Start Control in Cycling Saccharomyces cerevisiae Cells1

START Control in Cycling

Saccharo rnyces cerevisiae Cells'

HANS K ~ N T Z E LHANS,~ WERNERROTTJAKOB, ANGELIKAS C H W E D AND WERNERZWERSCHKE Max-Planck-lnstitutfur Experimentelle Medizin 37075 Giittingen, Germany I. G I Phase and Cell-Cycle START . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A. Ras and Adenylate Cyclase . . . . . . .

C. Swi4 and Swi6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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........... V. A Regulatory Network Operating at START VI. Saccharomyces cerevisiae Gene Symbols . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . ..............

2 2 3 4 4 5 8 9 11 11 11 13 16 16 19 20 21 24 25

Abbreviations: ARS, autonomously replicating sequence; BI, bud initiation; CAMP,adenosine 3',5'-cyclic monophosphate; Cap, cyclase-activated protein; cA-PK, CAMP-dependent protein kinase; CDC, cell-division cycle; IIAG, 'diacylglycerol; CK, cytokinesis; DSG, DNA synthesis genes; GAP, GTPase-activating protein, IP,, inositol 1,4,5-trisphosphate; MBF, MCB-binding factor; MCB, MZuI cell-cycle box; ND, nuclear division; PKC, protein kinase C: PEST, peptide domain rich in Pro, Ser, Thr and acidic residues; PIP,, phosphatidylinositol4,5hisphosphate; PI-PLC, PIP,-specific phospholipase C (phosphoinositidase); SBF, SCB-binding factor; SCB, Swi4-dependent cell-cycle box; SH, Src homology region; SPB, spindle pole body; TC, transcriptional complex; UAS, upstream activating sequence. The three-letter codes of yeast genes are given in italicized uppercase (wild-type alleles, e.g., CDC25) or lowercase letters (recessive mutant alleles, e.g., cdc25-I). The respective gene products (proteins) are written with an initial uppercase letter followed by two lowercase letters (e.g., Cdc25). A glossary of S. cereoisim gene symbols is given in Section VI 2 To whom correspondence may be addressed. Progress in Nucleic Acid Research and Molecular Biology, Vol. 48

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Copyright D 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

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HANS KUNTZEL ET AL.

The budding yeast Saccharomyces cerevisiae is an excellent model organism for studying the molecular biology of the cell cycle. This is mainly due to the availability of a large number of temperature-sensitive cell-division-cycle (cdc) mutants, which were selected for their property to arrest at various defined morphological states (1). A now classical review from 1981describes a functional model of the cell cycle, based on the analysis of 51 cdc mutants (Z), and most of these 51 CDC genes are now cloned, sequenced, and functionally characterized. One of the most complex stages of the yeast cell cycle is the transition from the “decisive” G1 phase to the “committed” S phase. Dnring G1 the cell has the option to leave the mitotic cycle and to enter either a quiescent state (at limiting nutrient supply) or a mating-competent state (in the presence of pheromones of opposite mating type), whereas after passing the G U S border the cell is committed to the next round of mitotic division. At the G U S transition step, three parallel pathways are initiated, leading to the replication of chromosomes, the duplication of the spindle pole body (SPB), and the formation of a new bud (2). This article attempts to discuss only some aspects of the G U S control network, including growth control by nutrient signaling, the activation of Cdc28 kinase by G U S cyclins, and the coordinated expression of DNA synthesis genes.

1. G1 Phase and Cell-Cycle START

A. Morphological G1 Events Mitosis ends with a dramatic phase (the landmark of late nuclear division) characterized by the collapse of the elongated telophase spindle and the reorganization of daughter nuclei (2). One of the first morphologically recognizable G1 events is the formation of a primary septum at the mother/ daughter neck, leading to cytokinesis (3).Septum formation depends on the performance of mitosis and nuclear division (4) and requires the product of the chitin synthase gene CHS2 (5). Another morphological event of cytokinesis is the deposition of cortical actin dots at the neck region (6). The next landmark, cell-separation, requires the action of the CTSIencoded chitinase (7) as well as a CHSI-encoded chitin repair enzyme (8). The newly separated cells immediately start to reorganize their actin cytoskeleton (S), and to mark their new bud sites by the deposition of the SPA2encoded protein (9). The single SPB of the nuclear envelope is first distal to the new bud site, with cytosolic microtubules already oriented toward the Spa2 patch, and the nucleus then turns around to have the SPB proximal to the Spd-marked site (9). At the cell separation step, the daughter cell is smaller than the mother

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cell, and the daughter cell must grow up to a critical size (i.e., that of the mother cell) before entering the S phase (2, 10, 1 1 ) . Therefore, the period between cell separation and GUS transition is always longer for daughters than for mothers ( 1 1 ) . Finally, a late G1 event is the formation of an SPB satellite (12). The GUS transition step itself is difficult to measure by morphological parameters: the two events of SPB duplication and bud emergence occur during the S phase, and DNA replication may even be completed at the time a new bud is visible (2).

B. The Concept of START During the G1 phase, cells monitor extracellular signals such as nutrients or mating pheromones, and respond to these signals by entering a GO-like quiescent state, if essential nutrients are limiting (starvation response), or by entering a mating-competent gamete state characterized by a “shmoo”-like projection, if mating pheromones of the opposite mating type are present (13). Diploid cells do not respond to mating pheromones, hut enter the quiescent state at starvation, or initiate the meiotic pathway under special nutrient supply (e.g., absence of glucose and nitrogen source, presence of potassium acetate as carbon source) to produce ascospores (14). The response to extracellular signals is correlated with the onset of G l / S transition during the G1 phase; this decisive step has been termed START (2). The START phase must be completed to initiate a new cycle of mitotic division. Some of the cdc mutants originally isolated by Hartwell ( 1 ) were characterized as START mutants, since their arrest morphology (unbudded cells with a G1 nucleus) resembles that of starved or pheromone-arrested cells. They were further classified as START II/START A mutants arresting as nongrowing stationary-like cells without an SPB satellite, or as START I/ START B mutants arresting as growing cells having the morphology of pheromone-arrested mating-competent cells, including an SPB satellite (2, 15). The former group includes mutant alleles of CDC25 and CDC35, two genes involved in the control of CAMP-dependent protein kinase (see Section II), and the latter group includes the original START mutant allele cdc28-1. The CDC28 gene encodes a protein kinase (16),which corresponds in structure and function to the Cdc2 protein kinases from the fission yeast Schizosuccharomyces pombe and from animal cells (13). The Cdc28/Cdc2 kinase is now recognized as a master regulator of the eukaryotic cell cycle: the kinase must be activated by G U S cyclins to promote the G U S transition, or START, and by G2 cyclins to promote the G2/M transition (13).Therefore, one of the critical events of START is the transient accumulation of GUS cyclin inRNAs in daughter cells at a late G1 period, after the critical size has been reached by nutrient-controlled growth (see Section 111,B).

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HANS KUNTZEL ET AL.

II. Growth Control by a Nutrient-Signaling Complex A. Ras and Adenylate Cyclase One of the group A START mutants, cdc35-1, was found to be allelic with the temperature-sensitive adenylate cyclase-deficient mutant cyrl (17, 18), and the CDC35ICYRl gene product was soon identified as the catalytic subunit of adenylate cyclase, a large protein of 2026 residues (19, 20). The protein contains a central domain composed of a repeating 23-amino-acid leucine-rich peptide, and the catalytic center is located within the C-terminal 400 residues (20, 21). The yeast Saccharomyces cerevisiae appears to be unique among eukaryotes in that adenylate cyclase is the downstream effector of the small GTP-binding Ras proteins (22).Neither of the two Ras genes R A S l and RAS2 is essential for viability, but the disruption of both genes is lethal to the cell (23, 24). The activation of adenylate cyclase requires either Rasl or Ras2 in the GTP-bound form (22, 25), and both Ras proteins appear to recognize two specific regions of the Cyrl protein, although a physical interaction between Ras and Cyrl has not been directly demonstrated. A plasmid expressing a catalytically inactive adenylate cyclase interferes dominantly with the Rasdependent activation, and the interfering region has been mapped to the central leucine-rich repetitive domain of the Cyrl protein (26).More recently, another region of the Cyrl protein, a 14-amino-acid segment between the leucine-rich repeat and the catalytic site, has been suggested to be involved in the Ras-dependent activation of adenylate cyclase, since antibodies against this epitope can mimic the action of Ras proteins (27). The Ras-responsive adenylate cyclase complex contains a regulatory cyclase-associated protein (Cap) of 526 residues, which binds to a small segment near the C-terminus of the Cyrl protein (28),and which is encoded by the essential CAPISRV2 gene (29,30).The Cap protein is not required for the in vitro or in vivo activation of adenylate cyclase by wild-type Ras proteins (27-29), but is involved in some other aspects of Ras-dependent signaling, such as the response of adenylate cyclase to mutationally activated Ras proteins (28). While the N-terminal domain of the Cap protein is required for the Cyrl interaction, the C-terminal domain appears to interact both with the actin cytoskeleton and with phosphatidylinositol 4,5-bisphosphate (PIP,), a phospholipid component of the plasma membrane important for a mitogenic signaling pathway (31-33). The latter function of Cap is suggested by the morphological and nutritional defects associated with the deletion of the C-terminal domain; these defects are compensated by overexpressed profilin, a protein known to interact both with the actin cytoskeleton and with PIP, (33, 34). Therefore, Cap appears to provide a link between nutrient-

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dependent signaling pathways (involving adenylate cyclase and phosphoinositidase as effectors) and the reorganization of the cytoskeleton at the early G1 phase (6, 33).

B. Cdc25 1. THE RAS-ACTIVATING DOMAIN Another important link between nutrient responsiveness and Rasdependent adenylate cyclase activity is the product of the START A gene CDC25, a protein of 1589 residues (35,36). The Cdc25 protein functions upstream of the Ras/Cyrl/cAMP-dependent protein kinase control chain, since cdc25 mutants have a low CAMP content (35) and are rescued by dominant-activating R A S alleles (36, 37), and by multicopy genes encoding the catalytic subunit of CAMP-dependent protein kinase (38, 39). The Cdc25/Ras/Cyrl chain is required for the nutrient-dependent “growth to critical size” step during the G1 phase (40). A Cdc25 protein map deduced from DNA sequence data (35, 36) is shown in the upper part of Fig. 1. Deletion mapping and mutational analysis have defined a growth-essential domain (E) of about 450 residues at the C-terminal part of Cdc25 (35,36,4143), which promotes the GDP/GTP exchange on Ras proteins (44)by stabilizing a transitory nucleotide-free state of Ras (43).The E domain interacts physically with the GDP-bound Ras2 protein, as shown by a two-hybrid binding assay (43, and exhibits two regions conserved in GDP/GTP exchange proteins of other organisms, such as Drosophila, mammals, and the fission yeast Schizosaccharornyces pornbe (43, 46-48). One o f the two regions (shown as a black box in Fig. 1) is conserved not only in Ras-activating Cdc25 homologs, but also in Bud5, another putative GTP/GDP exchange factor of S. cerewisim, which interacts with a small G protein (BudURsrl) involved in the control of cell polarity (49). The replacement of two adjacent residues (1462 Tyr and 1463 Leu) within the hydrophobic core of this region by a pair of charged amino acids (Asp and Arg) is lethal to the cell (42), and the same mutation (cdc25-hl)completely prevents the physical interaction with the Rase protein in a dual-hybrid assay (T. Munder and P. Furst, personal communication). The more upstream region (shaded box in Fig. l),which is conserved in Ras-activating members of the Cdc25 family but not in Bud5 (49),may have a function in discriminating Ras proteins from other members of the small G protein family (43). All known temperature-sensitive cdc25 mutant alleles map within the E domain between the two conserved regions (50). The CDC25 gene can be disrupted by the marker gene URA3 within the essential E domain (mutant allele cdc25-d4), without affecting viability (51). However, the d4 disruption strongly impairs nutrient-depending functions such as the glucose-induced transient hyperactivation of adenylate cyclase (51, 52), and also prevents the

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HANS KUNTZEL ET AL.

N

C

E

I

1 2

4 4 44

SH3

I

Ste6 I

sos I Ras-GRF I

I

/h

VA

m

I GDP

CDC25 Ste6 SOS Ras-GRF

,

3 4

I

1 GTP

tt

I R G G T K E A L I E H L T S H E L L D A A F N V T M L I T F R S DVACVPFFGVYLSDLTFTFVGNPDF I K T A T L V F I I N Y L L R T D I D S T F F T T I F L N T Y A S VLPCVPFLGVYFTDLTFLKTGNKDN IKGATLCKLIERLTYHIYADPTFVRTFLTTYRY NPPCVPFFGRYLTNILHLEEGNPDL I R Y A S C E R L L E R L T D L R F L S I D F L N T F L H S Y R V DPPCVPYLGMYLTDLAFLEEGTPNY 0 00 000 00 0 00. 000 om0 0 .om0 00 .om

FIG.1. The Cdc25 protein family. (Top) A map of the S. cereoisiue Cdc25 protein (1589 residues) with three major domains (N, E, and C; see Section II,B,2). Numbered arrows denote the positions of the following mutations: 1, cdc25 ; : URA3-d4 (42);2, cdc25-2, cdc25-5 (1328 Glu+Lys), cdc25-10 (1328 Glu+Val); 3, cdc25-1 (1403 Al-Val) (50);and 4, cdc25-hl (1462 TywAsp, 1463 L e w A r g ) (42).Black circles indicate potential cA-PK or PKC phosphorylation sites (42), and the open triangle indicates a potential “membrane-seeking” a-helix (residues 519-536). The boxed region at the N-terminus contains an SH3 domain (residues 65-129) potentially interacting with the actin cytoskeleton (53, 54). The shaded and black boxes within the essential E domain show regions with sequence similarity to the Cdc25 honrologs Ste6 (Schizosacchnroniycespornbe, 911 residues) (46),SOS (son of sevenless, Drosophila, 1595 residues) (47), and Ras-GRF (Ras-specific guanylatereleasing factor from rat, 1244 residues) (48). (Bottom) Sequence alignments of conserved regions probably involved in Ras-specific interactions (left, see shaded boxes above), and in catalyzing the GDPIGTP exchange or GDP release (right, see black boxes above). Amino acids conserved in all four sequences are indicated by black circles; those conserved in only three sequences, by open circles.

“return to the cycle” in a cdcl5-2 background after release from thermal arrest (A. Schwed and H. Kiintzel, unpublished data; see Section V). 2. NUTRIENT RESPONSE The N domain of the Cdc25 protein is dispensable for viability (35, 36, 41), but controls a number of nutrient-dependent properties, such as growth on nonfermentable carbohydrates, sporulation (of homozygous diploids), and glucose-induced transient accumulation of CAMP(41, 42, 52). Interestingly, an SH3 domain is found close to the N-terminus of Cdc25 (53);SH2 and SH3

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elements are involved in protein-protein interactions of membraneassociated signaling proteins and may also control cytoskeletal interactions (54). A Cdc25 homolog of insect and mammalian cells (mSOS) is docked to transmembrane receptors via the SH2/SH3 protein Grb2 (55). The small C domain (containing the last 38 residues) is required for feedback inhibition of adenylate cyclase, since its deletion prevents the rapid decrease of cAMP following the glucose-induced hyperactivation of adenylate cyclase (52). Recent evidence suggests that the Cdc25 protein controls a second nutrient-induced signaling pathway involving phosphoinositidase as an effector (56).The evidence is based on the observation that the cellular level of the second messenger inositol 1,4,5-trisphosphate (IP,), a product of the phosphoinositidase-catalyzed cleavage of PIP,, increases 3- to 4-fold within a few minutes after the addition of a nitrogen source (e.g., ammonium sulfate or amino acids) to starved yeast cells. The elevated IP, content persists for at least 1 hour, in contrast to the glucose-induced transient increase in CAMP. Both induction effects appear to be nutrient-specific, since glucose does not induce IP,, and nitrogen does not induce cAMP in starved cells, and both effects depend on an intact Cdc25 protein. Interestingly, the conditionally lethal cdc25-I mutation (mapping at position 2 within the essential domain; see Fig. 1) affects the formation of IP, more than that of CAMP: the IP, level in cdc25-1 drops to zero in starved cells, and nitrogen induction of IP, is completely abolished even at permissive temperatures. The second product of PIP, hydrolysis, diacylglycerol (DAG), is also induced (a %fold increase within 2 minutes) by nitrogen feeding of starved yeast cells (56),although most DAG is expected to derive from other sources (e.g., hydrolysis of phosphatidylcholine or dephosphorylation of phosphatidic acid (57).Indeed, the molar ratio of DAG to IP, extractable from growing yeast cells is about 30 : l (51, 56). The nucleotide sequence of the recently cloned PLCl gene predicts a homolog of mammalian phosphoinositidase (PI-PLC), showing a special similarity to enzymes of the 6 class (58-59a). The PLC1 gene is important for growth, since its deletion causes temperature sensitivity (growth arrest above 37°C) (59,59a)or even lethality, depending on the genetic background (58).The PLCl gene encodes a Ca,+-dependent PIP,-specific PI-PLC controlling osmoregulation and nutritional responses (5%). Interestingly, Plcldeficient haploid cells lose their viability upon nitrogen limitation (59a), suggesting that Plcl is involved in nitrogen sensing and in the Cdc25dependent nitrogen-induced formation of IP, and DAG. 3. MEMBRANE ASSOCIATION

In uitro studies have suggested that Cdc25 forms a membrane-associated ternary complex with Cyrl and Ras (60, 61). The catalytically active Cyrl

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HANS KUNTZEL ET AL.

protein itself is found both in. the cytosol and peripherally bound to the plasma membrane (62), and the membrane-bound form depends on the presence of Ras proteins being anchored to the plasma membrane by Cterminal fatty acylation (63).The Cyrl protein appears to be relocalized to the membrane in the absence of Ras proteins, if the Cdc25 protein is overproduced, suggesting a direct interaction between Cyrl and Cdc25 under these conditions (61).The 180-kDa Cdc25 protein is detectable by Western blot analysis in cells containing the CDC25 gene on multicopy plasmids; however, cellular localization studies have produced conflicting results. The protein was found in the insoluble cytoskeleton fraction (51, 64),tightly bound to the membrane fraction as an intrinsic membrane protein (65) or distributed between the cytosol and membranes (44, 66).

4. FEEDBACK INHIBITION The CAMP levels of mutants affected in the Cdc25/Ras/Cyrl/cAPK control chain can vary over at least a 10,000-fold range, and the analysis of these mutants has revealed a rigorous feedback control loop that depends on the presence of Cdc25, Ras, and an active CAMP-dependent protein kinase (67). A Cdc25- and Ras-dependent transient accumulation of CAMP is observed, if starved yeast cells are fed with glucose (42, 52, 68, 69), and the drop in CAMP following the hyperactivation of adenylate cyclase could be explained by a feedback inactivation of Cdc25 and/or Ras upon CAMP-dependent phosphorylation. Indeed, the membrane-bound Cdc25 protein was shown to be phosphorylated during glucose induction, leading to a partial release into the cytosol(66). On the other hand, the elimination of two potential cA-PK/PKC target sites from the C-terminal Cdc25 domain prevents the transient hyperactivation of adenylate cyclase, instead of affecting only the feedback drop in CAMP, and the deletion of potential cA-PK sites from the N-terminal half of the Cdc25 protein (see Fig. 1)is phenotypically neutral (42). Perhaps other protein kinases (recognizing Cdc25 target sites to be identified) are involved in a glucose-induced CAMP-dependent phosphorylation cascade.

C. Ira1 and Ira2 The CAMPcontrol pathway is negatively regulated by the IRA1 and IRA2 genes, which encode very large proteins of 2938 and 3079 residues, respectively (70, 71).Disruption of both genes does not affect viability, but renders cells sensitive to heat shock and nitrogen starvation, and both Ira proteins act additively on Ras proteins by stimulating the intrinsic GTPase activity of Ras (72). A central domain of the Iral protein has sequence similarity to mammalian GTPase-activating protein (GAP), and overexpressed bovine GAP is a suppressor of iru mutants. On the other hand, the deletion of a C-terminal domain (residues 2515-2938) from the Iral protein is sufficient to suppress

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the lethality of CDC25 disruptions (70).Thus, Iral acts as an antagonist to the GDP/GTP-exchanging Cdc25 protein, but the C-terminal domain is apparently more important for Ras GTPase stimulation than the central GAP-homologous domain. The C-terminal Iral domain is also required for feedback inhibition of adenylate cyclase following a glucose-induced hyperactivation (70). The C-terminal Iral deletion leads to an elevated membrane-bound adenylate cyclase activity, whereas a central disruption of IRA1 (removing most of the protein, including the central GAP-related domain) has an opposite effect on adenylate cyclase: the membrane-bound activity is strongly reduced, and most of the activity is found in the cytosol (73).Thus, the Iral protein is apparently required to anchor the Cyrl protein to the membrane, in addition to stimulating the Ras GTPase activity. Indeed, the lRAl sequence predicts at least five potential transmembrane domains to suggest a receptor-like serpentine topology (70, 73).

D. Nutrient-Signa Iing Pathways Figure 2 summarizes some features of the Cdc25-dependent nutrientsignaling system, with at least seven protein components (Cyrl, Cap, Rasl/ Ras2, Cdc25, and Iral/Ira2) forming the core of a membrane-associated complex. The catalytic subunit of adenylate cyclase (Cyrl) appears to be anchored to the inner side of the plasma membrane by interacting both with the transmembrane protein Iral (or Ira2) and with Ras proteins, which are attached to the lipid phase through their C-terminal fatty acids.

glucose nitrogen

GDP

ATP

CAMP

1

Ca2+

FIG. 2. Nutrient-signaling pathways in S. cereuisiae. Details of this schematic representation are discussed in Section 1I.D.

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HANS KUNTZELETAL.

The N-terminal domain of the bifunctional Cap protein probably interacts both with Cyrl and Ras, while the C-terminal domain controls the nutritional response of adenylate cyclase (possibly by interacting with Cdc25) as well as the cytoskeletal organization (by interacting with actin filaments or with actin-binding profilin). The Cdc25 protein interacts with GDP-bound and/or nucleotide-free Ras, and may also interact with profilin, since temperature-sensitive cdc25 mutations confer lethality at 25°C to profilindeficient strains (H. Kuntzel, unpublished observation). The Cdc25dependent signaling complex possibly includes Plcl, a PIP2-specific phosphoinositidase, and may also contain other yet unidentified nutrient-sensing proteins (e.g., transporters or receptors for glucose and nitrogen sources) acting upstream of Cdc25. Addition of glucose to starved cells leads to a Ras-dependent transient hyperactivation of adenylate cyclase (Cyrl), whereas nitrogen sources (e.g., ammonium or amino acids) stimulate the formation of the second messengers IP, and DAG, products of PLC-catalyzed PIP, hydrolysis (DAG also derives from other sources). Both nutrient effects depend on the intact Cdc25 protein, suggesting the involvement of the N-terminal domain as a nutrient sensor (see Section 11,B). The PLC1-encoded phosphoinositidase is involved in nitrogen sensing and probably acts downstream of the Cdc25 protein. Little is known about the in uiuo functions of protein kinases activated by the nutrient-induced second messengers CAMP and DAG. While there are only three known isoforms (Tpkl, Tpk2, and Tpk3) of CAMP-dependent kinases (39),the number and identity of DAG-activated kinases are still open. The latter group may include a yeast homolog (Pkcl) of mammalian DAGactivated protein kinase C (74), although Pkcl activation by DAG has not yet been demonstrated. A few stress genes (e.g., SSA3 and C T T I ) containing a CAMP-responsive cis element in their upstream activating sequence are negatively controlled through nutrient-induced phosphorylation and inactivation of gene activators (75, 76), and Pkcl appears to trigger a phosphorylation cascade involving a group of protein kinases (+Bckl+Mkkl/2+Mpkl) related to mammalian mitogen-activated protein kinases (77-79). It remains to be shown whether the second messenger IPS triggers a release ofcalcium ions from intracellular stores (e.g., vacuoles or endoplasmic reticulum) into the cytosol, as in higher eukaryotes (80).The PLCl-encoded phosphoinositidase may indeed be involved in the control of intracellular calcium, since the temperature-sensitive and chromosome missegregation phenotype of plcl-1 cells has been shown to be partially suppressed by exogenous calcium (59). Furthermore, Plcl-deficient strains are much less sensitive to the growthinhibitory effect of high exogenous CaC1, than wild-type strains ( 5 9 ~ ) .

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111. Transcriptional Control of G1/S Genes

A. Periodic Fluctuation of mRNAs Cell-cycle-specific gene expression depends on various parameters that are only partially understood. Transcriptional regulation involves the temporal activation and/or derepression by trans-activating and DNA-binding proteins recognizing specific promoter sequence elements (cell-cycle boxes). However, the transient accumulation of mRNAs also depends on the RNA turnover rate, which itself could vary during the cell cycle. Furthermore, the protein products of fluctuating mRNAs may be additionally regulated in a cell-cycle-specific manner by posttranslational modification (e.g., by phosphorylation/dephosphorylation), affecting subcellular targeting, turnover, and functions during their execution points. Since a transient gene activator itself may be regulated at the transcriptional and/or posttranslational level at specific phases of the cell cycle, the availability of key components executing cell-cycle-specific functions may depend on activating cascades, which could start well ahead of the final execution points. A compilation of periodically transcribed genes is presented in Table I. Most of these genes are transiently expressed at the late G1 phase and are functionally involved in the control of GUS transition, or START. The group of G U S genes can be further subdivided into a smaller group encoding GUS cyclins (stage-specific activators of the Cdc28 protein kinase) (81-83), and a larger group encoding proteins involved in DNA synthesis (84-87). The two cyclin genes CLB5 and CLB6 could also be placed into the DNA synthesis group, since the Clb5- and Clb6-activated Cdc28 kinase controls some aspects of DNA replication (83).Similarly, the DBF4 gene product has a cyclin-like function for DNA synthesis, since it is a stage-specific cofactor of the Cdc7 protein kinase, an enzyme required for the initiation of replication (2, 84). Not all transcripts of GUS-controlling genes accumulate at late G1, as shown in Table I. The genes SW15 and ACE2 are expressed mainly at G2 (88, 89), but their products are required to activate the mother-specific endonuclease gene HO (90)and the chitinase gene CTSl (89), respectively, at late G1. The Swi4 protein is required to activate the genes C T S l , HO, C L N l , and CLN2 at late G I , but is made earlier, since the SW14 mRNA maximum precedes the HO mRNA maximum (91).

B. The Cyclin (Cln) Proteins The cyclin genes C L N l , CLN2, and CLN3 encode stage-specific activators of the Cdc28 protein kinase, which are required only for the GUS phase transition and are rapidly degraded during the S phase (reviewed in 13). The

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HANS KUNTZEL ET AL.

TABLE I PERIOD~CALLY TRANSCRIBED GENES Gene code

Gene function

Phase of mRNA accumulation

Reference

CTSl HO CLNl CLN2 HCS26 CLB5 CLB6 DBF4 CDC2l CDC8 RNRl RFAl RFA2 RFA3 POL1 POL2 DPB2 DPB3 POL3 POL30 PRIl PR12 CDC9

Chitinase Endonuclease (mating type switching) GUS cyclin GUS cyclin GUS cyclin GUS cyclin GUS cyclin Cofactor of Cdc7 kinase Thymidylate synthase Thymidylate kinase Ribonucleotide reductase subunit Origin-binding protein Origin-binding protein Origin-binding protein DNA polymerase I DNA polymerase I1 DNA polymerase 11 subunit B DNA polymerase I1 subunit C DNA polymerase 111 PCNA (replication factor) DNA primase I DNA primase I1 DNA ligase

Late G I Late G I Late G1 Late GI Late GI Late G1 Late GI Late GI Late G1 Late GI Late G I Late G I Late G1 Late G1 Late G1 Late G1 Late G I Late GI Late G1 Late G I Late G1 Late G I Late G I

89 90 81 81 82 83 83

CLB3 CLB4 H2A H2B

Mitotic cyclin Mitotic cyclin Histone 2A Histone 2B

S S S

92 92 93 93

CLBl CLB2

Mitotic cyclin G2 cyclin Activator of HO Activator of CHSl Transmembrane protein Initiation of replication

G2 G2 G2 G2

Protein kinase Initiation of replication Function unknown Activator of HO, C L N l , CLN2, and HCS26

G21M M/G1 Early G I Early G1

sw15

ACE2 MSTl CDC46 DBF2 CDC6 EGTl

s w14

S

62 G2

84

85 85 86 87 87 87 85 85 85 85 85 85 85 85 85

94 94

88 89 95 96

97 98 95 91

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deletion of all three genes is lethal, whereas cells expressing only one of the three genes are viable, suggesting overlapping functions. However, the regulation and function of the CLN3 gene are quite different from those of the rather equivalent gene pair CLNl ICLN2. CLN3 is expressed throughout the cell cycle, whereas CLNl and CLN2 are transiently transcribed at the late G1 phase (81, 82, 95, 99). Cln3 is a much rarer protein than Clnl or Cln2, and the Cdc28/Cln3 complex has a weaker histone H1 kinase activity than the two other complexes (99). Furthermore, the Cdc28/Cln3 kinase is obviously not required beyond START (99), whereas the Cdc2WClnl and/or Cdc28/Cln2 kinases are involved in post-START pathways such as bud formation (see Section V). The Cln3 protein has been suggested to be crucial only for the mother cell, which does not require a size-control step for START (11). However, more recent data (99) suggest an important function of Cln3 in daughter cells as well: the transcription of other GUS cyclin genes, such as CLB5, ORFD, and HCS26, depends on Cln3 in the absence of Clnl and Cln2, and Cln3 is proposed to be a general upstream activator of START-catalyzing GUS cyclins. Since the constitutively made Cln3 has a short half-life, its critical abundance may depend on growth rate, protein synthesis, and cell size, and Cln3 could provide a link between “growth to critical size” and performance of START (99). A dominant mutant allele (CLN2-I, encoding a C-terminal deleted longlived Cln2 protein) prevents a G1 arrest upon nitrogen starvation and triggers a premature GUS phase transition at a small size (100).These and other observations (13)suggest that Cln proteins must be absent at the onset of the nutrient-dependent growth-control step, and establish the temporal order: nutrient-dependent growtbCln3-induced CLNlICLN2 expression+Cdc28 activation-start of S phase.

C. Swi4 and Swi6 The cell-cycle-specific transcription of CLNl and CLN2 genes involves two regulatory proteins, Swi4 and Swi6 (82, 101).The SWZ4 gene is essential for haploid cell viability at 37°C and for diploid cells at all temperatures, and the temperature sensitivity of haploid m i 4 cells is suppressed by multicopy plasmids containing the GUS cyclin genes CLNl, CLN2, and HCS26 (82). Cells lacking a functional SW16 genes are viable, but the deletion of both SWZ4 and SW16 is lethal, and both functions are required to activate CLNl and CLN2 (101). The Swi4ISwi6 complex specifically binds to a cis element of the CLNl and CLN2 promoter regions termed SCB (Swi4ISwiG-dependent cell-cycle box), having the consensus sequence CNCGAAA (82, 101). The Swi4/Swi6 complex or SCB-binding factor (SBF) was originally identified as the activa-

14

HANS KUNTZEL ET AL.

tor of the HO gene, which encodes an endonuclease involved in mating type switching (90, 102). The Swi4 protein contains an N-terminal DNA-binding domain recognizing the SCB element, whereas the C-terminal domain interacts with the Swi6 protein (103-105). Swi6 does not bind directly to SCB, but controls the accessibility of the sequence-specific Swi4 DNA-binding domain within the complex; however, the DNA-binding function of Swi4 does not depend absolutely on the Swi6 protein, at least if Swi4 is overproduced (105). The cell-cycle-specific transcription of CLNl and CLN2 not only requires the Swi4/Swi6 complex, but also depends on a functional CDC28 gene (106, I O V , suggesting that Clnl and Cln2 proteins promote their own synthesis by a positive feedback control loop involving a Clnl/ClnZ-activated Cdc28 protein kinase (82,106,107).The Cln3 protein is made throughout the cell cycle and does not promote its own synthesis. However, Cln3 is required to stiinulate the expression of nonfunctional clnl and cZn2 reporter genes in the absence of functional CLNl and CLN2 genes (106).Thus, the activation of the Cdc28 kinase by Clnl, Cln2, or Cln3 is sufficient to promote the Swi4/SwiG-dependent positive feedback loop. Most of the periodically expressed DNA synthesis genes contain one or several cis elements termed MCB (MZuI cell-cycle box, consensus sequence sequence ACGCGTNA) in their promoter regions, generally between 90 and 250 b p upstream of the start codon (85). For some of these genes (e.g., POLl, CDCS, and CDC2l) the periodic transcription has been demonstrated to depend on the presence of at least one MCB (108, l o g ) , and a single MCB can confer GUS-specific transcription of reporter genes, if placed into a suitable context (110, 111). The periodic transcription of many, if not all, MCB-containing genes requires SWIG function (112-114). The Swi6 protein is a component of an MCB-recoghizing complex termed either MBF (MCB-binding factor) or DSCl (DNA synthesis control), together with DNA-binding p120 protein component (112). A 17-kDa MCB-binding protein (108)has been suggested to derive from the DSCl complex by proteolysis (85). The periodic transcription of MCB-containing genes does not necessarily depend only on these MCB elements alone, as has been shown in the case of the SWZ4 gene (115).The SW14 upstream activating sequence contains three MCBs, but the deletion of all three elements does not abolish periodicity. On the other hand, the periodic fluctuation of SWZ4 mRNA remains dependent on SWIG function, suggesting that other Swi6-dependent cis elements conferring periodicity must be present on the SWI4 promoter (115). Figure 3 summarizes some features of the Swi4- and Swi6-dependent gene expression at late G1 (START). The Swi6 protein is a common trans-

S. cerevisiae

15

CELLS

0 Swi6

t

0 Swi6

__..________.._......--------........---------

I I

I

I

FIG. 3. Transcriptional activation of START-inducible genes by Swi6 and Swi4. Shown are three genes (coding regions boxed) containing either MCBs (SWZ4 and POL1) or SCBs (CLN2) in their promoter regions. A transcriptional complex (TC)initiating gene transcription (wavy arrows) is induced either by the MCB-binding factor (MBF, containing p120 and Swi6)or by the SCB-binding factor (SBF, containing Swi4 and Swi6). The Cdc28/CIn2 (or Cdc28/Clnl) conplex is believed to stimulate the transcription of MBF- and SBF-controlled genes by phosphorylating SwiG and Swi4.

activating component of the two complexes SBF (Swi6/Swi4) and M B F (Swi61p120) recognizing the cell-cycle boxes SCB (CTCGAAA) and MCB (ACGCGT), respectively. The CLN2 gene represents the group of SBFcontrolled genes (including HO, C L N l , and HCS26), whereas the DNA polymerase gene POL1 represents the large group of MBF-controlled DNA synthesis genes (also including the G1/S cyclin genes CLB5 and CLBG and the SW14 gene). Most of the SBF- and MBF-controlled genes are coordinately expressed at late G I , whereas the SW14 gene is expressed at an earlier stage. Both Swi4 and Swi6 may be phosphorylated and activated by the Clnl/Cln2-associated Cdc28 protein kinase during the positive feedback loop. Indeed, Swi6 i s a phosphoprotein and can be phosphorylated in citro by Cdc28 protein kinase (113).The nucleotide sequence of the SWIG gene

16

HANS KUNTZEL ET AL.

(11 5 4 predicts several potential Cdc28 phosphorylation sites (13)in addition to potential targets for cA-PK, PKC, and casein kinase 11, suggesting the possibility of multiple stage-specific phosphorylations during the cell cycle.

D. Clb5 and Clb6 The large group of MBF-controlled genes includes the two recently discovered cyclin genes CLB5 and CLBG (83, 116, 117). The two gene products constitute a new subgroup, together with the pairs Clbl/Clb2 and ClbNClb4 (92, 94) of the B-type cyclin family. However, despite their structural homologies, the Clb5/Clb6 proteins differ functionally from the other G2-specific mitotic cyclins, since they are involved in the control of S phase entry and initiation of DNA replication (83). The two genes are not essential for viability, and the deletion of CLBG has no phenotypic consequences. However, strains lacking CLBS have a %fold extended S phase in comparison to the wild type, and cells lacking both CLBS and CLBG start their S phase with a delay of at least 30 minutes relative to bud emergence (83).These phenotypic properties suggest that the punctual initiation of replication, rather than DNA chain elongation, is controlled by Clb5 and Clb6, although it remains open how replication is triggered in the absence of the two cyclins. The Clb5/Clb6 proteins are suggested to be activators of the Cdc28 protein kinase (83),as are all other known cyclins (13, 81, 83, 92, 94). The promoter regions of the CLBS and CLBG genes do not contain SCBs, as do those of CLNl and CLN2, but contain clusters of four and three MCBs, respectively, having a single mismatch to the MCB consensus in all but two elements. Indeed, the GUS-specific transcription of CLB5 and CLBG is controlled by MBF, together with DNA synthesis genes, but is independent of the Swi4-containing SBF (83). The GUS-specific regulation of CLBSI CLBG also involves the Cln3-activated Cdc28 kinase as a common trigger of G I cyclin transcription (99), and the Clnl tCln2-activated Cdc28 kinase not only participates in a positive feedback loop amplifying CLNl ICLN2 transcription (82, 106, 107), but also stimulates transcription of the CLB5ICLB6 gene pair (83).

E. Cdc46 and Cdc6 Two members of the “DNA synthesis” gene group, CDC46 and CDC6, deserve special comment, because they are expressed at earlier stages of the cell cycle. The Cdc46/Mcm5 protein controls the initiation of replication together with three other proteins (Cdc45, Cdc47, and Cdc54), probably by interacting with autonomously replicating sequence (ARS) regions of the genome

S . cerevisiae

CELLS

17

(96, 118, 119). The Cdc46 protein shows sequence similarity to McmS and Mcm3, two other proteins important for ARS activity, and is not only transcriptionally, but also posttranslationally, regulated in a cell-cycle-specific manner: the gene is expressed at the G2 interval, the protein stays in the cytosol during M phase, quickly moves into the nucleus as mitosis is completed, and persists there until its execution point is reached at early S phase (96). The Cdc46 sequence predicts an N-terminal PEST region, which is found in proteins of higher turnover rate (120), including the Cln proteins (100). A single MZuI recognition site is present in the CDC46 upstream region, but it is not known whether this potential MCB is important for the periodic transcription. The CDC6 gene product is another protein involved in the control of initiation of replication (2, 121). Two observations suggest that the Cdc6 protein interacts with ARS regions: cdc6 mutants show a high rate of a 1 : 0 minichromosome loss (122), and the minichromosome loss in cdc6 strains is suppressed by extra copies of ARS elements (123).The Cdc6 protein appears to have another function as a suppressor of nuclear division, indirectly inhibiting the activation of the mitotic form of Cdc28 kinase (124).This latter observation implies that the Cdc6 protein must be removed soon after its execution point at early S phase. The sequence of the cloned CDCG gene (125, 126) predicts a 58-kDa protein containing the consensus elements for a purine nucleotide binding site, a central hydrophobic domain, several potential Cdc28 kinase target sites, and a C-terminal cysteine-rich domain related to metallothionein. Two potential nuclear localization signals and two PEST regions are found at the N-terminal domain, which may direct a cell-cycle-specific nuclear import and turnover similar to that of the Cdc46 protein. The deletion of 12 residues within the central hydrophobic domain is lethal for the cell, suggesting an important role for catalytic hnctioiis and/or nuclear membrane interactions. Furthermore, a bacterial-made Cdc6 fusion protein binds and hydrolyzes both ATP and GTP in vitro (127). The CDC6 transcript fluctuates during the cell cycle, like other MCBcontrolled genes (98). However, our recent data (see Section IV and Fig. 4) indicate that the CDCG gene is not coordinately expressed at the GUS transition point, as previously suggested (98), but is transcribed already at late mitosis (127).The maximuin of CDCG mRNA precedes that of CLN1 by about 15 minutes, if cells are synchronized either by release from pheromone arrest or by release from thermal arrest in cdc15 strains. Arrested cdc15-2 telephase cells (containing an elongated spindle and separated chromosomes) accumulate CDC6 mRNA, but not C L N l mRNA, and CDC6 mRNA disappears at late G1, when CLNl mRNA reaches its maximum. The

G1

M

S

G2

G1

S

+++

+ + +

ND CK BI 100

M

NDCKBI

@

80 +-

e

%

60

40 20 0

-

.-

a,

30

2

Q

?

s

- 20

\

Q

v

a

2

4

10

C

0

30

60

90

120

150

180

210

minutes FIG. 4. Periodic fluctuation of cAMP and mRNAs encoding Clnl and Cdc6 in synchronized cdcl5 cells. A MATa, cdc15-2, ura3, his3, add2 strain was grown to early exponential phase at 25”C, incubated for 4 hours at 37°C and released from the thermal arrest by shift to 25°C (zero time). Aliquots of cell suspensions were removed every 15 minutes to determine morphological parameters (A), the cellular content of cAMP (B) and the level of transcripts of CLNl (C) and CDCG (D). The upper part (A) shows the percentage of anaphase cells with an extended nuclear spindle (0 - O),glusulase-resistant large-budded cells (0- 0),and smallbudded cells (A - A), defining the landmarks of nuclear division (ND), cytokinesis (CK), and bud initiation (BI). The CAMPcontent was determined by itsing an Amershain radioassay kit (52).Standard methods were used for morphological measurements and for the determination of CLNl and CDCG mRNAs by Northern blot hybridization (see, e . g . , 95).

S . cerevisiae CELLS

19

differential timing of CDCG and CLNl expression is also evident during the second cycle upon cdcl5 release: again, CDCG mRNA appears before nuclear division, whereas CLNl mRNA accumulates around cell separation. A similar fluctuation pattern is seen after release from a G1 pheromone arrest, the CDCG mRNA again accumulating at late mitosis (124, 127). The upstream activating sequence region of the CDCG promoter contains two MCBs (ACGCGA and ACGCGT) separated by 6 bp (98),whereas only a single MluI site (ACGCGT) is present at the CDC46 upstream sequence (1 18). The MCB-containing region of the CDCG promoter confers periodic transcription of reporter genes (128) and interacts with an MCB-binding protein complex as efficiently as the corresponding regions of POLl, CDC2, and CDC21 (108).These observations suggest that the CDCG gene belongs to the group of MCB-controlled DNA synthesis genes, although its transcription starts already at late mitosis.

F. The Sit4 Protein Phosphatase The activation of the Cdc28 kinase by GUS cyclins during START not only depends on the Swi4/Swi6-controlled transcription of CLNl ICLN2 at late G1, but also requires the presence of the SIT4 gene product, a protein phosphatase of unknown substrate specificity (129-131). The Sit4 protein is associated with two phosphoproteins (pp155 and pp190) in two separate complexes during the S, G2, and M phases. Sit4 dissociates from these complexes at early G1 and reassociates at the GUS transition point (130). The Sit4 protein is required for the accumulation of SW14, CLNl, CLN2, and HCS26 mRNAs at late G1, but also for DNA synthesis and bud emergence, probably by dephosphorylating components of the positive feedback loop (e.g., Swi6). Since the Sit4 requirement for the accumulation of CLNl and CLN2 mRNAs is at least partially via Swi4, the primary action of Sit4 could be the stimulation of SW14 expression, which in turn would induce CLNl and CLN2 (131). The Sit4 phosphatase is essential for viability only in strains having a certain allele of the polymorphic SSDIISRKI gene (129, 132). The Ssdl protein has no similarity to known protein phosphatases, but probably activates an unidentified phosphatase acting downstream of or in parallel to the Sit4 protein (129, 130). The SSDIISRKI gene is also a suppressor of mutations leading to a constitutive or hyperactive CAMP-dependent protein kinase, such as bcyl (lacking the regulatory subunit of cA-PK) or pde2 (lacking CAMP phosphodiesterase) (129, 132), and the sit4 deficiency is lethal in a bcy l background (129). These genetic interactions suggest that both Sit4 and Ssdl interfere with cA-PK, and that the Sit4 function during late G1 requires a down-regulated cA-PK. One of several possibilities would be a CAMP-dependent phospho-

20

HANS KONTZEL ET AJ.,.

rylation of the Sit4 cofactors pp155 and pp190 at early G1, causing them to dissociate from Sit4. The reassociation with Sit4 at late G 1 could be regulated by removing phosphate groups from the cA-PK target sites, and by phosphorylation through another protein kinase (e.g., Cdc28) (129). Alternatively, a constitutive cA-PK could interfere with Sit4 by phosphorylating and inactivating a common substrate of both enzymes (e.g., Swi6) at START (see Section V).

IV. Periodic Fluctuation of cAMP The nutrient-dependent activation of cA-PK via the Cdc25/Ras/Cyrl control chain plays an important role for the “growth to critical size” step in daughter cells at the early G1 phase (40), and the same protein kinase may have to be down-regulated during the Sit4-dependent transcriptional activation of CLNl and CLN2 genes at late G1. Such a differential modulation of cA-PK during the G1 phase appears to be supported by the observed periodic fluctuation of the intracellular cAMP level in synchronized cells. Small unbudded daughter cells enriched by centrifugational elutriation have a high CAMPcontent, whereas a minimum is reached at the start of a new division cycle, between cell separation and bud initiation (133). We have studied the cAMP fluctuation in yeast cells synchronized by release from thermal arrest of a cdc15-2 train. Figure 4 shows the result of such an experiment. A c d d 5 strain was shifted from a permissive (25°C) to a restrictive (37°C) temperature, leading to the accumulation of cells arrested at late mitosis (telophase state with extended nuclear spindle and chromosomes distributed between mother and daughter) (2). After release from the arrest state (shift back to 25”C), aliquots of the cell suspension were taken every 15 minutes to analyze morphological parameters such as spindle morphology, cytokinesis, and bud initiation, as well as the cellular level of CAMP and the content of CLNl and CDCG mRNAs. The mitotic spindle of cdcl5 cells reproducibly collapses between 30 and 60 minutes after release and reaches a new maximum after 135 minutes. After 60 minutes (first cycle) and 150 minutes (second cycle), 50% of the cells have undergone cytokmesis, followed by the formation of small buds 10 to 15 minutes later. The cAMP level is high in cdcI5-arrested telophase cells and then declines in two steps between 0 to 30 minutes and 60 to 75 minutes to reach a minimum around the G l / S transition. During the S and G2 phases, the cAMP level increases again to reach a maximum after 150 minutes at late mitosis of the second cycle. The bottom of Fig. 4 shows the fluctuation of CLNl (Fig. 4C) and CDCG (Fig, 4D) mRNAs. The CLNl mRNA level is low in arrested telophase cells

S. cerevisiae C E L L S

21

and accumulates around 60 and 150 minutes, similarly to what was previously described (9S), whereas the CDC6 mRNA accumulates already in arrested cells and also precedes the CLNl mRNA maximum at the second cycle by about 15 minutes (see Section 111,E). A comparison of the CAMPand CLNl mRNA fluctuation patterns reveals that cAMP has dropped to an intermediate plateau around the first CLNl mRNA maximum, and that the cAMP minimum is reached at the end of the CLNl expression phase. However, the intermediate cAMP plateau of the second cycle is delayed in relation to the second CLNl mRNA maximum, indicating that both parameters (levels of CLNl mRNA and CAMP)are not strictly correlated throughout the measured time period. Furthermore, the CAMP fluctuation profile of Fig. 4 differs considerably from that of cells synchronized by centrifugational elutriation (133).In this case, small daughter G1 cells collected from an asynchronous culture have a high cAMP content even during the first budding period, whereas the second budding period starts at the cAMP minimum. Thus, the observed cAMP fluctuation patterns seem to depend strongly on the conditions of synchronization. On the other hand, both experiments indicate a transient downregulation of the CAMP-generating system (at nonlimiting nutrient supply) around the start of a new cycle, followed by a rise in cAMP during the S and G2 phases. A high cAMP content in arrested cdcl5 cells has been reported independently (134). In addition, it was shown that a decrease in the cellular cAMP level (e.g., by deleting RAS2 or overexpressing the cAMP phosphodiesterase gene PDE2) is sufficient to rescue the cdclS lesion, suggesting that cells containing a nonfunctional Cdcl5 protein kinase can traverse the M/G1 border by down-regulating cA-PK (134).

V. A Regulatory Network Operating at START Figure 5 summarizes some essential features of the START control network, as described in the previous sections. A central event during GUS phase transition is the temporal transcription of cyclin genes encoding cofactors and stage-specific activators of the Cdc28 protein kinase (CLNl, CLN.2, CLBS, and CLBG). Another group of genes coordinately expressed around START includes DBF4 (encoding a stage-specific activator of the Cdc7 protein kinase) and a large number of genes required for DNA replication (symbolized as DSG, or DNA synthesis genes). The DBF4 gene could also be considered a member of the DSG group, since the Dbf4-activated Cdc7 kinase is required for the initiation of DNA synthesis. All START-inducible genes shown in Fig. 5 contain either SCBs or MCBs

22

HANS KtfNTZEL ET AL.

high cAMP

low cAMP

[clnsl

b

growth to critical size

z"1.

---P early G1 phase

DSG

START

budding

initiation of replication

DNA synthesis

S phase

FIG.5. A START-controllingnetwork. START-induciblegenes (SWZ4 and DBF4) or groups of genes (CLN1,2:CLN1 and CLN2; CLB5,6: CLB5 and CLB6; DSG, DNA synthesis genes) are boxed. pp, Phosphoprotein cofactors of Sit4 (130). For further details see Section V.

as cell-cycle-specific cis elements in their promoters (see also Fig. 4). The permanently made Swi6 protein plays a central role in the transient induction of START genes, since it is a common component of the two activating complexes SBF (Swi4/Swi6, recognizing SCB) and MBF (X/Swi6, recognizing MCB). The DNA-binding p120 component of MBF has not yet been defined genetically, and MBFs controlling the large group of MCBcontaining genes (SWZ4, CLB5, CLB6, and DNA synthesis genes) may contain more than one gene product as MCB-recognizing components. An oversimplification of interactions between inducible genes and activating factors is unavoidable in a schematic representation like that of Fig. 5. For example: (a) the CLNl promoter contains both SCB- and MCB-like elements (85) and may be recognized by both SBF (Swi4/Swi6) and MBF (X/Swi6); (b) the Swi4/Swi6 complex (SBF) controls the temporal transcription of other GUS-specific genes (e.g., HO and HCS26) in addition to CLN2 (all having only SCBs in their promoters); (c) the transient SWZ4 mRNA accumulation precedes that of other MBF- and SBF-induced transcripts, and Swi6 controls the temporal transcription of SW14 by interacting with other cis elements after deletion of MCBs (98).

S . cerevisiae

CELLS

23

The permanently made Cdc28 protein associates with the permanently made Cln3 protein, as well as with the START-included cyclins Clnl, Cln2, ClbS, and Clb6, to form distinct species of activated protein kinases. The Cdc28/Cln3 complex is suggested to act as a common inducer of CLN1, CLN2, CLB5, CLB6, and SW14,possibly by phosphorylating proteins required for the basal transcription of these genes. The Cdc28ICln3 kinase may also be involved in the induction of other MBF-controlled DNA synthesis genes, although this remains to be tested. The Clnl/Cln2-activated Cdc28 kinase is not only responsible for the positive feedback loop of SBF-controlled CLNl ICLN2 transcription, but is also required to stimulate transcription of the MBF-controlled CLB5, CLBG, SW14,and DNA synthesis genes (13, 103). This stimulatory action is probably mediated by phosphorylation of SBF and MBF components, and a likely candidate substrate would be the Swi6 protein as a common trans-activator of START-inducible genes. In the absence of Clnl and Cln2, the expression of Clb5 and Clb6 depends on the Cdc28/Cln3 function, explaining why Cln3 is essential in the absence of Chi1 and Cln2 (99). Cells lacking Clnl and Cln2 show a delayed start of S phase and are also delayed in budding (83),whereas cells lacking ClbS and Clb6 are only delayed in entering the S phase (103).Thus, the Cdc28/Clnl and Cdc28/Cln2 kinases appear to promote budding in addition to stimulating DNA replication, whereas the Cdc28/ClbS and Cdc28/Clb6 kinases are more directly involved in DNA replication, and may control bud initiation only in the absence of Clnl an Cln2. The Cdc28/Clb5 kinase specifically controls the initiation of replication and, together with the enzyme activated by the mitotic cyclins Clb3 and Clb4, the formation of the mitotic spindle (103). The START control network involves another important protein, the Sit4 protein phosphatase, which is made throughout the cell cycle, but is modulated in a stage-specific way by two phosphoprotein cofactors. The Gl/Sspecific Sit4 phosphatase complex is functionally comparable with the GUSspecific Cdc28/Cln1,2 protein kinase complex: both enzymes stimulate the transcription of SW14,C L N l , CLN2 (and possibly other MBF-controlled genes), and both enzymes are probably involved in the modification of transcriptional factors such as Swi6. Two observations suggest that CAMP-dependent phosphorylation may be down-regulated during START: (a) the Sit4 function is counteracted by a constitutive (CAMP-independent) cA-PK in strains lacking the regulatory Bcyl subunit (synthetic lethality of sit4 and hcyl), and (b)the CAMPcontent drops to a minimum around START. The permanently made regulatory proteins Cln3 and Swi6 contain skveral potential cA-PK target sites (115, 135) and are possible candidates for monitoring the growth status of G1 cells, being modulated by stage-specific phosphorylation/dephosphorylation

24

HANS KUNTZELETAL.

events (99, 112). During the high CAMP stage at late mitosis and early G1, the Swi6 protein may be kept in a START-incompetent form by CAMPdependent phosphorylation, and the Gl/S-specific Sit4 complex could be required to activate the protein by dephosphorylation. Although CAMP-dependent protein phosphorylation is a central feature of nutrient-dependent growth control, we know very little about the in vivo functions of cA-PK. Similarly, the roles of the various cyclin-associated Cdc28 protein kinase species must be studied further, to better understand the complex regulatory network operating at the GUS phase transition. The identification of in uivo substrates for these protein kinases will certainly be a major task of future cell biology research.

VI. Succhuromyces cerevisiue Gene Symbols ACE BCK BCY BUD CAP CDC CHS CLB CLN CTS CYR DBF DPB EGT H2A H2B HO IRA MCM MKK MPK M ST ORF PDE PFY PLC POL

activation of CUP expression bypass of C kinase bypass of cyclic AMP deficiency bud formation cyclase-associated protein cell division cycle chitin synthase cyclin B cyclin chitinase adenylate cyclase deficient dumbbell former DNA polymerase B early G phase transcription histone 2A histone 2B homothallic switching inhibitor of ras minichromosome maintenance deficiency mitogen-activated protein kinase kinase mitogen-activated protein kinase mitose-specific transcription open reading frame phosphodiesterase (CAMP) profilin phospholipase C DNA polymerase

25

S. cerevisiae CELLS PRI

RAS RFA RNR

RS R SIT S PA SRV SSD SWI TPK

URA

primase homologous to RAS proto-oncogene replication factor A ribonucleotide reductase ras related suppression of initiation of transcription spindle pole antigen suppressor of Ras-Val19 suppressor of SIT deletion homothallic switching deficient threonine protein kinase uracil requiring

ACKNOWLEDGMENTS We thank D. Gauss and T. Munder for comments on the manuscript. H.-W.R. and W. Z. were supported by a grant of the Deutsche Forschungsgemeinschaft.

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