responseto glucose are components of a large nuclear assemblage (gene expression machine) that is tethered to nuclear pores (dashed lines) via multipleinteractions with the Nup84 subcomplex. Red arrows depict repressed target genes, and green arrows depict activated targets. Inactivation ofDNA-bound regulators (Mig1, Mig3, and Nrg1) by the Snf1 kinase is predicted to involve a subtle conformational alteration that remains obscure.Many of these glucose regulatory factors, including components of the RNA polymerase II Mediator, are bifunctional factors that can eitherrepress or activate transcription in a context-dependent fashion (shown here for Rap1). This facile switching between transcriptional readoutsunderscores the mechanistic subtlety of regulatory partnering and/or conformational specificity. Association between nuclear pores and theGcr1/Gcr2 complex is required for transcriptional activation by both Rap1 and Nup84 subcomplex components (229). The perinuclear Rap1/Gcr1/Gcr2 assemblage thus allows productive access to GEMs by glucose-induced (glycolytic and RP) genes tethered at the nuclear periphery.



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stream factors in glucose repression, Mig1 (Ssn1) and Ssn6; theproducts of the other six SSN alleles associate biochemicallywith RNA polymerase II and participate in the Mediator-based mechanism of glucose repression by the Ssn6/Tup1 core-pressor: Ssn2, Ssn3, Sin4 (Ssn4), Srb8 (Ssn5), Rox3 (Ssn7),and Ssn8.

Thus, of all these Snf and Ssn loci, the only protein identifiedto date that exhibits specific DNA binding activity is the zincfinger protein Mig1. Mig1 (and its paralogs Mig2 and Mig3)bind to a GC core element upstream from SUC2 and otherglucose-regulated genes. Mig1 and Mig3, like most of the othertranscription factors identified in Snf and Ssn screens, areknown to function as both repressors and activators. Never-theless, Mig1 is presumed not to participate in activation ofSUC2, which it binds to and represses in the presence of glu-cose. This conclusion is based on three assumptions that arewell worth revisiting. First, SUC2 is derepressed normally inthe absence of MIG1 (and/or MIG2). However, the redun-dancy of factors that bind to similar GC recognition sites inDNA makes it difficult to construct a strain in which all regu-lators that could bind and activate transcription are known tobe eliminated. Thus, it is quite possible that MIG deletionuncovers a cryptic activator function in the SUC2 promoter.Second, Mig1 relocates to the cytoplasm in the absence ofglucose. However, the Mig1 molecules remaining in the nu-cleus are sufficient to occupy binding sites upstream of Mig1target genes (see above). Third, removal of the Mig1 recogni-tion motif in the promoter does not impair SUC2 derepression.However, this argument rests on the assumption that the re-cruitment model of transcriptional activation is correct, andthat once binding of those factors to their respective DNArecognition sites is blocked, they no longer remain in the vi-cinity of (i.e., tethered to) their respective target genes. Incontrast, the reverse recruitment hypothesis postulates that agiven regulator may remain in the vicinity of a “tethered”target gene and continue to influence its expression by alteringthe behavior of associated GEMs. This leaves open the possi-bility that Mig1 and other factors originally identified as re-pressors (e.g., Rgt1) might, in contrast to previous expecta-tions, undergo a repressor-to-activator switch and activatetheir target genes under derepressing conditions (Fig. 9).

Reverse recruitment thus provides a viable alternative to anincreasingly untenable prediction of the diffusion-based re-cruitment model that a disparate collection of activators, re-pressors, coactivators, corepressors, and GEM components arebrought to each promoter as needed to provide the appropri-ate regulatory input. An improved understanding of nuclearsubstructure and its reception of regulatory signals from theplasma membrane and cytoplasm is therefore a critical prereq-uisite to constructing an accurate global wiring diagram forglucose signaling in S. cerevisiae and other eukaryotes. Reas-sessment of the regulatory relationships in other systems willlikewise become necessary if reverse recruitment is as wide-spread an explanation for gene induction and repression asnow seems plausible.

ACKNOWLEDGMENTS

I am extremely grateful to Kristine Willis for figure design andpreparation, to Youping Deng and Venkata Thodima for the graphicaldepiction of the ClustalW alignment of Gcr1 and Rpo21 with their M.

invertens homolog, to Kellie Barbara for graciously contributing themicroarray data shown in Fig. 5, to Kellie Barbara and Nicole Thomp-son for help with literature searches, and to Kristine Willis, Jeong-HoKim, Kelly Tatchell, Lucy Robinson, and Brenda Andrews for review-ing the manuscript, as well as for their keen insight and tolerance. Mostimportantly I thank the current and past members of the Santangelolaboratory for their perspiration, inspiration, and helpful suggestions.Finally, kudos and thanks to the many fine biomedical researchers inMississippi with whom I have collaborated during my tenure as Direc-tor of the Mississippi Functional Genomics Network (mfgn.usm.edu/mfgn).

Work in my laboratory is supported by National Institutes of Healthaward RR16476.

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