Yeast (Molecular and Cell Biology) || Yeast Growth and the Yeast Cell Cycle

7Yeast Growth and the Yeast Cell Cycle7.1Modes of Propagation

As already briefly indicated, yeast can follow two modes ofreproduction: (i) asexual budding, the most common modeof vegetative reproduction in yeasts, or (ii) mating of haploidcells of opposite mating-type that can propagate vegetativelyor – under starving conditions – be induced to sporulate. Inbudding cells, the chromosomes are duplicated in a mitoticcycle, and distributed between mothers and daughters fol-lowed by cell separation, while sporulation involves meiosisto generate four (haploid) ascospores. Various uniqueaspects of these lifestyles of yeast, including budding, cellpolarity, spindle formation, cytokinesis, cell division, andsporulation, have been intensively studied at the cellular andmolecular levels.

7.1.1Vegetative Reproduction

Budding is the most common mode of vegetative growthin yeasts and multilateral budding is a typical reproductivecharacteristic of ascomycetous yeasts, including Saccharo-myces cerevisiae. The eukaryotic cell cycle involves bothcontinuous events (cell growth) and periodic events (DNAsynthesis and mitosis). Commencement and progressionof these events in yeast can formally be distinguished intopathways for DNA synthesis and nuclear division, spindleformation, bud emergence and nuclear migration, andcytokinesis. However, from a molecular viewpoint theseprocesses are intimately coupled.

The cell cycle can be defined as the period between divi-sion of a mother cell and subsequent division of its daugh-ter progeny. The regulatory mechanisms that order andcoordinate the progress of the cell cycle have beenintensely studied (overviews: Mata and Nurse, 1998;Futcher, 2000; Lauren et al., 2001). The cell cycle (Fig-ure 7.1) consists of two separable phases – interphase andmitosis. While in interphase three sections (G1, S, and G2

phase) are distinguished, mitosis comprises four sections– prophase (chromosome condensation), metaphase (chro-mosome alignment), anaphase (chromosome separation),and telophase (chromosome decondensation).

7.1.1.1 BuddingBudding can occur in both haploid and diploid yeast cells. Inthis context, it has to be recollected that a and a cells exhibitan axial budding pattern (the mother cell buds immediatelyadjacent to its last daughter; the daughter cell buds towardits mother), whereas diploid cells exhibit a bipolar buddingpattern (the mother cell can bud at or near either of its poles;the daughter buds away from its mother). In the case of axialpattern, the polarized cell growth may facilitate matingbetween cells of the opposite mating-type, whereas in thebipolar pattern it may allow cells to grow away from eachother (Figure 7.2).

Yeast buds are initiated when mother cells attain a criticalsize, at the same time starting with DNA synthesis. This isfollowed by localized weakening of the cell wall and allowsextrusion of cytoplasm in an area bounded by a newly syn-thesized cell wall separating the bud from the mother cell.The cleavage plane, which bisects the spindle axis at cyto-kinesis, is defined by a ring of proteins (the septins) and by achitin ring. Once mitosis is complete and the bud nucleusand other material have migrated into the bud, cytokinesisensues, and a septum is formed in the isthmus betweenmother and daughter, and the chitin ring after cell divisionleaves a characteristic bud scar.

Budding in S. cerevisiae has been intensively studied by IraHerskowitz and collaborators and by many others. Buddingturned out not to be a randomized but a highly controlledprocess (Horvitz and Herskowitz, 1992; Roemer, Vallier, andSnyder, 1996; Herskowitz, 1997; Madden and Snyder, 1998;O’Shea and Herskowitz, 2000; Vogel and Snyder, 2000).The establishment of cell polarity in S. cerevisiae is governedby a “morphogenetic hierarchy” involving the interplay ofvarious genes which determine the orientation of cyto-skeletal elements.

Establishment of cell polarity is triggered either byinternal signals from the cell cycle engine (budding) or byan external signal cascade commencing with a pheromonegradient (mating) (Figure 7.3). In response to cell cyclesignals or mating pheromone stimulation, the essential,small GTPase Cdc42p and the actin cytoskeleton becomepolarized – Cdc42p is accumulated as a “polar cap,” andactin cables become oriented in a polarized actin networkwith patches concentrated near the growth site and cables

Yeast: Molecular and Cell Biology, Second Edition. Edited by Horst Feldmann.# 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

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oriented into the direction of growth in order to achievetargeted secretion towards these caps. Nucleation of actincables is thought to be stimulated by GTP-Cdc42p, thusaugmenting the transport of more Cdc42p along thesecables towards the critical sites. Either this process leadsto bud formation or to a mating projection (“shmoo”)(Slaughter, Smith, and Li, 2009).

Concentrating on bud site selection, all key molecularcomponents governing this process (Chant and Herskowitz,1991; Chant et al., 1991; Park, Chant, and Herskowitz, 1993;Stamnes et al., 1995; Sanders and Herskowitz, 1996; Zahner,Harkins, and Pringle, 1996; Gray et al., 1997; Takizawa et al.,1997), polarized cell growth (Chenevert et al., 1992; Zimanet al., 1993; Chenevert, Valtz, and Herskowitz, 1994; Chantet al., 1995; Herskowitz et al., 1995; Valtz, Peter, andHerskowitz, 1995; Halme et al., 1996; Valtz andHerskowitz, 1996; Amberg et al., 1997; Park et al., 1997;Park, Sanson, and Herskowitz, 1999; Sheu, Barral, andSnyder, 2000; Bidlingmaier et al., 2001; Ni and Snyder,2001), and septin ring formation (see Section 7.1.1.2) havebeen characterized.

Fig. 7.1 Yeast cell morphology during the

cell cycle.

Fig. 7.2 Bud site selection patterns in S. cerevisiae.

Fig. 7.3 Establishment of cell polarity. (After Slaughter, Smith, and Li,

2009.)

176j7 Yeast Growth and the Yeast Cell Cycle

Several genes for bud site selection (BUD genes) areinvolved in orienting the actin fibers. Genes for bud forma-tion (e.g., Cdc42p, Cdc24p, and Bem1p) also direct cell sur-face growth to the developing bud. These processes requirenumerous small GTPases (cf. Section 6.1.2). Rsr1p (Bud1p)is a Ras-like GTPase required for the first step within thebudding machinery, bud site selection, that links the budscar signal with the Cdc42 polarization module. RSR1 wasfirst identified as a high-copy suppressor of a temperaturesensitive mutation in the guanine nucleotide exchange factor(GEF) for Cdc42p, Cdc24p. Mutations in RSR1 result in arandom budding pattern rather than the usual axial patternin haploids and bipolar pattern in a/a diploids. Rsr1p is alsorequired for morphological changes in response to matingpheromone, and for efficient cell fusion. Rsr1p shows signif-icant similarity to many proteins in the Ras superfamily,especially to mammalian Rap-GTPases. The GTPase-activat-ing protein (GAP) for Rsr1p is Bud2p that appears to nega-tively regulate Rsr1p, while Bud5p, the GEF, positivelyregulates it. This GTPase complex is associated with theplasma membrane through Rsr1p. Rsr1p also has beenfound to interact with Cdc24p when it is in its GTP-boundstate, but with Bem1p when it is in its GDP-bound state. Thefunction of this interaction may be to localize Bem1p,Cdc24p, and also Cdc42p at the nascent bud site where theycan reorganize the actin cytoskeleton to establish polariza-tion, because Bem1p helps to establish the cellular polaritythat is required for both bud and shmoo formation.

Bem1p is a protein with two SH3 domains that bindsCdc24p. As Bem1p also binds Ste5p and Ste20p, which arecentral components of the mating pathway, Bem1’s role maybe to connect the mating signal to the proteins that inducethe appropriate changes to the actin cytoskeleton. Myo4p isrequired for the proper regulation of mating-type switchingthrough its general role in transporting mRNAs to the tip ofthe bud, one of which is responsible for the production ofAsh1p, a zinc finger inhibitor of HO transcription, whosemRNA is localized and translated in the distal tip of ana-phase cells, resulting in accumulation of Ash1p in daughtercell nuclei. Ash1p is a potential substrate of Cdc28p. ThesemRNAs associate with Myo4p via the She2p and She3padapter proteins. She3p links Myo4p to its cargo. She2pbinds to ASH1 and IST2 (a gene encoding a plasma mem-brane protein that may be involved in osmotolerance (Taki-zawa et al., 2000)) for mRNAs, while She3p binds to bothShe2p and Myo4p. The corresponding ribonucleoprotein(RNP) complexes are thought to translocate to the bud cortexalong actin cables because perturbing actin cables withmutants or drugs disrupts the localization of ASH1 andIST2mRNAs to the bud.

Like other eukaryotic Rho-type GTPases, Cdc42p hasdownstream effects that include protein kinase-dependentinduction of transcription. At the restrictive temperature,temperature-sensitive cdc42 mutants fail to bud but continueto grow and then arrest as large, unbudded cells. TheGTPase activity of Cdc42p is stimulated by the GAPs

Bem3p, Rga1p, and Rga2p. Once GTP is hydrolyzed, a GEF(Cdc24p) promotes the exchange of GDP for GTP.

The establishment of cell polarity involves several othersmall GTPases in the Rho/Rac subfamily of Ras-likeGTPases, such as Rho1p, Rho2p, Rho3p, and Rho4p. Rho1pregulates protein kinase C (encoded by PKC1) and the cellwall synthesizing enzyme b-1,3-glucan synthase (encoded byFKS1 and GSC2). Rho1p is also localized to the plasmamembrane at sites of growth such as incipient bud sites, budtips, and the bud neck during cytokinesis. The GTPase activ-ity of Rho1p is positively regulated by the GAPs Bem2p,Sac7p, Bag2p, and Rdi1p, and negatively regulated by theGEFs Rom1p and Rom2p.

Rho2p may play a role in the establishment of cell polarityas well as in microtubule assembly. Deletion of RHO2causes increased sensitivity to the microtubule depolymeriz-ing drug benomyl. Overexpression of RHO2 suppresses thetemperature sensitivity of Dcik1 or Dkar3 mutants, wherebyCik1p and Kar3p represent microtubule-associated proteins.The GTPase activity of Rho2p is positively regulated by theGAP Bem2p, and negatively regulated by the GEFs Rom1pand Rom2p.

Rho3p is a nonessential small GTPase. Temperature-sen-sitive rho3 mutants lose cell polarity at the restrictive temper-ature; a dominant rho3 allele causes cold sensitivity andaberrant cell morphology. RHO3 interacts genetically withSEC4, which encodes a Rab-type small GTPase (cf. Sec-tion 6.1), suggesting that Rho3p may regulate polarizedsecretion. The GTPase activity of Rho3p is positively regu-lated by the GAP Rgd1p.

Rho4p is another nonessential small GTPase in theRho/Rac family. In the establishment of cell polarity, Rho4pregulates interaction between Bnr1p and Hof1p, two pro-teins implicated in cytoskeletal organization. The GTPaseactivity of Rho4p is positively regulated by the GAP Rgd1p.

A decisive role in determining cell polarity is taken by thepolarisome – a complex including a number of components(Bni1p, Spa2p, Bud6p, Pea2p, Msb3p, and Msb4p) that actsas a focal point for polymerization of actin monomers intoactin cables; the complex is required for the proper initiationof bud growth and the proper shape of vegetative buds ormating shmoos (Tolliday, Bouquin, and Li, 2001). Spa2p actsas a scaffold for the Mkk1p and Mpk1p cell wall integrity sig-naling components. Bud6p is an actin- and formin-interact-ing protein, involved in actin cable nucleation. Pea2p is acoiled-coil polarisome protein required for polarized mor-phogenesis and cell fusion. Msb3p and Msb4p are similar toeach other, and function as GAPs mainly for the Rab-GTPaseSec4p, whereby the action of Sec4p regulates exocytosis andproper actin organization.

Localization of Bni1p is dynamic – in small-budded cellsBni1p moves along the bud cortex, becoming more abundantat the bud tip and bud neck as growth proceeds. In addition,Bni1p is found as abundant cytoplasmic speckles throughoutthe cell cycle; these speckles are associated with actin cablesand are not visible upon loss of polymerized actin.

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Although bni1 null mutants are viable, they exhibit ashorter replicative lifespan than wild-type cells. They are par-tially deficient in cytokinesis, and homozygous diploids arepredominantly round in shape, exhibit a random buddingpattern, and are defective for filamentous growth in nitrogenstarvation conditions. Null mutants also display reducedmating efficiencies in response to pheromone treatment,and detailed genetic studies indicate Bni1p plays a role inpolarized recruitment of Ste5p and the consequent activationof Fus3p during mating response.

7.1.1.2 Septins and Bud Neck FilamentsThe septins were first discovered in the budding yeast, S.cerevisiae, by Hartwell and colleagues and named after theirrole in cytokinesis and septum formation (Hartwell, 1971a;Hartwell, 1974; Hartwell et al., 1974a). Work on septins hasbeen continued by many researchers, such as the groups ofJ. Pringle and M. Snyder. Septins are now known to behighly conserved cytoskeletal elements in fungi, mammals,and all eukaryotes examined thus far, with the exception ofplants. The septins have a highly conserved structure. Theycontain a central GTP-binding domain flanked by a basicregion at the N-terminus, and most septins contain a coiled-coil domain at the C-terminus. In yeast, five septins, Cdc3p,Cdc10p, Cdc11p, Cdc12p, and Sep7p, plus two, Spr3p andSpr28p, in the forespore membrane (FSM), are known. Cdc3and Cdc12 are essential for growth at all temperatures,whereas Cdc10 and Cdc11 are required only at elevated tem-peratures. Cells containing temperature-sensitive mutationsin either CDC3, CDC10, CDC11, or CDC12 delay at a G2

checkpoint and arrest at the restrictive temperature, formingextensive chains of highly elongated cells (Table 7.1).

In yeast, the septins form a series of 10-nm filaments thatin vegetatively growing cells assemble into a ring (Byers andGoetsch, 1976) on the inner surface of the plasma mem-brane at the mother bud neck (Figure 7.4). Assembly anddisassembly of the ring is regulated by phosphorylation anddephosphorylation, respectively. In S. cerevisiae, the septinsCdc3p, Cdc11p, and Sep7p are major targets for SUMOyla-tion. Proper assembly of the septin ring is monitored by anunknown mechanism at the morphogenesis checkpoint,which acts at the G2/M-phase boundary of the cell cycle.

SUMOylation of the septins occurs during mitosis beforeanaphase and the modifications disappear abruptly atcytokinesis.

Septins function as a scaffold to recruit proteins to the budneck and to act as a boundary limiting diffusion during bud-ding and cytokinesis. Evidence has been provided that yeastseptins are involved in a broad range of dynamic membraneevents and participate in a variety of other cellular processes,including cell morphogenesis (Ford and Pringle, 1991), budsite selection (Flescher, Madden, and Snyder, 1993; Casa-mayor and Snyder, 2002), chitin deposition (DeMarini et al.,1997), cell cycle regulation (Barral et al., 1999; Johnson andBlobel, 1999; Longtine and Bi, 2003), cell compartmentaliza-tion (Barral et al., 2000), cytoskeleton organization (Barralet al., 1999; Finger, 2002), and spore wall formation (Fares,Goetsch, and Pringle, 1996). Since septins participate in somany cellular processes, it is not surprising that the organi-zation and function of septins are highly regulated, and that

Table 7.1 Diversity of septin expression and function.

Gene Function Localization Biochemistry

CDC3, CDC10,CDC11,CDC12

essential for cytokinesis and polar-bud growth control; Cdc3p andCdc12p required for viability, but not Cdc10p and Cdc11p insome backgrounds; required for proper regulation of the Gin4pand Hsl1p kinases

bud neck, site of budemergence, base ofshmoo

found in a 370-kDa complexthat can form filamentsin vitro

SHS1 nonessential septin control of polarized budgrowth

SEP7 required in vivo for proper regulation of Gin4p kinase bud neck, site of budemergence

can complex with Cdc3p,Cdc10, Cdc11p, Cdc12p

SPR3 sporulation efficiency FSM no dataSPR28 no obvious phenotype FSM no data

Fig. 7.4 Bud neck filaments in budding yeast.


a diverse set of proteins is associated with the yeast septincytoskeleton (Table 7.2) (Longtine et al., 2000; Gladfelter,Pringle, and Lew, 2001; Cid et al., 2002; Faty, Fink, andBarral, 2002; Roh et al., 2002; Casamayor and Snyder, 2003;Lew, 2003; Versele and Thorner, 2005; Douglas et al., 2005).

In multicellular organisms, septins are found at the cleav-age furrow and other cortical locations. Consistent with theirlocalization, septins have been shown to be required for cyto-kinesis in yeast, Drosophila melanogaster, and mammaliancells.

7.1.1.3 Spindle Pole Bodies and their DynamicsThe spindle pole body (SPB) is the sole site of microtubuleorganization in the budding yeast S. cerevisiae. SPBs areembedded in the nuclear envelope throughout the yeast lifecycle, and are therefore able to nucleate both nuclear andcytoplasmic microtubules. The small size of the yeast SPB,its location in a membrane, and the fact that nearly all genesinvolved in SPB function are essential have presented signif-icant challenges in its analysis. Nevertheless, the SPB is per-haps the best-characterized microtubule organizing center(MTOC).

Nucleation of microtubules by eukaryotic MTOCs isrequired for a variety of functions, including chromosome seg-regation during mitosis andmeiosis, cytokinesis, fertilization,cellular morphogenesis, cell motility, and intracellular traffick-ing. Analysis of MTOCs from different organisms shows thatthe structure of these organelles is widely varied even thoughthey all share the function of microtubule nucleation. Despitetheir morphological diversity, many components and regula-tors ofMTOCs, as well as principles in their assembly, seem tobe conserved (Pereira and Schiebel, 2001; Segal and Bloom,2001; Cheeseman and Desai, 2004; Jaspersen and Winey,2004;Maekawa et al., 2007).

The SPB is a cylindrical organelle that appears toconsist of three disks or plaques of darkly staining material(Figure 7.5): an outer plaque that faces the cytoplasm and isassociated with cytoplasmic microtubules, an inner plaquethat faces the nucleoplasm and is associated with nuclearmicrotubules, and a central plaque that spans the nuclearmembrane. One side of the central plaque is associated withan electron-dense region of the nuclear envelope termed thehalf-bridge. This is the site of new SPB assembly becausedarkly staining material similar in structure to the SPB

Table 7.2 Budding yeast septin–protein interactions.

Protein Function Septin-dependentlocalization

Interactions Localization

Gin4p,Hsl1p,Kcc4p

protein kinases that function in septin localizationand cell cycle progression

yes Gin4p with septins bud neck, site of budemergence

Bni4p required for normal chitin deposition andmorphology

yes two-hybrid interactionwith Cdc10p and Chs4p

bud neck

Chs3p,Chs4p

required for normal chitin synthesis yes Chs4p, Cdc12p bud neck, site of budemergence

Yck1p,Yck2p

casein kinase I homologs, required for septinlocalization, cytokinesis, morphogenesis, andendocytosis

no unknown bud neck, sites of polarizedgrowth, plasma membrane

Bud3p,Bud4p

bud site selection yes unknown bud neck, site of budemergence

Spa2p polarisome component; scaffold for Mkk1p andMpk1p cell wall integrity signaling components

Cdc10p

Bni1p formin; cytokinesis and morphogenesis Cdc12p bud tipMyo1p type II myosin; plays a role in cytogenesis yes unknown bud neckArf1p morphogenesis during mating Cdc12p base of mating projections

Fig. 7.5 SPB in S. cerevisiae. (Modified from Jaspersen and Winey, 2004.)


accumulates on its distal, cytoplasmic tip during the G1

phase of the cell cycle. Careful analysis of SPB size and struc-ture indicates that the SPB is a dynamic organelle. In haploidcells, the SPB grows in diameter from 80 nm in the G1 phaseto 110 nm in mitosis. The molecular mass of a diploid SPB,including microtubules and microtubule-associated pro-teins, is estimated to be 1–1.5GDa; most components of themitotic SPB have been identified to date (Table 7.3).

In S. cerevisiae, the mitotic spindle must orient along thecell polarity axis, defined by the site of bud emergence, toensure correct nuclear division between the mother anddaughter cells (Karsenti and Vernos, 2001; Segal and Bloom,2001; Wittmann, Hyman, and Desai, 2001). Spindle polaritydictates this process, and relies on the concerted control ofspindle pole function and generates a precise program thatoriginates from the cell cortex regulating the cytoplasmicmicrotubule attachments during spindle morphogenesis.This cross-talk with the machinery is responsible for budsite selection, indicating that orientation of the spindle ismechanistically coupled to the positioning of a polarity axisand the division plane (Segal and Bloom, 2001). Among thecortical components implicated in spindle orientation areBni1p, a target of the polarizing machinery essential in budsite selection and spindle orientation, and the actin inter-actor Aip3p/Bud6p initially localized to the bud tip (Amberget al., 1997). Other cortical elements (e.g., Num1p) are

restricted initially to the mother cell during spindle assembly(Farkasovsky and Kuntzel, 1995).

Spindle morphogenesis in yeast is initiated by START atthe G1/S transition of the cell cycle. Progression throughSTART triggers bud emergence, DNA replication, and theduplication of the MTOC – the SPB (see Figure 7.5)(Jaspersen and Winey, 2004). Actin filaments, either as cyto-skeletal cables or as cortical membrane patches, are sub-jected to dynamic changes during the cell cycle (Takizawaet al., 1997). The polymerization and depolymerization oftubulin (the major microtubular protein) and microtubules’motility are assisted by mechanochemical enzymes or motorproteins that are necessary in spindle morphogenesis: cyto-plasmic dynein and the kinesin-like proteins Kip2p andKip3p, as well as Kar3p, are involved in regulating micro-tubule dynamics, mediating nuclear migration to the budneck and facilitating spindle translocation (Pellman et al.,1995).

Microtubule dynamics is regulated by microtubule-associ-ated proteins of the XMAP215/Dis1 family, Stu1p and Stu2p.Stu1p is a component of the mitotic spindle that binds tointerpolar microtubules via its association with b-tubulin(Tub2p); in this way, the interpolar microtubules provide anoutward force on the spindle poles. Stu2p interacts with theSPB component Spc72p, thus regulating spindle orientationand metaphase chromosome alignment. As indicated, themicrotubules emerge from the SPBs toward the new budand orientate the nucleus and intranuclear spindle at mito-sis. The nuclear membrane in yeast remains intact (contraryto that in mammals, for example) throughout mitosis withthe mitotic spindle forming intranuclearly between twoSPBs embedded in the nuclear envelope. Once the genomeduplicates, the spindle aligns parallel to the mother bud axisand finally elongates to supply one nucleus to both motherand daughter.

Factors mediating the process of microtubule attachmentwith the bud cell cortex are Bim1p and Kar9p (DeWulf,McAinsh, and Sorger, 2003). Bim1p can directly bind tomicrotubules and is required for the high dynamic instabilityof microtubules that is characteristic of cells before spindleassembly. Kar9p has been implicated in the orientation offunctional microtubule attachments into the bud during veg-etative growth. It is delivered to the bud by a Myo2-depen-dent mechanism presumably tracking on actin cables.Interaction of the two factors, Bim1p and Kar9p, appears toprovide a functional linkage between the actin and micro-tubule cytoskeletons. In addition, Bud3p, a protein for axialbudding of haploid cells, accumulates at the bud neck and isrequired for the efficient association of Bud6p with the neckregion.

SPB duplication has to occur prior to cell division. Regula-tors of SPB duplication and function associate with the SPBduring all or part of the cell cycle. Mps1p, a conserved pro-tein kinase, is required for multiple steps in SPB duplicationand also for the spindle checkpoint; its substrates includeSPB proteins Spc42p, Spc110p, and Spc98p, mitotic exit

Table 7.3 Yeast SPB components.

Component Location in SPB Function

Tub4p g-tubulincomplex

MTnucleation

Spc98p g-tubulincomplex

MTnucleation

Spc97p g-tubulincomplex

MTnucleation

Spc72p OP, HB g-tubulin binding proteinNud1p OP, satellite MEN signalingCnm67p IL1, OP, satellite spacer, anchors OP to CPSpc42p g-tubulin

complexstructural SPB core

Spc29p CP, satellite structural SPB coreCmd1p CP structural Spc100p binding

proteinSpc110p CP to IP spacer, g-tubulin binding proteinNdc1p SPB periphery membrane protein, SBP insertionMps2p SPB periphery membrane protein, SBP insertionBbp1p SPB periphery SBP core, HB linker to membraneKar1p HB membrane protein, SBP

duplicationMps3p HB Membrane protein, SBP

duplicationCdc31p HB SBP duplicationSfi1p HB SBP duplicationMpc54p MP replaces Spc72p in meiosis ISpo21p MP replaces Spc72p in meiosis II

OP: outer plaque; IP: inner plaque; CP: central plaque; HB: half-bridge; IL1:inner layer 1; IL2: inner layer 2; MP: membrane protein; MT: microtubule.


network (MEN) protein Mob1p, and checkpoint proteinMad1p (Winey et al., 1991; Botstein et al., 1997).

SPB duplication (Figure 7.6) can be divided into threesteps: (1) half-bridge elongation and deposition of satellitematerial, (2) expansion of the satellite into a duplication pla-que and retraction of the half-bridge, and (3) insertion of theduplication plaque into the nuclear envelope and assembly ofthe inner plaque. Following completion of SPB duplication,the bridge connecting the side-by-side SPBs is severed, andSPBs move to opposite sides of the nuclear envelope (4). Therequirements for various gene products in each step areshown in Figure 7.6. Spc72p, Nud1p, and Cnm67p are prob-ably required for the second step. SPBs are not synthesizedde novo. Consequently, every time a cell divides it must dupli-cate its SPB, as well as its genome, to ensure that both themother and daughter cell contain one copy of all 16 chromo-somes and one SPB. SPB duplication occurs in the G1 phaseof the cell cycle; however, defects in SPB duplication are notdetected until mitosis when cells fail to form a functionalbipolar spindle. Generally, SPB defects cannot be reversed atthis point, so cells will eventually attempt chromosome seg-regation with a monopolar spindle, which results in progenywith aberrant DNA content and/or SPB number. Therefore,accurate SPB duplication during G1 is essential to maintaingenomic stability (Jaspersen and Winey, 2004).

7.1.2Sexual Reproduction

Many yeasts have the ability to undergo sexual reproduction.Best understood are the underlying processes in S. cerevisiaeand Schizosaccharomyces pombe. We will follow here onlythe mating behavior of S. cerevisiae, as the sexual life cycle inS. pombe is different.

In S. cerevisiae, mating affords the conjugation of two hap-loid cells of opposite mating-type (a and a). A prerequisitefor mating is that the two cells synchronize each others’ cellcycles at START in response to the secreted mating phero-mones (a- and a-factor). Conjugation is preceded by“shmoo” formation that generates specialized surface projec-tions as contact regions (cf. Figure 7.3). Shmoo formation

involves a similar reorganization of the cytoskeleton (actincables and microtubules) and some of the components (budsite proteins) that have been outlined for budding, except theneck filaments (cf. Section 7.1.3). After conjugation, the mat-ing cells fuse their plasma membranes to form a commoncytoplasm. Nuclear fusion (karyogamy) can then proceedand results in a diploid set of chromosomes. The stable dip-loid zygote will continue mitotic cell cycles in rich growthmedia. Under starving conditions or growth in nonferment-able carbon sources (such as acetate or ethanol), the cells areinduced to undergo meiosis. Following meiotic nuclear divi-sions, a diploid mother cell differentiates into an ascus, nor-mally containing four haploid ascospores (2a and 2a). In richmedia, the spores can germinate (normal mitosis) and mateonce again to form diploids.

Sporulation is attractive to cell biologists because it repre-sents a mode of cell division that is different from typicalmitotic cell division; namely, plasma membranes for fourdaughter cells (haploid spores) have to be constructed withinthe mother cell cytoplasm (Shimoda and Nakamura, 2004;Moreno-Borchart and Knop, 2003). More details are dis-cussed in Section 7.2.5.

7.1.3Filamentous Growth

Filamentous growth in yeasts may be considered as an alter-native method of vegetative propagation. It is found innumerous yeast species and can adopt different morpholo-gies, such as pseudohyphae or true hyphae. Pseudohyphaeare chains of budding cells that have elongated withoutdetachment. The elongated budding period leads to symmet-ric cell division and synchronous re-entry to the buddingcycle. Hyphae are formed by branched or unbranched fila-mentous cells formed from germ tubes. In this pattern,asymmetric distribution of the vacuole occurs betweenthe apical and subapical cells, so that the G1 phase in thesubapical cell is reached before branch emergence, while theapical cell starts its next cell cycle immediately. Hyphal andpseudohyphal growth are subject to different developmentalpathways, and represent an adaptation by yeasts to scarce

Fig. 7.6 SPB duplication pathway. (After

Jaspersen and Winey, 2004.)


nutrition, particularly when there is a shortage of nitrogencompounds. Generally, filamentation in yeast is reversibleand leads back to unicellular growth, as soon as growth con-ditions improve. In S. cerevisiae, filamentous growth has

been studied in some detail (Robertson and Fink, 1998;Conlan and Tzamarias, 2001; Pan and Heitman, 2002) andthe signaling pathway is documented in Section 10.2.2. Sev-eral gene products (Table 7.4) are involved in filamentous

Table 7.4 Yeast gene products involved in pseudohyphal and invasive growth.

Protein Function Interaction

Ach1 CoA transferase activity (succinyl-CoA to acetate); has minor acetyl-CoA hydrolaseactivity; phosphorylated; required for acetate utilization and for diploidpseudohyphal growth

Ash1 GATA-like transcription factor; acts to specify daughter cell fate in mating-typeswitching in haploid cells and in pseudohyphal growth in diploid cells deprived ofnitrogen

activates FLO11/MUC1

Dfg5 putative mannosidase, essential GPI-anchored membrane protein required forcell wall biogenesis in bud formation, involved in filamentous growth,homologous to Dcw1p

Dfg10 probable polyprenol reductase; catalyzes conversion of polyprenol to dolichol;involved in filamentous growth

Dfg16 multiple transmembrane protein, involved in diploid invasive and pseudohyphalgrowth upon nitrogen starvation; required for accumulation of processedRim101p

Dia1 protein of unknown function, involved in invasive and pseudohyphal growthDia2 origin-binding F-box protein; involved in invasive and pseudohyphal growthDia3 protein of unknown function, involved in invasive and pseudohyphal growthDia4 probable mitochondrial seryl-tRNA synthetase, mutant displays increased invasive

and pseudohyphal growthFlo1, Flo5,Flo9, Flo10

lectin-like cell surface proteins, aggregate cells into “flocs” by binding to mannosesugar chains on the surfaces of other cells (Guo et al., 2000)

Flo11(Muc1)

GPI-anchored cell surface glycoprotein (flocculin); required for pseudohyphalfilament formation; similar to StuA – an Aspergillus nidulans developmentalregulator (Douglas et al., 2007)

regulated by the MAP kinase pathway (viaSte12p and Tec1p) and the cAMP pathway(via Flo8p)

Flo8 transcription factor; required for flocculation, diploid filamentous growth, andhaploid invasive growth; S288C and most laboratory strains have a mutation inthis gene

Gpg1 proposed g-subunit of the heterotrimeric G-protein that interacts with thereceptor Gpr1p; involved in regulation of pseudohyphal growth

Hms1 basic helix–loop–helix (bHLH) protein with similarity tomyc family transcriptionfactors

overexpression confers hyperfilamentousgrowth and suppresses the pseudohyphalfilamentation defect of a diploidmep1 mep2homozygous null mutant

Mep2 ammonium permease; member of family of NH4 transporters; expression isunder the nitrogen catabolite repression regulation

Mga1 protein similar to heat-shock transcription factor multicopy suppressor of pseudohyphalgrowth defects of ammonium permeasemutants

Msn1 transcriptional activator; involved in regulation of invertase and glucoamylaseexpression, invasive growth and pseudohyphal differentiation, iron uptake,chromium accumulation, and response to osmotic stress; localizes to the nucleus

Phd1 transcriptional activator; enhances pseudohyphal growth; recruits Tup1p to itstargets; regulates expression of FLO11

Tup1–Cyc8 complex; Cdc28p

Pgu1 polygalacturonase, pectolytic enzyme that hydrolyzes the a-1,4-glycosidic bonds inthe rhamnogalacturonan chains in pectins

Sfg1 nuclear protein, putative transcription factor required for growth of superficialpseudohyphae (which do not invade the agar substrate) but not for invasivepseudohyphal growth; may act together with Phd1p; potential Cdc28p substrate

Phd1p; Cdc28p

Sfl1 transcriptional repressor and activator; involved in repression of flocculation-related genes and activation of stress-responsive genes

negatively regulated by cAMP-dependentPKA subunit Tpk2p

Vip1 IP6 and IP7 kinase; IP7 production is important for phosphate signaling; involvedin cortical actin cytoskeleton function and invasive pseudohyphal growth

Vps60 cytoplasmic and vacuolar membrane protein involved in late endosome to vacuoletransport; required for normal filament maturation during pseudohyphal growth

Vta1p


growth, either as regulators (Ash1p, Flo8, Gpg1p, Mep2,Mga1p, Phd1p, Sfg1p, and Sfl1p) or as structural compo-nents that are required to maintain a sort of agglutinated cellstate (Dfg5p, Dfg10p, Dfg16p, Flo11p/Muc1p, and Pgu1p)that is connected with filamentation.

The invasive growth pathway – manifest by the phenome-non of cells growing into the agar layer – is activated whenhaploid cells are limited for carbon (whereas diploid pseudo-hyphal growth is stimulated by nitrogen limitation). The car-bon limitation activates Ras2p, which stimulates either of twopathways: a mitogen-activated protein (MAP) kinase cascadegoverned by Kss1p or the cAMP-dependent protein kinase(protein kinase A (PKA)) pathway. Kss1p is phosphorylatedand activated by Ste7p, leading to activation of the hetero-meric transcription factor Tec1p–Ste12p, and induction of tar-get genes that provide Pgu1p, Muc1p, and the cyclin Cln1p.In its inactive unphosphorylated form, Kss1p binds to Ste12pand prevents it from activating genes involved in invasivegrowth. This same cascade functions to maintain cell wallintegrity during vegetative growth (cf. Section 10.2.3).

Pseudohyphal and invasive growth is enhanced by thedeletion of particular genes, one of which is SFL1, encodinga transcriptional repressor of flocculation-related genes (Rob-ertson and Fink, 1998; Galeote et al., 2007); it is normallyrequired for cell surface assembly in vegetative growth. Sfl1pcontains two domains homologous to Myc oncoproteins andthe yeast heat-shock transcription factor Hsf1p. The N-termi-nal region of Sfl1p reveals homology to the DNA-bindingdomain of Hsf1p. Sfl1p interacts specifically with Tpk2p, thecatalytic subunit of PKA, which negatively regulates Sfl1pfunction (Conlan and Tzamarias, 2001). Phosphorylation byPKA relieves Sfl1p-mediated repression by prohibitingdimerization and DNA binding by Sfl1p, and in Dtpk2strains, the levels of Sfl1p protein associated with specificpromoter elements increase.

Sfl1p interacts directly with Ssn6p in the Ssn6–Tup1 com-plex (cf. Section 10.3.1) that inhibits the transcription ofmany diversely regulated genes. Sfl1p activity is linked toSsn2p, Ssn8p, Sin4p, and Rox3p, suggesting that Sfl1p mayinteract with Srb/mediator proteins to inhibit transcriptionby the RNA polymerase II holoenzyme. Sin4p and Ssn3p,components of specific RNA polymerase II subcomplexesrequired for Ssn6p–Tup1p repression activity, are alsorequired for Sfl1p repression function, indicating a possiblemechanism for Sfl1p-mediated repression via Ssn6p–Tup1pand specific subunits of the RNA polymerase II holoenzyme.

7.1.4Yeast Aging and Cell Death

Aging and the death of yeast cells is a subject of interest foryeast researchers as well as for biotechnologists. Leavingaside the practical aspects for microbiologists in the food,fermentation, and healthcare industries, yeast has served asa useful model in studying phenomena such as cellularaging and apoptosis (see also Section 7.1.4.2).

As we will explain later (Section 13.3), yeast has also beenused extensively as a model to study age-related disorders (inaddition to Alzheimer’s, Parkinson’s, Huntington’s disease,or, for example, diabetes type II, cancer, and cardiovasculardisorders) to help define molecular cues underlying thesephenomena.

7.1.4.1 Yeast LifespanThe age, or more accurately, the lifespan of a yeast cell isdefined by the number of times it undergoes division. Themaximum lifespan of a particular yeast strain therefore cor-responds to the highest number of cell divisions it canundergo. Genetic and environmental factors will of coursedetermine lifespan. Usually, between 13 and 30, but also upto 50 divisions can be found. Beyond these limits, no repro-duction is possible and cells will enter a senescent state lead-ing to death. As many of the morphological changes inbudding yeasts affect the cell wall, the number of chitin-accu-mulating bud scars (detected by scanning electron micros-copy) may serve as a measure for the age of cells.

With regard to aging, numerous investigations havebeen based on the fact that yeast cells analyzed under dif-fering conditions show variations in lifespan. Mostongoing studies are attempting to address aging in yeastcells in terms of longevity, whereby one has to clearly dif-ferentiate between replicative lifespan (RLS; the numberof daughters produced by each dividing mother cell) andchronological lifespan (CLS; the capacity of stationary (G0)cultures to maintain viability over time) (Piper, 2006). Thiswork provided a means of analyzing the longest lifespansthat will be most informative about the determinants oflongevity and yield results most relevant to aging in morecomplex systems.

Studies initiated in the mid-1990s by Guarente and col-laborators (Sun et al., 1994; Kennedy et al., 1995; Smealet al., 1996; Sinclair, Mills, and Guarente, 1997; Sinclair,Mills, and Guarente, 1998) demonstrated that aging inyeast mother cells, which undergo a finite number of divi-sions before cessation of cell growth, is accompanied by anumber of morphological changes whereby the underlyinggenetic alterations can extend or decrease the number ofdivisions. Observed phenomena were, for example,increase in cell size, onset of sterility, enlargement andfragmentation of the nucleolus, and redistribution of theSir3p and Sir4p proteins from telomeres and HML orHMR loci to the nucleolus. Deletion of the yeast SGS1gene, the homolog of the human BLM (Bloom’ssyndrome) or WRN (Werner’s syndrome) genes, reducesyeast lifespan by 60% and shows other signs of acceleratedaging, thus paralleling defects observed in Werner’ssyndrome. Aging yeast cells also accumulate extrachromo-somal rDNA circles (ERCs) generated by homologousrecombination of tandemly arrayed copies of rDNA(Larionov, Kouprina, and Karpova, 1984), indicatingthat ERCs are a cause of aging (Sinclair and Guarente,1997).


Yeast cells kept under nutrient starvation can survive forprolonged periods of time in stationary phase. This survivalrequires the activity of copper/zinc superoxide dismutase,Sod1p (Longo et al., 1997), illustrating the importance ofdetoxification of oxygen radicals during this period. Suchcells did not display any of the “aging defects.” However,when nutrients were returned to allow resumption of celldivision, survivors exhibited all phenomena of aging and amuch shorter replicative lifespan than nonstarved controls(Ashrafi et al., 1999).

Overexpression of SOD1 was confirmed both to prolongchronological lifespan in stationary cells and to elevate thelevels of Sod1p activity 6- to 8-fold in vegetative culturesprovided that high copper concentrations were applied orwhen CCS1, the gene encoding the chaperone necessaryfor copper loading to Sod1p, was overexpressed simulta-neously (Harris et al., 2005). However, if SOD1 was over-expressed alone or in the absence of high copper, bothchronological and replicative lifespan were shortened, andthe cells became abnormally sensitive to endogenous oxy-gen stresses.

Lifespan in yeast (CLS and RLS) was also found to be pro-longed in deletion mutants for Sch9p, Ras2p, and Tor1p(Cheng et al., 2007a), all of which are involved in stress sig-naling. The analysis of microarray expression data with motifand chromatin immunoprecipitation (ChIP)-on-chip data inwild-type and mutant cells revealed that at least three stressresponse transcription factors, Msn2p, Msn4p, and Gis1p,are activated in all three mutants as a prerequisite for longev-ity. At present, however, the mechanisms that lead to down-regulation of the Sch9, PKA, or TOR (target of rapamycin)pathways are not completely known.

Recent advances in aging research suggest thatlongevity-determining pathways have been evolutionarilyconserved from yeast through mammals. High-through-put, genome-wide approaches were used to identify a largefraction of the nonessential, single-gene deletion muta-tions that confer increased longevity on yeast (Kaeberleinand Kennedy, 2005). The characterization of conservedgenes that regulate the aging process aims at an improvedunderstanding of the causes of human aging and providepotential therapeutic targets for drug discovery. The onlyexperimental manipulation known to extend the lifespanof a number of organisms is calorie restriction (Bordoneand Guarente, 2005). These recent findings may aid indeciphering the mechanisms by which calorie restrictionfosters longevity and the reduction of the incidence ofage-related disorders.

Very interesting genes determining yeast longevity are thelongevity assurance genes LAG1 (D’mello et al., 1994) andLAG2 (Childress et al., 1996; Liu et al., 2009a; Sieriejuk et al.,2009).

Lag1p is a ceramide synthase component, involved insynthesis of ceramide from C26(acyl)-coenzyme A anddihydrosphingosine or phytosphingosine and functionallyequivalent to Lac1p. Both genes were identified as having

a decreasing expression with increasing age of yeast cells.Deletion of LAG1 results in a 50% increase in the lifespanof the mutated cells. Double deletions (Dlag1–Dlac1) arelethal. The human counterparts have been cloned and cancomplement lag1 or lac1 deletants. The proteins have beenlocalized to the endoplasmic reticulum (ER) and arethought to participate in the transport from the ER to theGolgi of glycosylphosphatidylinositol (GPI)-anchoredproteins.

Lag2p is a protein that negatively regulates the SCF E3ubiquitin ligase by interacting with and preventing rubyla-tion (NEDDylation) of the cullin subunit, Cdc53p (Liuet al., 2009a). Lag2p inhibits association of Cdc34p to theSCF complex and at the same time inhibits the conjuga-tion of Rub1p to Cdc53p in competition with Dcn1p; thelatter reaction is specific for Cdc53p. When in lag2mutants either dcn1 or jab1 are deleted, growth of yeastcells is repressed. The property of Lag2p as a longevitydeterminant has been recognized to reside in its preferen-tial expression in young cells; this behavior is similar tomammalian CAND1.

7.1.4.2 Yeast ApoptosisThe finding, more than a decade ago, that S. cerevisiae canundergo apoptosis (Madeo, Frohlich, and Frohlich, 1997)opened the possibility to investigate this mode of pro-grammed cell death in a model organism that is a smalleukaryote, but offers extreme technical advantages. Sincethen, numerous exogenous and endogenous triggers havebeen found to induce yeast apoptosis, and multiple yeastorthologs of crucial metazoan apoptotic regulators have beenidentified and characterized at the molecular level.Apoptosis-relevant orthologs include proteases such as theyeast caspase as well as several mitochondrial and nuclearproteins that contribute to the execution of apoptosis in acaspase-independent manner. In addition, aspects of agingand failed mating behavior have disclosed how apoptosis istriggered in yeast (Carmona-Gutierrez et al., 2010).

While using the term “apoptosis” in yeast at the beginningled to harsh criticism, it is now well established that manycriteria applied to document programmed cell death inmammalian systems are also applicable to yeast. It is note-worthy that several morphological features (e.g., reduction ofcellular volume, chromatin condensation, or nuclear frag-mentation) have to be always considered together to docu-ment cell death. In this regard, yeast does fulfill thesedemands.

7.1.4.2.1 External Triggers of Yeast Apoptosis Apoptosis inyeast can be induced exogenously and endogenously. Exog-enous stimuli are hydrogen peroxide or acetic acid. Highdoses of hydrogen peroxide lead to a necrotic phenotype,but low doses induce apoptosis. In this latter process, anumber of factors are involved, such as the yeast caspaseYca1p and the apoptosis-inducing factor Aif1p. Inductionalso depends on the small GTPase Rho5p, which interacts


with the thioredoxin reductase Trrp1p – a key componentof the cytoplasmic thioredoxin antioxidant system. Aceticacid treatment leads to mitochondrial cytochrome c releaseand depends on proteins that render the mitochondrialouter membrane permeable through the so-called perme-ability transition pore (PTP).

Further external agents that cause apoptosis in yeastinclude hypochlorous acid, high salt, UV irradiation, or heatstress. Even “normal” nutrients, such as glucose, sorbitol,copper, manganese, and iron, may trigger apoptosis, when-ever they are supplied at supraphysiological concentrations.The lethal action of cadmium depends on the yeast caspaseand glutathione synthesis. Even calcium may critically beassociated with yeast cell death (cf. Section 8.3.3.3);a-factor-induced apoptosis raises Ca2þ levels, leading tomitochondrial fragmentation in a process depending onthe yeast suicide protein (Ysp1p). Further, the Ca2þ level ismediated by the calcineurin/calmodulin system, which inturn is connected to ER stress regulation.

Izh2p, one of the four yeast genes involved in elevatedexpression in zinc-deficient cells (Lyons et al., 2004) (cf. Sec-tion 8.3.4.3), mediates the effects of the tobacco antifungalprotein osmotin, which induces yeast to undergo apoptosis(Narasimhan et al., 2005). Izh2p binds osmotin at the plasmamembrane and overexpression or deletion of IZH2 causesincreased or decreased osmotin sensitivity, respectively.Genetic interactions with RAS2 suggest that IZH2 and RAS2act in the same pathway for osmotin-induced apoptosis.

Yeast pheromones act as natural triggers of yeastapoptosis – exposure of haploid cells to low doses of theircorresponding mating hormone causes apoptosis, when suit-able mating partners are not available. The signaling cascadeleads to an increase in intracellular Ca2þ, a rise in mitochon-drial activity and cytochrome c release, followed by apoptosis.More recently, Ste20p activity has been linked to chromatincondensation during apoptosis. Similar to what is seen inapoptotic mammalian cells, hydrogen peroxide-induced celldeath in S. cerevisiae requires chromatin condensation result-ing from H2B phosphorylation. Upon treatment with hydro-gen peroxide, Ste20p translocates to the nucleus and directlyphosphorylates Ser10 of histone H2B (encoded byHTB1 andHTB2), even though there is no apparent nuclear localizationsignal present in the kinase.

7.1.4.2.2 Endogenous Triggers of Yeast Apoptosis There areseveral cellular processes in which defects will cause apoptosis:failures in N-glycosylation, chromatid cohesion, mRNA stabil-ity, or ubiquitination. DNA damage (mainly caused throughfailures in oxygen metabolism and by reactive oxygen species(ROS) generation) and replication errors can stimulate yeastcell death. Intriguingly, unusual players have been identified,such as the tRNA methyltransferase 9 (Trm9p), which acts asa tRNA modification enzyme that positively regulates theexpression levels of the major DNA damage response pro-teins. The same holds true for the peroxiredoxin Tsa1p, whichis a key peroxidase that can suppress genome instability.

Although autophagy mostly serves as a mechanism of cel-lular adaptation and survival, under specific circumstances itmight mediate a certain type of programmed cell death,defined as autophagic cell death.

Recently, it was reported that the yeast protein Bxi1p(encoded by YNL305c) is a bona fide member of the Baxinhibitor superfamily (Cebulski et al., 2011). The absence ofthis protein renders mutants relatively more susceptible toheat shock than wild-type controls. Though Dbxi1 cells havesimilar growth rates to their wild-type counterparts at 30 �C,they show higher sensitivity both to ethanol-induced and toglucose-induced programmed cell death. Significantly, aBxi1p-GFP colocalizes with the ER-localized protein Sec63p-RFP, suggesting that yeast Bax inhibitor-1 functions in theER like its mammalian counterparts. Additionally, Dbxi1cells were more sensitive to drugs that induce ER stress, butalso have a decreased unfolded protein response (UPR) asmeasured with a unfolded protein response element(UPRE)::lacZ reporter. Finally, deleting BXI1 diminishes thecalcium signaling response in response to the accumulationof unfolded proteins in the ER as measured by a calcineurin-dependent response element (CDRE)::lacZ reporter. In all,the data suggest that Bxi1p, like its eukaryotic homologs, isan ER-localized protein that links the UPR and programmedcell death in yeast.

7.1.4.2.3 Regulation of Yeast Apoptosis There are threesmall signaling molecules involved in inducing yeast apopto-sis: ROS, nitric oxide (NO), and ammonia. The significanceof ROS as cell death regulators has been widely recognized;potential sources are considered as the respiratory chain, ER,and iron-coupled reactions. NO is produced by an arginine-dependent mechanism in hydrogen peroxide-induced apo-ptotic cells and S-nitrosates GAPDH. Inhibition of NO syn-thesis promotes the survival of cells, because the effects ofNO are decreased. Ammonia is critical to older cells. Duringthe development of multicellular colonies, ammonia accu-mulates in the center of the colonies and will kill these cells,while younger cells at the periphery can exploit the “newnutrients.” Cdc48p, which is involved in ER-associated deg-radation (ERAD) (cf. Section 8.1.3.3), is antiapoptotic. How-ever, in impaired ERAD, the ER undergoes stress, inductionof the UPR, resulting in ROS production and consequentlycell death.

Although Yca1p is a “metacaspase” that cleaves its sub-strates after a basic residue (arginine or lysine) instead of anaspartate residue as in caspases, it has to be considered afunctional caspase homolog and a pivotal player in cell deathexecution. Not all apoptotic activity, however, needs a partici-pation of caspase. For example, defective N-glycosylation incells lacking Ost2p, independently from Yca1p leads to apo-ptosis. The same is true for the yeast apoptosis-inducing fac-tors Aif1p or Nuc1p.

The yeast nuclear factor Nma111p (nuclear mediator ofapoptosis) is another protease that may be involved in apo-ptosis, because Nma111p is able to cleave the only known


inhibitor of apoptosis in yeast, Bir1p. Bir1p disruption afteroxidative stress results in higher death rates. We havealready discussed above the effects of H2B phosphorylation.

Mitochondrial factors substantially contribute to apo-ptosis. Like its mammalian counterpart, yeast Aif1pundergoes mitochondrial–nuclear shuttling upon apo-ptotic induction (by superoxide, acetate, aging). The yeastNADH dehydrogenase Ndi1p, which catalyzes the oxida-tion of mitochondrial NADH, being localized to the innermitochondrial membrane (cf. Section 8.3.7.2) is believedto be implicated in yeast cell death. Disruption of NDI1reduces ROS production and hence extends chronologicallifespan. The participation of yeast cytochrome c in apo-ptosis remains under debate, while in mammalian cells itis clear that cytochrome c release provokes a direct cas-pase activation. Nuc1p, another death-inducing factor, isalso located to yeast mitochondria; upon apoptosis induc-tion it translocates to the nucleus. Mitochondrial frag-mentation is hazardous to yeast cells, similar to fission ofmitochondria in mammals. In yeast, Cnm1p is the factorthat promotes apoptosis after mitochondrial fragmentationor degradation. Dnm1p interacts with the proteins Mdv1p,consistently promoting cell death, as well as Fis1p, whichseems to exert a positive role in cell survival. Finally, thereis a yeast ortholog of the human translationally controlledtumor protein TCTP that is involved in apoptosis, Mmi1p.This factor translocates to mitochondria upon oxidativestress.

Other organelles contributing to induction of apoptosisare the yeast vacuole and the yeast peroxisomes. After oxi-dative stress, for example, the RNase Rny1p is releasedfrom the vacuole to the cytosol where it directly promotescell death independent from its enzymatic activity. Dele-tion of PEX6 in yeast peroxisomes can induce necrotic celldeath.

7.2Cell Cycle

7.2.1Dynamics and Regulation of the Cell Cycle

7.2.1.1 Some Historical NotesEarly attention toward the “biology of the cell cycle” wasdrawn by a book written by J.M. Mitchison that appeared in1971 (Mitchison, 1971). It was Lee Hartwell who made thedecisive step in cell cycle research by introducing the bud-ding yeast, S. cerevisiae, as an experimental system into thisfield and by characterizing a number of genes involved incell division and cell cycle control (Figure 7.7 and Table 7.5),dubbed CDC genes (Hartwell, Culotti, and Reid, 1970a;Hartwell, 1971b; Hartwell, 1974; Hartwell et al., 1974b). Heand his collaborators arrived at this issue after research onprotein synthesis and ribosome synthesis in yeast (Hartwell,1967; Hartwell and McLaughlin, 1969; Hutchison, Hartwell,and McLaughlin, 1969; McLaughlin and Hartwell, 1969;Hartwell et al., 1970b; Hartwell, McLaughlin, and Warner,1970c) as well as on studies of mating and mating phero-mones in yeast carried out between 1967 and 1970.

Research on the yeast cell cycle included work on synchro-nization of haploid yeast cells as a prelude to conjugation(Hartwell, 1973; Wood and Hartwell, 1982; Hartwell andWeinert, 1989; Paulovich, Toczyski, and Hartwell, 1997). Itwas these issues that lead Hartwell to think about the ques-tion as to what research on the yeast cell cycle could contrib-ute to an understanding where the controls of cell growthand division are defective in cancer (Hartwell, 1992). In1980, Kim Nasmyth, one of the early coworkers of PaulNurse, whose fields comprised the mating phenomenon inS. cerevisiae and the cell cycle, succeeded in isolating cellcycle genes by molecular cloning (Nasmyth and Reed, 1980).

Fig. 7.7 Pathway of selected gene-

controlled events in the S. cerevisiae cell

cycle. Numbers refer to CDC genes

encoding the respective Cdc products.


With his colleagues, Hartwell extended his work on the cellcycle to the chromatin field at the beginning of this century(Emili et al., 2001).

Soon after Hartwell’s approach, the group of Paul Nursechose to introduce the fission yeast, S. pombe, to this field asanother simple model organism (Nurse, Thuriaux, andNasmyth, 1976), which marked the beginning of an extremelysuccessful period of cell cycle research. Thus, it was straight-forward to followHartwell’s approach by isolating cdcmutantsin fission yeast. The first mutants collected were mainly defec-tive in the events of mitosis and cell division, and subsequentscreening carried out together with Kim Nasmyth identifiedmore defective mutants in the S phase (Nurse, Thuriaux, andNasmyth, 1976).

One important early finding was the presence of a yeasthomolog in S. pombe, cdc2, which later was shown to func-tionally correspond to the yeast CDC28 gene (Beach,Durkacz, and Nurse, 1982). Furthermore, there was the S.pombe wee1 gene that acted in G2 and controlled the cell cycletiming of mitosis (Nurse and Thuriaux, 1980). Surprisingly,further experiments disclosed that cdc2 was unusual in beingrequired twice during the cell cycle, first in G1 for onset of theS phase and then again in G2 for the onset of mitosis (Nurseand Bissett, 1981). Obviously, cdc2 had a central role in con-trolling the fission yeast cell cycle. In G1, it was required toexecute the onset of the S phase and in G2 it acted as a majorrate-limiting step determining the onset of mitosis. Next, thecdc2 gene product was identified as a kinase (Simanis andNurse, 1986) and shown to undergo tyrosine phosphorylationat the G2/M transition (Gould and Nurse, 1989). Anotherimportant player in controlling the cell cycle turned out to bethe cdc13 cyclin, the level of which varied during the cellcycle, and which was required for cdc2 protein kinase activa-tion (Moreno, Hayles, and Nurse, 1989).

The proposition that cell cycle control was conserved inyeast and humans, and probably in all eukaryotes, was sub-stantiated by isolating and characterizing equivalents forcdc2 (Lee and Nurse, 1987). The speculation was that humanCDC2 might act at two points in the cell cycle – at the “G1

restriction point” known to operate in mammalian cells andat the G2/M transition where it served as “maturation pro-moting factor” (MPF) known to control the M phase in meta-zoan eggs and oocytes (Nurse, 1990). These two functionsaccentuate the importance of cyclin-dependent kinases(CDKs) in regulating the orderly progression through theS phase and mitosis during the cell cycle. The onset of theS phase is thought to require two sequential steps – the firstone is operative only if CDK activity is absent (i.e., inearly G1), while the second requires the presence of CDKactivity, which later appears at the G1/S boundary, thus allow-ing progression through step 2 and bringing about the initia-tion of the S phase (Wuarin and Nurse, 1996). During G2,the continued presence of CDK activity prevents step 1 fromoccurring again and this blocks onset of a further S phase. Atthe G2/M boundary, a further increase in CDK activity trig-gers mitosis. Exit from mitosis and the termination of the

cell cycle therefore requires destruction of CDK activity, andbecause the subsequent G1 cells lack CDK activity, they areable to carry out the first step for the S phase and the wholeseries of events can be repeated (Stern and Nurse, 1996).

In the long run, the findings from the two yeast modelsobviously had to be complemented by observations made inother organisms. An understanding of what actually “drives”the cell cycle came from very important discoveries inaquatic organisms such as clams, sea urchins, frogs, andstarfish. In 1980, Wu and Gerhart purified the MPF fromXenopus eggs (Wu and Gerhart, 1980). Tim Hunt, whostarted research on the cell cycle the same year, was soonable to show the existence of cyclins in sea urchins (Evans,Hunt, and Youngblom, 1982; Evans et al., 1983). In his NobelLecture (Hunt, 2001), Hunt stated that he was fascinated bythe amphibians and the “glamour of MPF” to investigate thecell cycle. None of the classical cdc mutations in buddingyeast corresponded to cyclins and were identified only later.Similarly, the crucial CDK-activating kinase was not detect-able by genetic approaches. Thus, it appears a very fortunatesituation that contributions to cell cycle research accumu-lated from several sources and places within a remarkablynarrow time window.

Although many research groups contributed to this field,Hartwell, Hunt, and Nurse were honored for their pioneer-ing work and outstanding discoveries in cell cycle researchby awarding them the Nobel Prize in Medicine in 2001(Hartwell, 2002; Hunt, 2001; Nurse, 2001). A concise butextremely informative summary of the events during the cellcycle has been formulated (Nurse, 2000). The eukaryotic cellcycle consists of two separable phases – interphase and mito-sis. While three sections (G1, S, and G2 phase) are distin-guished in interphase, mitosis comprises four sections:prophase (chromosome condensation), metaphase (chromo-some alignment); anaphase (chromosome separation), andtelophase (chromosome decondensation). Most importantfor the cell cycle to operate serenely are three control points,which have to be successfully passed:

i) The first is localized to the late G1 phase (START inyeast or the “restriction point” in mammals). BeforeSTART, cells have the option to either enter themitotic cycle, provided they meet adequate nutri-tional conditions and have reached a critical size, orunder starvation conditions to initiate a sexualprogram.

ii) The second control point localizes to the late G2 phase:during the G2 phase, cells have to ensure that com-plete DNA replication has been achieved without caus-ing any damage to the DNA.

iii) The third control to be passed lies prior to anaphase.Entry of anaphase will depend on correct chromosomealignment and proper spindle formation. Wheneverthese checkpoints fail (e.g., if the order of events isincorrect) cell division will lead to genetically aberrantprogeny.

7.2 Cell Cyclej187

7.2.1.2 Periodic Events in the First Phases of the Cell CycleProgression through the cell cycle is a carefully regulatedprocess that is conserved throughout eukaryotes. Periodicactivation of CDKs is required for this process; the critical

CDK involved in cell cycle progression in yeast is Cdc28p(also termed Cdk1p). (Kin28p, Pho85p, Ssn3p, and Ctk1p,the other CDKs in yeast, cooperate with other cyclins and arenot involved in cell cycle regulation.) The periodic events

Table 7.5 Yeast Cdc proteins.

Component Features/functions

Cdc2 DNA synthaseCdc3 septin, required for bud site selection, morphogenesis, and cytokinesis; contains GTP-binding domain; essentialCdc4 F-box containing proteinCdc5 polo-like kinase; required for mitotic exitCdc6 required for assembly and maintenance of the pre-replicative complex; loading factor for Mcm2–7pCdc7 essential serine/threonine protein kinase, required throughout the S phase; Cdc7p–Dbf4p complex interacts with origins of

replication (Orc2) and Mcm proteinsCdc8 thymidylate synthaseCdc9 DNA ligase, joins Okazaki fragments during DNA replication; also active in DNA repair; nonredundant paralog is Dnl4pCdc10 septin, required for bud site selection, morphogenesis, and cytokinesis; contains GTP-binding domain; nonessentialCdc11 septin, required for bud site selection, morphogenesis, and cytokinesis; contains GTP-binding domain; nonessentialCdc12 septin, required for bud site selection, morphogenesis, and cytokinesis; contains GTP-binding domain; essentialCdc13 interacts with different binding proteins; telomere capping function established by interaction with Stn1p and Ten1p (essential

for telomere length regulation); activates Est1p and Est2pCdc14 protein phosphatase, essential for mitotic exit: coordinates inactivation of mitotic cyclins, proper spindle disassembly,

completion of cytokinesisCdc15 protein kinase active in mitotic exit; relieves the inhibition of the protein phosphatase Cdc14p by Net1p, thereby allowing exit

from mitosisCdc16 (Apc6) component 6 of APC (E3 ubiquitin ligase of the APC); Cdc16p localizes to the centrosomes and the mitotic spindleCdc19 pyruvate kinase, catalyzes final step in glycolysis; also involved in cell cycleCdc20 F-box protein; activator of APC, mediates degradation of Pds1p, Clb5p, and Clb3pCdc21 dTMP synthaseCdc23 (Apc8) component 8 of APC (E3 ubiquitin ligase of the APC); Cdc23p localizes to the nucleus, kinetochores/microtubule ends, and

mitotic spindle in budding yeast; interacts with Mnd2p, Cdc16p, Cdc27p, and Clb2pCdc24 GEF for Cdc42pCdc25 regulated by glucose; Cdc25p activity is not necessary for growth in glucose, but is essential for growth in galactose and

nonfermentable carbon sourcesCdc26 subunit of APC, heat-shock protein for growth at high temperatureCdc27 (Apc3) component 3 of APC (E3 ubiquitin ligase of the APC); helps regulate the metaphase/anaphase transition and exit from mitosis/

G1 entry by ubiquitination of various substrates: sister chromatid separation inhibitor Pds1p, the Kip1p and Cin8p motorproteins, Cdc5p, and the spindle disassembly factor, Ase1p; contains TPR protein–protein interaction motif

Cdc28 catalytic subunit of main CDKCdc31(centrin)

component of TREX-2 complex with Sac3p, Sus1p, and Thp1p

Cdc34 ubiquitin-conjugating enzyme; catalyzes the transfer of activated ubiquitin (SCF) to the target protein; contact between Cdc34pand substrate by SCF protein complexes; regulated by phosphorylation (casein kinase 2), autoubiquitination, and self-association

Cdc36 (Not1) component of CCR4–NOTcomplex, which has multiple roles in regulating mRNA levels including regulation of transcriptionand destabilizing mRNAs by deadenylation; basal transcription factor

Cdc37 critical role in activating CDKs, cooperates with Hsp82pCdc39 (Not2) component of CCR4–NOTcomplex, which has multiple roles in regulating mRNA levels including regulation of transcription

and destabilizing mRNAs by deadenylation; basal transcription factorCdc40(Ppr17)

essential mRNA splicing factor

Cdc42 small GTPase in the Rho/Rac family, involved in establishment of cell polarity; localized to plasma membrane at sites of growthCdc45 involved in DNA replicationCdc46 equivalent to Mcm5pCdc47 equivalent to Mcm7pCdc48 AAA proteinCdc53 scaffolding subunit (cullin) for various RING-typeE3 ubiquitin–ligase complexes, also termed Skp1–cullin–F-box (SCF)

ubiquitin ligases; regulated by “NEDDylation” (conjugation with Rub1p in yeast)Cdc54 equivalent to Mcm4pCdc60 probably cytosolic leucyl-tRNA synthetaseCdc73 required for modification of some histones; telomere maintenance


occur in four phases (cf. Figure 7.7): a presynthetic gap (G1

phase), DNA synthesis (S phase), a postsynthetic gap (G2

phase), and mitosis (M phase). For division, yeast cells mustreach a critical size. The key point in control of the cell cycleis START – the transition that initiates processes like DNAsynthesis in the S phase, budding, and SPB duplication.Once cells have passed START, they are irreversibly commit-ted to replicating their DNA and progressing through the cellcycle. START thus coordinates the cell cycle with cell growth.Nutrient starvation as well as induction of mating blocks pas-sage through START. There are additional checkpoints thatarrest cells during the cell cycle to avoid DNA damage or celldeath due to events occurring out of order. Situated at theG1/S and G2/M boundaries, these control points function as

internal regulatory systems that arrest the cell cycle if prereq-uisites for progression are not met. After having passed thecell-size dependent START checkpoint, the level of cyclins(Cln, Clb) dramatically increase.

7.2.1.2.1 CDK and Cyclins Cyclins are periodicallyexpressed to function as the regulatory subunits activatingCDKs at the appropriate time in the cell cycle; CDKs are onlyactive when associated with a cyclin. In fact, as we have seenabove, they were named for their cyclical accumulation dur-ing particular phases of the cell cycle. There are at least 11different cell cycle-specific cyclins in yeast (Table 7.6) knownto form complexes with Cdc28p. The gene encoding Cdc28pis essential. Cdc28p is a 34-kDa protein that can bind and

Table 7.6 Cdc-associated proteins and components involved in cell cycle.

Component Features/functions

Apc1, Apc4, Apc5, Apc9,Apc13

components of APC/C complex

Apc2 component of the catalytic core of APC/C; has similarity to cullin Cdc53pApc10 processivity factor of APC/C complexApc11 catalytic subunit of APC/C complexCdh1 cell cycle-regulated activator of APC/C; targets the APC/C to specific substrates including Cdc20p, Ase1p, Cin8p, and

Fin1pCks1 protein associating with Cdc28p; regulates activity of Cdc28p/cyclin complex probably by targeting substrates;

homologs in S. pombe, Xenopus, and humanClb1, Clb2 B-type cyclins that activate Cdc28p to promote the transition from the G2 to the M phase of the cell cycle; expressed at

G2/M; promote spindle elongation; negatively regulate bud emergence; promote switch to depolarized bud growth;repress SBF-mediated transcription

Clb3, Clb4 B-type cyclins that activate Cdc28p to promote the transition from the G2 to M phase of the cell cycle; expressed inmid-S to G2; important for spindle formation

Clb5, Clb6 B-type cyclins that activate Cdc28p to promote initiation of DNA synthesis; expressed at START; can stimulate SBF-regulated gene transcription; prevent reinitiation on DNA replication origins that have already “fired;” have a possiblerole in spindle formation

Cln1, Cln2 cyclins involved in the G1 to S phase transition; closely related; expressed at START; accumulation of their mRNA inlate G1 depends on two transcription factor complexes, MBF (Swi6p–Mbp1p) and SBF (Swi6p–Swi4p), which bind toMCB and SCB promoter elements, respectively; stimulate Sic1p degradation; initiate localized growth leading tobudding; initiate SPB duplication; repress pheromone-induced transcription

Cln3 G1 cyclin; activates CLN1 and CLN2 transcriptionFkh1, Fkh2 members of the winged-helix/forkhead (FOX) transcription factor family regulating the expression of the CLB2

cluster of genes during the G2/M phase of the mitotic cell cycleLte1 essential for termination of the M phase at low temperaturesMbp1 Winged helix–turn–helix transcription factor involved in regulation of cell cycle progression from the G1 to the S

phase, forms a complex with Swi6p that binds to MluI cell cycle box regulatory element in promoters of DNAsynthesis genes

Mcm1 transcription factor involved in cell type-specific transcription and pheromone response; plays a central role in theformation of both repressor and activator complexes

Pcl1, Pcl2 Pcl1p and Pcl2p have some redundancy with Cln1p and Cln2p; required for cell cycle progression in the absence ofCln1p and Cln2p

Swe1 “Saccharomyces wee1;” protein kinase involved in regulating the G2/M transitionSwi4 DNA-binding component of the SBF complex (Swi4p–Swi6p) – a transcriptional activator that in concert with MBF

(Mbp1–Swi6p) regulates late G1-specific transcription of targets including cyclins and genes required for DNAsynthesis and repair

Swi5 transcription factor that activates transcription of genes expressed at the M/G1 phase boundary and in the G1 phase;localization to the nucleus occurs during G1 and appears to be regulated by phosphorylation by Cdc28p kinase

Swi6 DNA-binding component of the SBF (Swi4p–Swi6p) and MBF (Mbp1–Swi6p) complexesWhi3 RNA-binding protein that negatively regulates CLN3, both directly and indirectlyWhi5 cell cycle-regulated transcriptional repressor that inhibits both SBF (SCB-binding factor) and MBF (MCB-binding

factor)-mediated G1/S phase transcription

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hydrolyze ATP, and phosphorylate substrates at serine orthreonine in Ser/Thr–Pro motifs. Homologs have been char-acterized in many other eukaryotes; the crystal structure ofthe human counterpart, Cdk2, has been solved.

G1 cyclins (Cln1p, Cln2p, and Cln3p) are responsible forthe transition from G1 to S phase. Cln1p/Cdc28p andCln2p/Cdc28p are specifically able to repress pheromone-inducible transcription. While Cln1p and Cln2p are closelyrelated and similar in function, Cln3p, a particular G1 cyclinand the “oddest,” is a putative sensor of cell size, which actsby modulating the levels of other cyclins: Cln3p/Cdc28p hasa unique role in G1 as an activator of CLN1 and CLN2 tran-scription. Transcription of CLN3 itself is not strongly peri-odic with respect to the cell cycle, but there is a small rise atthe M/G1 border over its basal level.

In yeast, there are six B-type cyclin (CLB) genes involvedin the activation of the S, G2, and M phases of the cell cycle,expressed in three successive waves from START to the Mphase. These are pairs of homologous cyclin genes, the prod-ucts of which share common functions; regulation occursboth transcriptionally and post-translationally. Complex for-mation of Cdc28p has been established with Clb5p/Clb6p (Sphase), Clb3p/Clb4p (S/G2 phase), and Clb1p/Clb2p (Mphase, to promote cell cycle progression into mitosis). Alter-nation of cell cycle phases appears to be due to mechanismsthat one cyclin family succeeds another. The level of B-typecyclins are controlled by synthesis and programmed proteol-ysis by the ubiquitin–proteasome system. It was initially pro-posed that the Clb proteins play a role in the degradation ofthe G1 cyclins, but it was later shown that G1 cyclins areunstable in G1 phase, and Clb activity is not required fortheir degradation. These cyclins are often recognized by thepresence of a conserved domain, the “cyclin box,” a sequenceelement with a recognizable structural motif, the “cyclinfold.”

Both Clb5p and Clb6p promote progression into the Sphase. The corresponding genes are expressed periodicallythroughout the cell cycle and the cyclins are most abundantduring late G1. The CLB5 and CLB6 genes (Kuhne andLinder, 1993; Schwob and Nasmyth, 1993) are coexpressedwith CLN1 and CLN2, and could, in a sense, be classified asG1 cyclins. Importantly, Clb5p and Clb6p are kept in aninactive state by the CDK inhibitor (CKI) Sic1p up to a time-point when Cln1p/Cdc28p and Cln2p/Cdc28p activities haveappeared (see below).

Both gene promoters contain MCB (MluI cell cycle box)motifs, which are elements found in several DNA synthesisgenes. The transcriptional activator MBF (MCB-binding fac-tor), which is comprised of the Mbp1 and Swi6 proteins,bind to the MCB elements to activate transcription. Progres-sion through the cell cycle becomes highly coordinated. Rep-lication origin firing during the S phase is not random, butrather is under strict temporal and spatial control. Replica-tion forks cluster in discrete “replication factories” withinthe nucleus and components required for elongation associ-ate with nuclear structural components such as the lamina.

Definitely, early and late origins have to be distinguished (cf.Section 5.1.3). As it appears, Clb5p executes the origin firingprogram in both early and late origins, while Clb6p/Cdc28pcan only fire early replication origins. Further, the origin-firing program is subject to checkpoint controls, one ofwhich involves Rad53p as one of the essential players thatwill monitor successful execution of the program of DNAreplication during the S phase and coordinates a con-trolled arrest if problems are encountered. Rad53p alsoseems to be required for maintaining the level of nucleo-tides in the normal S phase.

Genetic interactions have shown that Clb3p and Clb4pmay both be involved in DNA replication and spindle assem-bly as well as in the G2/M-phase transition. CLB3 and CLB4transcripts arise near the beginning of the S phase (after theCLN1 and CLN2 peak) and remain high until late anaphase.Like the other Clb proteins, Clb3p and Clb4p contain adestruction box motif in their N-termini, which targets themfor ubiquitin-mediated degradation by the proteasome.Recent work has demonstrated that Clb4p/Cdc28p facilitatesspindle alignment by regulating the interaction of astralmicrotubules with subdomains of the bud cortex.

CLB1 and CLB2 are strongly periodic, peaking shortlybefore anaphase (Fitch et al., 1992); transcription isrepressed by the end of mitosis. The associated proteinkinase activity has a similar periodicity. Measurements ofabsolute levels of protein kinase activity indicate that Clb2p/Cdc28p constitutes the majority (85%) of Cdc28 activity inmitotically arrested cells. Clb1p and Clb2p cyclins aredegraded at the end of mitosis, by employing the destructionbox motifs in their N-termini, which target them for ubiqui-tin-mediated degradation by the proteasome. Expressionstudies indicate that Clb1p is the primary cyclin for the regu-lation of meiosis, while Clb2p is involved only in mitosis.

7.2.1.2.2 Regulation of the CDK/Cyclin System The abun-dance of the Cdc28p polypeptide is virtually unchangedthroughout the cell cycle. However, the activity of Cdc28p is,in addition to the cyclins, directly or indirectly influenced byabout 120 factors that act as positive and negative regulators,of which only a selection will be discussed here.

One such regulator is Cak1p – a CDK-activating kinaserequired for passage through the cell cycle, that activatesCdc28p by phosphorylating T169 of the Cdc28 protein andinduces a conformational change opening up the proteinsubstrate binding region and increasing the number of con-tacts between the Cdk and the cyclin (Thuret et al., 1996;Enke et al., 1999).

Another regulator is Cks1p – a small protein that physi-cally associates with the active (i.e., cyclin-associated) formof Cdc28p (Reynard et al., 2000; Morris et al., 2003). Cks1pmay perform a more subtle role in regulation of Cdc28p,such as targeting the Cdc28/cyclin complex to its substrates.Recent biochemical data argue strongly for a role as a CDK/cyclin assembly factor. The crystal structure of a human CDKbound to human Cks1 showed that this binding occurs at a


site adjacent to the catalytic site, which supports the model.Cks1p homologs have also been identified in S. pombe (suc1)and Xenopus (Xe-p9).

Important players in the regulation of the cell cycle areinhibitor proteins, known as CKIs, which block CDK activity(Figure 7.8). One such inhibitor is Sic1p, whose expressionis limited to the G1 phase (Schwob et al., 1994). Sic1p inhibi-tory activity is due to its ability to exclude substrates from theCdc28 active site. One task of Sic1p is to prevent prematureS-phase initiation until after Cln/Cdc28p levels have risensufficiently to complete bud initiation and SPB duplication –

a function that is performed by inhibiting Clb5p/Cdc28 andClb6p/Cdc28 complexes until Sic1p is destroyed. In fact,abolishing the inhibitory effect of Sic1p is a key mechanismby which the onset of proteolysis, which is induced throughits interaction with Cln/Cdc28p, phosphorylating Sic1p. Thedegradation of Sic1p then triggers the G1/S transition. A sec-ond function of Sic1p may be to assist programmed proteoly-sis at anaphase (see below).

A Cdc28p cyclin inhibitor that becomes active in responseto pheromone induction is Far1p that will be discussed below.

Swe1p inhibits the kinase activity of Cdc28p throughphosphorylation of a conserved tyrosine residue, Y19 (Gouldand Nurse, 1989). Y19 phosphorylation is reversed by thephosphatase Mih1p, which is homologous to CDC25 inother organisms. Swe1p-mediated inhibition of Cdc28p isimportant for delaying mitosis until all appropriate condi-tions for cell cycle progression are met (Lew and Reed,1995). It further appears that Swe1p regulates Clb/Cdc28pcomplexes to different degrees depending on which B-typecyclin is involved.

Swe1p is also important for delaying meiosis when thepachytene checkpoint is triggered. In addition to checkpoint

functions, a Swe1p-mediated G2 delay is employed duringfilamentous growth to promote bud elongation and invasivegrowth. Swe1p may also be required for reentry into the cellcycle after a G1 arrest caused by defects in ribosome bio-genesis or protein synthesis.

Swe1p expression is cell cycle regulated, with accumula-tion beginning in the S phase. As the cell cycle progresses,Swe1p undergoes a complex series of sequential phosphoryl-ations by a variety of kinases, including Cdc5p, Cla4p, andClb/Cdc28p, which result in hyperphosphorylation and sub-sequent ubiquitin-mediated degradation. Swe1p abundancealso increases transiently in response to ethanol stress. Over-expression of Swe1p leads to a G2 arrest, while in somestrain backgrounds null mutants enter mitosis prematurely.In premitotic cells, Swe1p localizes to the nucleus as well asto the daughter side of the mother-bud neck where it may bemarked for degradation.

Swe1p homologs have been identified in several orga-nisms, including S. pombe (wee1), Xenopus (Xwee1), andhumans (Wee1Hu), where they are required to govern entryinto mitosis and to delay cell cycle progression in response toDNA damage.

Cell Cycle-Specific Transcription Regulation of the cell cycleis dependent on the timely transcription of its elements.Generally, four waves of cell cycle-specific transcriptionaffecting Cdc28 activity are recognized: the genes at START(CLN1, CLN2, CLB5, and CLB6), at the M/G1 border (CLN3,CDC6, SIC1, and FAR1), at the S phase (CLB3 and CLB4),and at G2 (CLB1, CLB2, and many others).

Factors important for Cdc28p regulation and coordinatelyexpressed during the G2/M phase of the mitotic cell cycleinclude the cyclins Clb1p and Clb2p, and two transcription

Fig. 7.8 Simplified scheme of the yeast

cell cycle and regulation during START and

the mitotic phases.

7.2 Cell Cyclej191

factors, Swi5p (Nasmyth, Seddon, and Ammerer, 1987) andAce2p (Dohrmann et al., 1992), involved in the subsequenttranscriptional wave of cell cycle-regulated gene expressionin the M/G1-phase interval, as well as Cdc5p and Cdc20p(Shirayama et al., 1998), both of which become implicated inthe regulation of anaphase proteolysis. The correspondinggenes are summarized as the CLB2 cluster. SWI6 mRNA,encoding a transcription factor component involved inSTART, also shows a modest peak of accumulation in G2.The periodic transcription of the CLB2 cluster genes stemsnearly entirely from the activities of Mcm1p and SFF (Swifive factor) (Lydall, Ammerer, and Nasmyth, 1991), and isregulated by Fkh1p and Fkh2p (Zhu et al., 2000; Kumaret al., 2000; Hollenhorst, Pietz, and Fox, 2001; Morillonet al., 2003b).

Fkh1p and Fkh2p are members of the winged-helix/fork-head (FOX) transcription factor family, and appear to havepartially redundant roles. Promoter sequences responsiblefor restricting transcription of genes in this cluster to the lateS, G2, and M phases were first identified upstream of SWI5and CLB2, which possess upstream activating sequences(UASs) that contain binding sites for Mcm1p and for SFF –

an activity known to be involved in the formation of ternarycomplexes at these promoters in the presence of Mcm1p.Mcm1p is an essential, acidic transcription factor containingmultiple polyglutamine stretches. The N-terminal domain,known as the MADS box, is similar to that of other trans-criptional activators, that all bind a similar DNA element, CC(A/T)6GG (called the MCE (Mcm1 cell cycle element)). MCEsare found in the promoters of CLB1, CLB2, and SWI5 (Kuoand Grayhack, 1994), and each of these genes, as well asACE2, requires Mcm1p for its expression. The full expres-sion of SWI5, however, needs SFF (hence the name) as anadditional cofactor.

Fkh2p was subsequently identified as a component ofSFF. Inclusion of Fkh2p in the complex is facilitated byDNA bending induced by Mcm1p. The rate-limiting tran-scriptional coactivator Ndd1p is finally recruited to the chro-matin of G2/M-regulated promoters through interactionswith Fkh2p in a manner that is dependent on both the phos-phorylation of Ndd1p by the Cdc28p–Clb2p kinase complexand the phosphorylation of Fkh2p by one or more complexescontaining Cdc28p together with a B-type cyclin (Clb2p orClb5p). Fkh2p is also required for the recruitment of theCdc5p polo-like kinase, inducing the formation of a Fkh2p–Ndd1p–Cdc5p complex on CLB2 cluster promoters and lead-ing to the phosphorylation of Ndd1p by Cdc5p – an eventrequired for the proper temporal activation of CLB2 clustergenes during G2/M.

Distinct functions for Fkh1p and Fkh2p in the control ofG2/M-phase transcription and regulation of the cell cyclebecome evident from mutant strains. Strains deleted forFKH1 alone demonstrate enhanced transcription of CLB2throughout the cell cycle and have a slightly elevated rate ofprogression through the S and G2/M phases. By contrast,deletion of FKH2 exhibits reduced CLB2 transcription and a

reduced rate of progression through the cell cycle. Addition-ally, Fkh1p and Fkh2p in many cases compete for target pro-moter occupancy. Interestingly, Fkh1p cooperates with thechromatin-remodeling complex Isw1 to repress transcriptionof CLB2 during G2/M, whereas Fkh2p cooperates with thechromatin-remodeling complex Isw2 to repress CLB2 tran-scription during the G1 phase. Finally, Fkh1p and Fkh2passociate with the coding region of active genes where theyregulate transcriptional elongation and termination inopposing ways by affecting the phosphorylation status of theC-terminal repeat domain (CTD) of RNA polymerase II.Strains deleted for both genes display morphological altera-tions including defects in cell separation, budding, and theinduction of a nutrient-independent pseudohyphal-likegrowth phenotype.

During telophase in mitosis, several genes become acti-vated that will be important for CDK regulation during theG1 phase: CLN3, transcription factor SWI4, REM1 (an inhib-itor of sporulation-specific transcription and activator ofSTART-specific transcription), CDC6, FAR1, and the CKISIC1. CLN3 and SWI4 are controlled by Mcm1p that bindsat a site called the early cell cycle box (ECB) (McInerny et al.,1997).

Control of Cdc28p activity at START is perhaps the mostcritical event, since it determines whether the cell will enterinto a round of mitotic division or not. Start-specific tran-scription at this point in the cell cycle is therefore a keyevent, concerning the production of major Cdc28p regula-tors such Cln1p, Cln2p, Cln3p, Clb5p, and Clb6p. As wehave noticed, the transcription of other important Cdc28pregulators such as Swi4p or Swe1p is also strongly influ-enced by events at START. The expression of the correspond-ing genes is governed by the action of two relatedtranscription factors, SBF and MBF, which are responsiblefor most of the periodic, late-G1-specific mRNA production.SBF (SCB-binding factor) and MBF (MCB-binding factor)complexes both contain the Swi6p transcriptional coactivatorand either Swi4p (SBF) or Mbp1p (MBF) – two sequence-specific DNA binding proteins. These complexes bind eitherto SCB (Swi4/6-dependent cell cycle box; CACGAAAA ele-ment) or MCB (Mlu1 cell cycle box; multiple ACGCGTsequences) promoter sites to enhance the transcription ofhundreds of genes during the G1 phase, including additionaltranscription factors. In fact, SBF is the major factor involvedin CLN transcription, while MBF is dominantly responsiblefor CLB5/6 transcription.

Expression of SBF and MBF themselves is controlled by anumber of parameters, but the best understood is the controlby Cln3p/Cdc28p. This complex appears to represent theonly stimulator of START-specific transcription, activatingthe SBF and MBF complexes. In aberrant conditions, thissystem needs to be repressed. Inhibition is brought about bythe repressor protein Whi5p, which associates with G1-spe-cific promoters through direct interactions with both SBFand MBF complexes, and affects the onset of G1/S-phasetranscription. Cln3p/Cdc28p, thus adopting the role of a


key regulator of both MBF- and SBF-dependent geneexpression, hyperphosphorylates the Whi5 protein duringthe late G1 phase causing it to dissociate from SBF and exitthe nucleus, so that Whi5p remains in the cytosol until theend of mitosis. Whi5p re-enters the nucleus only at the endof mitosis, after CDK activity has been eliminated by theMEN (see Section 7.2.2.3).

Another regulator of CLN3 is Whi3p, an RNA-bindingprotein that negatively regulates the expression of this gene,both directly and indirectly. Whi3p contains a C-terminalRNA recognition motif (RRM) that binds CLN3 mRNA andlocalizes the mRNA into cytoplasmic foci, perhaps to locallyrestrict synthesis of this G1 cyclin. Further, the N-terminalCdc28-recruitment region of Whi3p interacts with Cdc28pand G1 cyclin/Cdc28p complexes. In this capacity, Whi3pacts as a cytoplasmic retention factor, sequestering Cdc28pand associated cyclins in the cytoplasm of early G1-phasecells, thereby restricting the nuclear accumulation of thesecomplexes to the late G1 phase. Indirectly, Whi3p thus regu-lates the critical cell size required for passage throughSTART, the normal mating response, filamentous growth,and meiosis.

Cdk1p, as well as being essential for the S phase, is alsoimportant in controlling entry into mitosis.

Cell Cycle-Specific Degradation Ubiquitination of Sic1p forproteasomal proteolysis at the G1/S-phase transition iseffected by the E2 conjugase Cdc34p (the only essential E2enzyme in yeast) in conjunction with the E3 ligase SCFCdc4,which also recognizes Far1p and Cdc6p as substrates. Theinteraction between the F-box protein Cdc4p and the scaffoldprotein Skp1p is stabilized by the eight WD40 repeats inCdc4p.

Ubiquitin-mediated proteolysis applies also to Cln1p andCln2p (but not Cln3p) that has to occur at the early S phase;in this case the substrate-specific interaction is broughtabout by SCFGrr1 – an E3 ligase that contains the F-box com-ponent Grr1p. Remarkably, SCFGrr1 cannot functionallyreplace SCFCdc4 nor does it interfere with SCFCdc4. The onlyrequirement for the interaction with Cln1p and Cln2p is thatthese have been phosphorylated. SCFGrr1 is also involved inthe regulation of glucose metabolism in yeast (cf. Section10.4.2) – a relationship that becomes immediately meaning-ful considering that glucose has a major influence on cellcycle progression. This is also an indication that SCF com-plexes might be regulated by environmental conditions.

As we have noted, Clb protein levels are periodic, withmaximum accumulations occurring in the post-G1 phase ofthe cell cycle and sharp declines occurring in anaphase(Amon, Irniger, and Nasmyth, 1994). The half-lives of Clb5p,Clb3p, and Clb2p are 1–2min during the G1 phase when itsproteolysis gets active. Throughout the S and G2 phases,Clb2p and Clb3p are stable, but Clb5p continues to turnover; however, it has a longer half-life, of 10–15min. As withthe START proteolysis substrates, the Clbs are proteolyzedvia ubiquitinated intermediates. As mentioned briefly,

features relevant to this process are the cyclin “destructionboxes” – short sequence motifs residing near the N-terminusof the Clb proteins. Other components and the mechanismsinvolved in this pathway are considered in Section 7.2.2.2,because they are related to chromosome segregation and dis-tribution during anaphase and mitotic exit.

Cell Cycle Checkpoints In addition to the normal pathway,cell cycle progression is controlled by the availability ofnutrients. Nutrient levels (e.g., glucose or nitrogenous com-pounds) regulate the intracellular concentration of cAMP viathe small G-protein, Ras. The so-called Ras–cAMP pathwayis well documented (see Section 10.1). Decreasing levels leadto G1 arrest, while increasing levels induce the cAMP-depen-dent protein kinase PKA, which then phosphorylates andthereby activates specific transcription factors involved inSTART.

We have discussed already how the cell cycle will bearrested or at least delayed in response to DNA damage orincomplete DNA replication, the failure to duplicate SPB,or to assemble a proper mitotic spindle. Cell cycle arrest isalso invoked at stress conditions. The factor induced uponstress or starvation during mitosis, and late in meiosis, isXbp1p, a transcriptional repressor that binds to promotersequences of the cyclin genes, whereby regulation of theCln3–Cdc28 activity seems to be a key event. The proteinis a member of the Swi4p/Mbp1p family and a potentialCdc28p substrate (Mai and Breeden, 1997, 2008). Whenmating pheromone is applied to yeast cells, they will com-plete their current cell cycle, but then arrest by the actionof Far1p – a CKI that mediates cell cycle arrest in responseto pheromone. Far1p is induced in the pheromone signal-ing cascade (cf. Section 10.2.1).

7.2.2Dynamics and Regulation of Mitosis

Mitosis is the longest phase of the cell cycle. During thisperiod, accuracy of DNA duplication has to be checked, andsister chromatids have to be aligned, prepared for segrega-tion, and distributed in an ordered fashion between motherand daughter before cell separation.

7.2.2.1 Sister Chromatids: CohesionAs long ago as 1879, Fleming noticed that “the impetus caus-ing nuclear threads to split longitudinally acts simulta-neously on all of them” (Fleming, 1879). Chromosomeseparation is irreversible and must therefore be highly regu-lated. Damage to chromosomes cannot simply be repaired byrecombination nor can aberrant chromosome alignments bereversed, once sister chromatids segregate. Rather, sisterchromatids have to be tied together after chromosome repli-cation during mitosis until all control mechanisms havebeen executed that guarantee the intactness of all replicatedchromosomes before they are disentangled in metaphaseprior to cell division in anaphase. The early “interaction

7.2 Cell Cyclej193

model” that involved topoisomerase I proved invalid.Koshland and Hartwell (1987) used SV40 minichromosomesto show that the sister molecules are properly segregatedwhen a cell cycle block is removed, arguing that sister mini-chromosome molecules need not remain physically inter-locked until anaphase in order to be properly segregated.Among other approaches, this system has been kept since tostudy how sister chromatids behave during segregation (Iva-nov and Nasmyth, 2005). To date, we have ample knowledgeof the molecular subtleties of how sister chromatids are kepttogether and separated at appropriate moments of cell divi-sion, largely stemming from yeast as a model system.

Cohesion finally turned out to be mediated by factorsforming a multisubunit complex called cohesin, which bindsto chromosomes at multiple sites, from telophase until theonset of anaphase in the next cell cycle (Hirano, 2000;Nasmyth, 2001). Cohesin consists of four core subunits:Smc1p, Smc3p, Scc1p, and Scc3p. Smc1p and Smc3p pro-teins (SMC complex) are characterized by 50-nm antiparallelcoiled-coils flanked by a globular hinge domain and an ABC-like ATPase head domain. While Smc1p and Smc3p hetero-dimerize via their hinge domains, the kleisin subunit Scc1pconnects their ATPase heads.

An early postulate was that cohesin connects sister DNAmolecules through the binding of its two heads to each sisterDNA molecule, thus forming a “glue” between the sisterchromatids (Toth et al., 1999; Anderson et al., 2002). How-ever, the finding that the N- and C-termini of Scc1p bind,respectively, to the Smc3p and Smc1p heads of the Smc1/Smc3 heterodimer (Haering et al., 2002) suggested that cohe-sin forms a large proteinaceous ring in which DNA strandscould be trapped (Figure 7.9). Cleavage of Scc1p by a cysteinprotease called “separin” or “separase” (Esp1 in yeast (Ciosket al., 1998; Uhlmann et al., 2000)) triggers poleward move-ment of sister chromatids at the metaphase-to-anaphasetransition (see below). Scc1p in turn recruits and binds thefourth cohesion subunit, Scc3p, which has two orthologs inmammals. From these findings, it was concluded that the

connection between sisters must be a topological rather thana chemical one (Nasmyth, 2002). This hypothesis turned outto be correct (Haering et al., 2002; Gruber, Haering, and Nas-myth, 2003); it was confirmed and refined by experimentsinvestigating the topological interaction between cohesinrings and a circular minichromosome (Ivanov and Nasmyth,2005).

With a diameter of close to 50 nm, the ring is suffi-ciently large to hold two sister DNA strands together evenwhen wrapped around histones. At anaphase onset, whenthe Scc1p subunit is cleaved by separase, which in turndisrupts the interaction between the Smc heads in cohe-sin, the ring is opened (Weitzer, Lehane, and Uhlmann,2003; Lengronne et al., 2004). Critical to the cleavage ofScc1p (or other members of the kleisin family) is that itsC-terminal cleavage product is quickly destroyed by tar-geted proteolysis (Rao et al., 2001) to prevent the Smcheads from interacting.

Additionally, more factors involved in the establishmentand maintenance of the cohesin complex were disclosed,such as Mcd1p, Irr1p, and Pds5p. Mcd1p associates prior tometaphase with centromeres and other discrete sites alongthe chromosome arms; this interaction depends on the cen-tromere protein Mif2p, the centromere binding complexCBF3 and Cse4p (see also Figure 7.11). Mcd1p must be pres-ent during the S phase, but is dissociated from chromo-somes at the metaphase-to-anaphase transition by the actionof separin. An incidental cleavage of Mcd1p by the caspase-like protease Esp1 promotes apoptosis in yeast (Yang, Ren,and Zhang, 2008). Cohensin’s binding to chromosomes wasalready shown some years ago to be accomplished by a “load-ing factor,” consisting of the proteins Scc2p and Scc4p(Ciosk et al., 2000). However, these factors are not stochio-metric subunits of cohesin, rather they are involved in estab-lishing sister chromatid cohesion even during double-strandbreak (DSB) repair via histone H2AX. Irr1p is another essen-tial subunit of the yeast cohesin complex, and required forsister chromatid cohesion in mitosis and meiosis. Reduced

Fig. 7.9 Cohesin rings keeping sister chromatids

together.


expression of IRR1 alters colony morphology and causesdefects in zygote formation and spore germination.

Pds5p is a protein that colocalizes with cohesin on chro-mosomes and may function as a protein–protein interactionscaffold; it is also required during meiosis (Zhang et al.,2005). Pds5p forms a complex with Scc3p and Rad61p (alsocalled Wpl1p), which will block the establishment of sisterchromatid cohesin (Kueng et al., 2006; Sutani et al., 2009).This intervention is induced by the acetylation of Smc3p atreplication forks and Mcd1p in response to double-strandedDNA breaks through the C2H2-type zinc finger acetyltrans-ferase Eco1p (Ctf7p) (Skibbens et al., 1999; Toth et al., 1999;Rolef Ben-Shahar et al., 2008). Furthermore, alternative chro-matin-remodeling complexes (RFCs) (Sjogren and Nasmyth,2001) as well as the nucleosome-remodeling complex (RSC)(Huang et al., 2004a) are required for efficient cohesionestablishment during the S phase.

Recently, it has been demonstrated that the kleisins (Scc1pand Scc3p) not only connect the Smc1p and Smc3p ATPaseheads, but also regulate their ATPase activity (Arumugamet al., 2006). For a long time, an unsolved problem was howthe two DNA strands are “entering” the cohesin ring during(or after) DNA replication (Lengronne et al., 2004). Could theDNA replication fork simply slide through the cohesin ringsthat were put around DNA before the S phase? This wouldleave two replication products trapped inside the same ringwithout further transport and at the same time provide anintrinsic solution to the crucial requirement to only establishsister chromatid cohesion between authentic replicationproducts and never between any other two sequences ofDNA.

Apparently, the problem of cohesin architecture has comeclose to solution. Gruber et al. (2006) showed that cohesin’shinges are not merely dimerization domains that are holdingtogether the cohesin ring by preventing kleisin’s dissociationfrom the SMC heads. Rather, entry of DNA into the cohesin

ring requires opening and transient dissociation of theSmc1p and Smc3p hinge domains (see also Shintomi andHirano, 2007).

The binding of condensin, the second sister chromatid-stabilizing complex, has similarities to cohesion (Lavoie,Hogan, and Koshland, 2002). Condensin, a 13S complex,consists of two Smc proteins, Smc2p and Smc4p, and con-tains three other essential subunits, one of which is homolo-gous to Scc1p; the newly found Smc5p–Smc6p complexpreserves nuclear integrity (Torres-Rosell, Machin, and Ara-gon, 2005). Just like cohesin, the topological structure of con-densin is a ring. Condensin associates with chromatinindependently of ATP, but ATP hydrolysis is needed for thebinding reaction. A particular feat of condensin is that chro-matin wraps around it, generating a torsion in the DNA(Wang et al., 2005a).

Thus, condensin is amenable of contributing to chromo-some compaction; it also participates in DNA repair (Chen,Sutani, and Yanagida, 2004). After partial removal of cohesinrings by the separase reaction, condensin replaces cohesin,both in mitosis and in meiosis (Yu and Koshland, 2005).

Recent studies have revealed that Smc5 and Smc6ptogether with the SUMO ligase Mms21p in a complex aregenerally involved in the structural maintenance of chromo-somes, and are required for growth and DNA repair (Onodaet al., 2004; Zhao and Blobel, 2005; Murray and Carr, 2008).

The ordered segregation and movement of chromatids toopposite poles of the cell is triggered by the fluctuation of themitotic B-type cyclins during the cell cycle (Evans et al., 1983)(Figure 7.10). At the commencement of anaphase at the lateG2 phase, CDK activity is destructed by proteolytic degrada-tion of the cyclins (Glotzer, 1991), but is not required for theseparation of sister chromatids (Surana et al., 1993; Dirick,Bohm, and Nasmyth, 1995). This finding suggested that pro-teolysis of further proteins is necessary for sister chromatidseparation (Holloway et al., 1993). The apparatus responsible

Fig. 7.10 Scheme of chromosome

segregation.

7.2 Cell Cyclej195

for targeting cyclin degradation turned out to be a highlyconserved multisubunit complex that possesses ubiquitin–protein ligase activity (Zachariae et al., 1998; Shirayamaet al., 1998). Initially named the cyclosome (Sudakin et al.,1995), the ligase moiety is called the anaphase-promotingcomplex (APC) in modern terms; it mediates the destructionof many proteins other than cyclins and was shown to beessential for the separation of sister chromatids (Irnigeret al., 1995; King et al., 1995; Zachariae et al., 1996; Guacci,Koshland, and Strunnikov, 1997; Michaelis, Ciosk, andNasmyth, 1997; Losada, Hirano, and Hirano, 1998;Zachariae and Nasmyth, 1999). We will come back to moredetails in the following sections, discussing the processes ofspindle assembly, APC activation, and exit from mitosis.

7.2.2.2 Spindle Assembly CheckpointIn the years following the discovery of the APC, it becameclear that its activity is controlled by the spindle assemblycheckpoint (SAC) – a surveillance mechanism shared bymost eukaryotic cells that prevents sister chromatid separa-tion when spindles are damaged or chromosomes fail toform spindle attachments (Amon, 1999; Nasmyth, 2002;Lew and Burke, 2003; Gillett, Espelin, and Sorger, 2004; Tan,Rida, and Surana, 2005; Fuller and Stukenberg, 2009). SACrestrains the onset of anaphase until all chromosomes areproperly attached to a bipolar spindle and develop tensionfrom the pulling forces exerted from either pole. Attachmentsites for the spindle microtubules are the kinetochores, spe-cialized regions on the chromosomes, which also act astransmitters of the SAC signal. The SAC is kept silent by twoindependent events that redundantly ensure all chromo-somes are properly attached to the mitotic spindle before theelimination of cohesion induces anaphase. The kinetochoreof a single lagging chromosome emits a signal capable ofblocking separation of all sister pairs.

The kinetochore is composed of protein assemblies thatcan be broadly classified into inner, central, or outer

kinetochore complexes (Figure 7.11 and Table 7.7). It wasdemonstrated (Ciferri, Musacchio, and Petrovic, 2007) thatthe outer kinetochore complex DAM1, composed of Duo1pand Mps1p (monopolar spindle 1) interacting complex, playsa crucial role in mediating the kinetochore–microtubularconnection and is regulated through phosphorylation by theIpl1p/Aurora B kinase. The central complex contains theIPL1, CFT19, NDC80, and MWT1 complexes that are associ-ated with both microtubules via the DAM1 complex andkinetochores via the inner complex; the most critical com-plex is CBF3 (centromere binding factor 3) (Joglekar, Bloom,and Salmon, 2010).

CBF3 consists of the essential proteins Ndc10p, Cep3p,Cft13p, and Skp1p, as well as a number of chromatin-spe-cific proteins, which are required to build up a kineto-chore at each centromere. The IPL1 (Aurora kinase)complex responds to the lack of tension in monotelicattachments and acts to resolve these inappropriate attach-ments, probably through its substrates. In the absence oftension, Ipl1p causes an increased turnover of kineto-chore–microtubule connections, perhaps by influencingthe Ndc80–DAM1 interaction. Experiments on buddingyeast have demonstrated that the tension resulting fromthe physical connection between bioriented kinetochoresand the activity of Ipl1p (rather than any specific chromo-somal architecture or kinetochore geometry) is sufficientfor the proper alignment of sister chromatids (Dewaret al., 2004).

The Aurora kinase complex (also named the chromosomalpassenger complex (CPC)) is an essential regulator of chro-mosome segregation, spindle checkpoint, and cytokinesis(Ruchaud, Carmena, and Earnshaw, 2007); its four membersare conserved from yeast to man. Ipl1p, the catalytic compo-nent, is a serine/threonine protein kinase. The other threecomplex members, Sli15p, Bir1p (Survivin), and Nbl1p(Borealin), are all essential genes in S. cerevisiae and arethought to play roles in Ipl1p localization, stabilization, and/or regulation. Bir1p further plays independent roles in chro-mosome stability and apoptosis (Owsianowski, Walter, andFahrenkrog, 2008).

Ipl1p function is required at many distinct locations andevents during cell division (Zich and Hardwick, 2010). Ipl1plocalizes to kinetochores from G1 to metaphase and to thespindle after metaphase. If the tension Ipl1p generates islost, it creates unattached kinetochores and activates theSAC. At late anaphase, Ipl1p relocalizes to the spindle mid-zone, where it ensures that cytokinesis completes only afterall chromosomes have migrated to the poles. Ipl1p is alsorequired for mitotic spindle disassembly; during this processIpl1p localizes to the plus ends of the depolymerizing spin-dle microtubules.

The dynamic localization of the Aurora kinase complexis carefully regulated by dephosphorylation of a noncata-lytic member of the complex, Sli15p, by Cdc14p. The mas-ter regulator, however, is the protein phosphatase Glc7pthat acts in opposition to Ipl1p by dephosphorylating

Fig. 7.11 Components of the yeast kinetochore.


Ipl1p targets (such as Cbf2p, Mif2p, and Tid3p; Ase1p,Mad3p, and Dam1p; condensin; histone H3; Sli15p),rather than by directly regulating Ipl1p itself (Bouck,Joglekar, and Bloom, 2008).

There are three Aurora kinase family members in Homosapiens: Aurora A, Aurora B, and Aurora C, which vary infunction and tissue specificity. Aurora kinases have beenimplicated in tumorigenesis, and Aurora kinase expression

Table 7.7 Components of the yeast kinetochore

Component/complex Characteristics/function

Inner complexCbf1p centromeric DNA binding factorMif2p AT-hook motifCse4p centromeric histone H3 equivalentCac1p chromatin assemblyHir1p chromatin assembly, WD40 domainSgt1p activates Ctf13pCBF3 all components essential for kinetochore assemblyNdc10pCep1pCtf13pSkp1pCentral complexesCTF19 localization dependent on CBF3 and Cse4p; Okp1p and Ame1p form the COMA subcomplexCtf19p Opk1pCtf3p Mcm16pChl4p Mcm21pIml3p Mcm22pAme1p Nkp1p

Nkp2pMTW1 also called MIND subcomplex; it is believed to promote biorientation that is monitored by Ipl1p kinaseMtw1p Nnf1pDsn1p Nsl1pNCD80 all components essential; localization dependent on CBF3 and Cse4p; this complex copurifies with SPBs;

complex required for kinetochore binding of the DAM1 complex, the kinase-related proteins Cin8p andKip1p, as well as microtubule-associated protein Stu2p

Ndc80p Spc24pNuf1p Spc25pIPL1 localization dependent on CBF3, requires Ipl1p kinase activity; Ipl1p/Aurora B, Bir1p, and Sli15p regulate

establishment of bipolar attachment of sister kinetochores to microtubules; substrates of Ipl1p includeNdc10p, Cse4p, Ndc80p, Dam1p, Spc24p, and Ask1p

Ipl1p Sli15pBir1p Nbl1pOuter complexesDAM1 localization of DAM1 is microtubule dependent and requires all kinteochore subcomplexes; regulation may

occur through Ipl1pDam1p Dad2pDuo1p Dad3pAsk1p Dad4pSpc34p Spc19p

Dad1pMicrotubule-associatedproteinsMps1p kinase activity and phosphorylation increase upon SCP activation; requires Ndc80 and Nuf2 for kinteochore

associationStu2p HEATrepeats, coiled-coilsSlk19p coiled-coilPlc1p phospholipase CGlc7p protein phosphataseGle2p WD40 domain proteinBik1p plus-end binding microtubule-associated protein, probably regulates plus-end microtubule dynamicsBim1p plus-end tracking microtubule-associated proteinKin1p, Kin3p, Cin8p kinesin-related motors

7.2 Cell Cyclej197

levels and activity have been shown to be upregulated inmany human cancers.

7.2.2.3 Chromosome SegregationNormally, once chromosomes have successfully bioriented,cells use an evolutionarily conserved machinery to initiateanaphase. As already mentioned above, rapid disjunction ofsister chromatids at anaphase requires cleavage of cohesinsubunit Scc1p by a cysteine protease called separase (Esp1pin budding yeast) inactive through most of the cell cycle byits association with an inhibitor, securin (Yamamoto et al.,1996a; Ciosk et al., 1998; Uhlmann, Lottspeich, andNasmyth, 1999). The metaphase-to-anaphase transition istriggered when securin (Pds1p in budding yeast (Cohen-Fixet al., 1996; Yamamoto et al., 1996b; Cohen-Fix and Kosh-land, 1997)) is degraded by the proteasome as a consequenceof ubiquitination by the multicomponent E3 ubiquitin ligaseknown as the APC. APC function, which is also responsiblefor ubiquitination and hence destruction of cyclin B, onset ofanaphase, and mitotic exit, is regulated (Figures 7.10 and7.12) by (i) phosphorylation and (ii) association of activatorproteins such as Cdc20p or Cdh1p, and the homolog Hct1p,which modulate the affinity of APC for different substrates(Peters, 2002).

Activation of the SAC invokes its “anaphase arm” andleads to inhibition of the ubiquitin-dependent proteolysis ofsecurin by the APCCdc20 (Hwang et al., 1998). It also blocksany further cell cycle progression, in order to prevent wrongcell division. In yeast, this process is triggered by therecruitment of a complex to the SAC, which contains the“mitotic arrest defective” proteins Mad1p, Mad2p (blockingseparase), Mad3p, and the tyrosine protein kinase Bub1p,whereby Bub1p is activated through Cdk1p. As a result ofthe phosphorylation reactions, Bub3p binds to the activatorCdc20p of the APCCdc20 complex, and thereby blocks ubi-quitination of both securin and cyclin B. In addition, pro-tein kinase Msp1p is required for modification of APCCdc20

and spindle pole duplication, as is subunit Ncd10p of thecentromere-binding complex CBF3. The MAD complex(Table 7.8) has been shown to be highly conserved amongother eukaryotes. The functions of APC and other factorsinvolved in regulating mitotic spindle disassembly havebeen described in detail (Buvelot et al., 2003; Pereira and

Schiebel, 2003; Schuyler, Liu, and Pellman, 2003; Tan,Rida, and Surana, 2005). In all, this signal transductionpathway is set into motion by the SAC to act on specificcellular targets in order to delay the onset of anaphase andmitotic exit in case of emergency.

Fig. 7.12 “Anaphase arm” of the SAC.

Table 7.8 Main proteins of the MAD complex.

Component Features

Mad1p contains coiled-coil domain and Q/N-rich regions for aggregationMad2p contains HORMA domain that recognizes chromatin states resulting from DNA damage, DSBs, or nonattached spindles;

recruits repair proteinsMad3p contains GLEBS motif recognized by Bub3p; N-terminal sequence similarity to Bub1pBub1p protein tyrosine kinase; GLEBS motif binds Bub3pBub2p functions both as a GAP and as a GTPase inhibitorBub3p contains WD40 domain; phosphorylated by Bub1pMsp1p protein (serine, threonine, tyrosine) kinase; essential function in spindle pole duplication and in proper chromosome

segregation in meiosis


7.2.2.4 Regulation of Mitotic ExitTo drive yeast cells into the mitotic phase, downregulation ofCdk by destruction of the B cyclins is essential. Additionally,an essential yeast phosphatase, Cdc14p, has been recognizedas a key regulator of mitotic exit. Cdc14p dephosphorylatesCdk, thereby activating the inhibitor Sic1p and its transcrip-tion factor Swi5p for the subsequent mitotic cycle, as well asa second APC activator Cdh1p (Visintin et al., 1998). (Cdksubstrates that are dephosphorylated by Cdc14p also includethe microtubule regulators Ase1p, Ask1p, Fin1p, andSli15p.) It was further noticed that activation of Cdc14p ledto an elongation of the spindle (Pereira et al., 2000).

Destruction of the major mitotic cyclin Clb2p occurs in abiphasic manner: APCCdc20 is responsible for the first phaseof Clb proteolysis that occurs at high cellular levels of Clbs.The resulting decrease in mitotic kinase (Cdk) activityinduces a net dephosphorylation of Cdh1p by Cdc14p, whichis the decisive step in Cdh1p activation. Activated APCCdh1

then mediates the second phase of Clb destruction and trig-gers mitotic exit (Yeong et al., 2000). The main role of Cdh1pappears to be to maintain a low mitotic kinase state in G1,because the extent of Clb2p proteolysis driven by APCCdc20

may already be sufficient to permit mitotic exit, even in theabsence of Cdh1p and the CKI Sic1p.

Meanwhile, it is accepted that activation of the Cdc14phosphatase takes place as early as to become a prerequisitefor sister chromatid segregation, and that two pathways inCdc14p activation can be distinguished: FEAR (Cdc fourteenearly anaphase release) when Cdk activity is still high(Stegmeier, Visintin, and Amon, 2002) and MEN (mitoticexit network) at low Cdk activity (Shou et al., 1999) which lat-ter is an essential element of mitotic exit. In a recent review(Queralt and Uhlmann, 2008a), the authors have summa-rized these pathways in a concise picture (Figure 7.13).

During most of the cell cycle, Cdc14p is sequestered in theRENT (regulator of nucleolar silencing and telophase) com-plex in the nucleolus by its inhibitor Net1p (Cfi1p) (Straightet al., 1999), also a component of the RENT complex. Phos-phorylation of Net1p during anaphase weakens its affinityfor Cdc14p, so that Cdc14p is released; initially it is distrib-uted within the nucleus and finally reaches the cytoplasm,where it localizes to SPBs. Although the main activation ofCdc14p occurs during MEN (which is discussed below), itstransient release from the nucleolus at early anaphase isinduced by FEAR in a MEN-independentmanner.

Important constituents of the FEAR network are sepa-rase (Esp1p), the polo-like Cdc5p kinase, the kinetochoreprotein Slk19p, and Spo12p, a nucleolar protein that regu-lates release of Cdc14p from the nucleolus in early ana-phase. In addition to its proteolytic properties, separasehas a second (nonproteolytic) capacity in that it suppressesthe activity of the phosphatase PPA2Cdc55, which normallycounteracts the phosphorylation of Net1p, thus supportingits sequestration in the nucleolus. This allows Clb2p/Cdk(whose activity is still high at this stage) to phosphorylateNet1p and to release Cdc14p. In suppressing PPA2,

separase cooperates with the two PPA2 regulators Zds1pand Zds2p (Queralt and Uhlmann, 2008b). The activity ofpolo-like kinase Cdc5p supports the directionality of thispathway.

The major pathway for mitotic exit, MEN, involvesmany more components than FEAR, although Cdc5p andPPA2 play essential roles in MEN as well. Most of theother constituents (including Tem1p, Cdc15p, Mob1p(Mah, Jang, and Deshaies, 2001), Dbf2p, Dbf20p, Bfa1p,Bub2p, and Lte1p (low temperature essential)) have beenknown for quite a time (Jaspersen et al., 1998). Tem1p(Shou et al., 1999; Lippincott et al., 2001) is a Ras-likeGTPase (with Lte1p as the GEF and the Bub2–Bfa1p com-plex (Wang, Hu, and Elledge, 2000; Hardwick, 1998), as adownregulating GAP); Cdc15p and Dbf2p are protein kin-ases, as is Cdc5p. A predominant effect of Cdc5p in thispathway is the inhibition of Bub2–Bfa1p, thus contribu-ting to activation of Tem1p. A further target for Cdc5p isprobably the Cdc14p inhibitor Net1p itself; but thisreaction depends on prephosphorylation by other kinases.From many observations, it is not expected that Cdc5p actsas a specific inducer of mitotic exit, but rather acts as anessential amplifier of generic phosphorylation. In thisregard, Cdc5p is not considered a regulator of onset ofmitotic exit, but may only indirectly contribute to it, all themore as its activity is abolished by APCCdh1-mediateddestruction at the end of mitosis; this may be sufficientfor resequestration of Cdc14p to the nucleolus. PP2ACdc55

counteracts the phosphorylation of Bfa1p during meta-phase, so that downregulation by PP2ACdc55 at anaphase

Fig. 7.13 Model for mitotic exit in yeast. Yellow, phosphatases; dark blue,

kinases; magenta, inhibitors. (After Queralt and Uhlmann, 2008a.)

7.2 Cell Cyclej199

onset not only directly promotes Cdk-dependent Net1pphosphorylation but also activates the MEN cascade.

The Bub2–Bfa1p GAP complex in yeast not only modu-lates the activation of the Tem1p GTPase and MEN signal-ing, but also is a direct target of regulation by the SAC.Bfa1p, Bub2p, and Tem1p associate with the SPB in a cellcycle-dependent manner. This and many other data indi-cate that the primary role of the Bub2–Bfa1p complex inthe SAC is to block the onset of mitotic exit until chromo-somes have segregated properly. This Bub2-dependent“exit arm” of the SAC also becomes essential when thenucleus fails to migrate into the bud. In this regard,another regulator becomes active, the mother cellrestricted kinase Kin4p, which suppresses the phosphoryl-ation of Baf1p by Cdc5p, in case the mitotic spindle failedto enter the daughter cell.

Released from the nucleolus, Cdc14p can dephosphoryl-ate the S- and M-phase mitotic cyclin substrates to coordi-nate the metaphase to anaphase transition (e.g., Cdc14preverses CDK-dependent phosphorylation of Cdh1p). Theaim of this complex interplay between the components ofthe MEN is the ultimate achievement of: (i) stabilization ofthe inhibitor Sic1p, (ii) inactivation of Cdc28p–Clb kinase,and (iii) triggering of cells into cytokinesis (Tan, Rida, andSurana, 2005). After mitotic exit, Cdc14p returns to thenucleolus.

7.3Meiosis

In eukaryotes, meiosis not only plays a central role in thelife cycle of sexual reproduction, but it is also essential forgenerating genetic diversity within species. Meiosis pro-duces haploid gametes (spores in yeast) from a diploidcell or zygote in two stages that in many ways resemblemitosis (Murakami and Nurse, 2000). In S. cerevisiae, spor-ulation is the only defined differentiation program forwhich a genomic reprogramming has to be started (Govinand Berger, 2009; Piekarska, Rytka, and Rempola, 2010).Two main events govern this program: (i) the execution ofa precisely reorganized transcription program and (ii) areorganization of the genome during the postmeitoticphase, when haploid spores compact their nucleus by adrastic chromatin compaction.

7.3.1Chromosome Treatment During Meiosis

Meiosis consists of a single round of chromosome dupli-cation, followed by two successive rounds of chromosomesegregation. The two successive nuclear divisions occurwithout the S phase, which reduces the number of chro-mosomes to half; on the other hand, the fusion of twogametes restores the full diploid chromosomecomplement.

Chromosome behavior during meiosis is, however,distinct from that in mitosis by several important features(Primig et al., 2000).

i) The “S phase” in meiosis, termed premeiotic DNAreplication, is 2–5 times longer than in mitotic cellcycles.

ii) Additional proteins are required for premeiotic DNAsynthesis compared to mitotic DNA synthesis. In S. cer-evisiae, Swi6p, Swi4p, and Mbp1p exhibit extensivehomology to cdc10, res1, and res2, respectively, whichhave been shown to be required in S. pombe for geneexpression in meiosis (preferably premeiotic replication).

iii) Cross-overs by homologous recombination betweenmaternal and paternal sister chromatids (detectedcytologically as chiasmata) occurs 100–1000 timesmore frequently than during mitosis.

An essential factor required for meiotic recombination isDmc1p (disruption of meiotic control); it was identified in ascreen for meiotically induced genes in yeast. In its absence,cells fail to repair the DSBs that are formed throughout thegenome and then arrest at pachytene during the first meioticdivision. Dmc1p exhibits sequence and functional homologyto the Escherichia coli recombinational repair gene RecA, aswell as to S. cerevisiae Rad51p. To reach full levels ofrecombination intermediates (Holliday junctions), Dmc1pactivity has to be coordinated with the other recombinationproteins, probably through interaction with Rdh54p/Tid1p.

The expression of Dmc1p is limited to early meiosis, con-sistent with the presence of the meiotic regulatory sequenceURS1 (upstream repression sequence 1) upstream in itsopen reading frame. On condensed chromosomes, discretecomplexes with Dmc1p can be visualized during meiosis. AsDmc1p is not expressed during mitosis, there is no effect ofit on mitotic growth or mitotic recombination. Orthologs ofthis protein have been identified in higher eukaryotes.

iv) In the first cell division of meiosis (meiosis I), sisterchromatids remain tied together; sister kinetochoresattach to microtubules from the same pole (monopolarorientation), causing maternal and paternal centro-mere pairs (and not sister chromatids) to be separatedand segregated to the opposite sides of a cell (i.e., thetwo copies of the same chromosomes segregate toopposite poles of the cell).

v) The second meiotic division (meiosis II) takes placewithout an intervening S phase; sister chromatid cohe-sion near centromeres has to be maintained through-out anaphase when cohesion along chromosome armsis beginning to be destroyed, a process that is regu-lated by polo-like kinases such as Cdc5p. Moreover,Cdc5p is required both for the formation of chiasmataand for cosegregation of sister centromeres at meiosisI (Clyne et al., 2003). The residual cohesion aroundcentromeres plays an essential role at division in


meiosis II when spindle microtubules from oppositepoles attach to sister chromatids.

7.3.2Regulation of Meiosis

7.3.2.1 Early, Middle, and Late Meiotic EventsApproximately 1000 genes are specifically expressed duringsporulation (Primig et al., 2000). They can be distinguishedin three groups: early, middle, and late genes; a more refinedtypification defines up to 10 clusters of genes. Notoriously,each of these genes is repressed during vegetative growthand sporulation, except at a limited time window of activa-tion (Figure 7.14). Constant repression of sporulation in dip-loid cells is absolutely required. In haploid cells, but also inartificial diploids (e.g., MATa/MATa cells) sporulation isrepressed by Rem1p, which acts on the master regulatorIme1p, whereby chromatin structure is modified such thatbinding of activators are prevented.

The earliest genes participate in meiotic replication,recombination, synaptonemal complex formation, and sisterchromatid cohesion during the first prophase; their maininducer is Ime1p. Middle genes are expressed before cellsstart the first meiotic division, preferably those acting in thepachytene checkpoint as well as all factors involved in celldivision during meiotic M phase. A major regulator duringthis period is Ndt80p. Late genes comprise all factors func-tioning in postmeiotic differentiation and spore wall forma-tion. The timing of gene expression has to be used as acriterion for typification, a fact that can be seen, for example,among the SPO genes (Table 7.8) – although most of themare involved in spore maturation, some of them are neededalready in meiosis.

Several early genes have to be repressed before they are“allowed” to participate in the sporulation program. The

repressor is Ume6p (unscheduled meiotic gene expression)– a zinc cluster DNA-binding protein that contains six con-served cysteines; it binds two Zn2þ ions to form a binuclearzinc cluster, but lacks an activation domain. Ume6p bindsspecifically to the URS1 sequence element (TAGCCGCCGA)that is located upstream from many early meiosis-specificgenes. During mitosis, Ume6p recruits the Sin3p/Rpd3p(histone deacetylase (HDAC)) complex to repress transcrip-tion of these genes by hypoacetylation of histone H3 and his-tone H4. Ume6p also recruits the chromatin-remodelingfactor Isw2 to establish and maintain a compact chromatinstructure. Among the Ume6p-repressed genes are those ofthe regulators Spo11p (a topoisomerase-like protein thatintroduces DSBs into DNA) (Keeney, 2001), Spo13p,required for sister chromatid cohesion, and Ime2p (themeiosis analogon of Cdc28p) (Guttmann-Raviv, Martin, andKassir, 2002).

For induction of meiosis, Ume6p displays the other sideof its dual regulatory role: upon entry into meiosis, Ume6pbecomes hyperphosphorylated by Rim11p and Mck1p.When phosphorylated, Ume6p interacts with the meiosis-specific transcriptional activator Ime1p. This interaction inturn leads to a rapid degradation of Ume6p during sporula-tion induction and reverses HDAC-mediated repression(Mallory et al., 2007). In addition to the regulation of meio-sis-specific genes, Ume6p has been implicated in the tran-scriptional regulation of genes participating in argininecatabolism (CAR1 and CAR2), peroxisomal function (FOX3),and DNA repair (PHR1). Like the early meiosis-specificgenes, these genes contain URS1 sequence elements.

Induction of sporulation is controlled by two regulators –Ime4p (Spo8p), having a minor effect, and Ime1p, the mas-ter regulator. IME4 (SPO8) is the only sporulation genewhose expression is independent from Ime1p, but consti-tutes one of the (rare) cases of functional antisense transcrip-tion in yeast (Hongay et al., 2006). Haploid yeast expressesthe antisense transcript, because of a stronger promoter, butin diploid cells, the MATa1/a2 heterodimer binds to a down-stream motif and blocks antisense transcription. Thus,under nitrogen starvation IME4 sense transcription can beinduced.

During early meiosis, several meiotic-specific genes haveto be activated (Figure 7.14). Ime1p is such an early meioticactivator that is expressed in response to genetic and nutri-tional signals (Vershon and Pierce, 2000). Ime1p can beviewed as amaster regulator of meiosis that acts as an activa-tor of other early meiotic genes, SPO11, DMC1, or ZIP1(required for recombination and synapsis), through interac-tion with Ume6p. It also activates Ime2p, an early meiosis-specific protein kinase that bears similarity to Cdc28p andreplaces Cdc28p in meiotic cyclin complexes. Ime2p willphosphorylate Ume6p, which then gets degraded by the 26Sproteasome (Kassir et al., 2003). The IME1 gene contains avery large (2.1 kb) upstream regulatory region that comprisesabout 10 different elements: two of these are controlled byRem1p and MATa1/a2, three are elements that repressFig. 7.14 Schematic representation of stages in meiosis.

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transcription by sensing glucose, three induce transcriptionby sensing acetate, and one is sensitive to nitrogen sourcesand represses expression of Ime1p.

The second wave of gene expression comprises middlesporulation gene products required for progression throughmeiosis such as Clb1p, Clb3p, and Clb4p, as well as prepara-tion of spore formation (e.g., Gip1p and Sps1p) (Carlile andAmon, 2008). This round is governed by a transcription fac-tor called Ndt80p (Chu and Herskowitz, 1998). NDT80 itselfis a meiosis-specific gene whose transcription is regulated intwo consecutive steps. First, NDT80 transcription occursafter early meiotic gene transcription, but before the middlesporulation genes are expressed; this process is under con-trol of both Ime1p and the kinase activity of Ime2p (Pak andSegall, 2002; Sopko, Raithatha, and Stuart, 2002). Ndt80 pro-tein then adopts the role as an activator of itself, binding tocis-elements in the NDT80 promoter, which have beentermed “middle sporulation elements.” In fact, the Ndt80protein becomes more abundant and activated by phospho-rylation, so that it binds to middle sporulation elementsupstream of many middle and late sporulation genes toinduce their transcription. The action of Ndt80p thus guar-antees the proper coordination of meiotic progression andascospore formation, because this single event – the induc-tion of Ndt80p – is sufficient to direct independent processessuch as exit from pachytene, the meiotic divisions, and thepackaging of haploid nuclei into spores.

One important regulatory branch involves the polo-likekinase Cdc5p, transcribed by Ndt80p, and responsible forpachytene resolution and exit, removal of Rec8p and supervi-sion ofmonopolin and shugoshin – two novel and conservedmeiosis-specific kinetochore proteins which take the role ofguardians (Watanabe, 2004; Watanabe, 2005). The yeastkinetochore-associated protein, Mam1p (monopolin), isessential for monopolar attachment (T�oth et al., 2000), whilethe meiosis-specific cohesin, Rec8p, is essential for main-taining cohesion between sister centromeres but not formonopolar attachment. The yeast shugoshin, Sgo1p, pre-vents removal of meiotic cohesin complexes from centro-meres during meiosis I and it appears that shugoshinprevents phosphorylation of cohesin’s Scc3-SA2 subunit atcentromeres during mitosis. This ensures that cohesin per-sists at centromeres until activation of separase causes cleav-age of its a-kleisin subunit (Rabitsch et al., 2003; Katis et al.,2004a; Katis et al., 2004b; McGuinness et al., 2005). In yeast,shugoshin also has a crucial role in sensing the loss of ten-sion at kinetochores and in generating the spindle check-point signal (Watanabe, 2005).

Accurate chromosome segregation in meiosis requiresCsm4p – a tail-anchored type II membrane protein with aC-terminal segment of polypeptide that serves as an endo-membrane system anchor. A Dcsm4 mutant undergoes bothmeiotic nuclear divisions and forms spores, but exhibitsmild chromosome missegregation and reduced spore viabil-ity. Apart from its function in diploid sporulation, Csm4pmay have an additional role in haploid cells during glucose

starvation, because the gene transcription is ADR1-regulatedunder these circumstances. Further, as an early meioticgene, CSM4 probably is under the control by chromatin-remodeling Iswi2p. Exit from meiosis II has been proposedto involve Cdc14p phosphatase activity, similar to its regula-tory role in mitotic exit.

7.3.2.2 SporulationMorphologically, at the end of meiosis II, the four haploidsets of chromosomes are distributed into lobes of thenucleus, which finally separate, each haploid nucleus beingencapsulated by a double membrane termed the “prospore”or “FSM.” The “prespore” contains the fragile, newly formedspore precursors (bounded by the FSM), and bears one hap-loid nucleus, a set of organelles, and some cytosol. Presporesthen mature through spore wall synthesis. Spore wall com-ponents are deposited in the lumenal space between theouter and inner leaflets of the FSM. The inner leafletbecomes the plasma membrane of the spore. The outer leaf-let covers the spore walls during early sporulation, but its fateis not known after spore maturation.

Prior to meiosis II, membrane vesicles carrying severalFSM components are synthesized within the cytoplasm ofmother cells. At meiosis II, the outer plaque of the SPB dif-ferentiates, recruiting several novel proteins and suggestingthat SPBs serve as the FSM-organizing centers. Vesiclesthen are assembled on the surface of the plaques and fuseto form a precursor to the FSM. t-SNAREs are alsorecruited, and the FSM grows continuously by fusion withvesicles supplied by the ER/Golgi, assuming a cup-likestructure. Chromosome segregation is coordinated with thegrowth of the FSM so that the divided nucleus is encapsu-lated by the FSM. The FSM eventually closes to form pre-spores. As previously mentioned, this triggers the synthesisof spore wall components, which accumulate in the lume-nal space of the FSM (Table 7.9).

The S. cerevisiae spore wall consists of four layers: twoinner polysaccharide layers composed of b-glucan anda-mannan, a central chitosan layer, and an outermost layerof cross-linked dityrosine. When a germinated spore com-mences polarized growth, the outer, electron-dense layer islocally disrupted and the inner layer protrudes, which indi-cates that the inner layer is structurally related to the vege-tative cell wall and that the outer layer is spore-specific. Asthe genes BGS2 and CHS1, which encode a b-glucan syn-thase and a chitin synthase, respectively, are required forformation of viable spores, b-glucan and chitin must benecessary components of spore walls (Coluccio et al., 2004;Suda et al., 2009).

7.3.3Checkpoints in Meiosis

In budding yeast, entry into meiosis is controlled by a num-ber of checkpoints controlling completeness of DNA repli-cation, possible DNA damage, and whether homologous


recombination has been successfully carried out. Thesecheckpoints are similar to those functioning at vegetativegrowth. A further checkpoint that is invoked in manyother organisms at meiosis – the control of nutritionalconditions – does not seem to be of importance in buddingyeast. It has been found that in G1, cyclin Cln-deficient cellsenter meiosis regardless of nutrient conditions and produceviable spores, indicating that Clns are not essential for mei-osis. Moreover, Clns downregulate levels of proteinsinvolved in triggering meiosis, such as Ime1p – a transcrip-tional activator of early genes involved in premeiotic DNAreplication, cross-overs and recombination. The negativeregulation by Clns may be a consequence of the fact thatmeiosis does not invoke bud formation (for which other-wise the action of Cln1p or Cln2p would be necessary).Instead, in meiosis, the targets of Sic1p seem to be thecyclins Clb5p and Clb6p. These findings also imply that theregulation of Sic1p degradation is different between mitosisand meiosis. In the mitotic cycle, Sic1p is phosphorylatedby Cln-dependent Cdk1p, which results in degradation ofSic1p, whereas in meiosis the degradation of Sic1p requiresthe meiosis-specific protein kinase Ime2p, which is

indispensable for premeiotic replication and may phospho-rylate Sic1p to target it for degradation.

The function of the DNA damage checkpoint has beendiscussed in Section 5.1.3.4. In addition to the Mec1–Rad9–Rad53 pathway, a DNA helicase that also functions as a DNA-dependent ATPase, Srs2p, seems to be responsible for thecorrect timing of meiotic recombination and a smooth tran-sition from meiosis I to meiosis II. Lcd1p is an essential pro-tein required for the DNA integrity checkpoint pathways; itinteracts physically with Mec1p and is a putative homolog ofRad9p.

In many organisms, cells defective in recombination orsynaptonemal complex formation arrest at the pachytenestage of meiotic prophase. This arrest is called therecombination checkpoint or the pachytene checkpoint(Figure 7.14). Pachytene is the stage of the meiotic cell cyclewhen premeiotic DNA synthesis is completed, the SPB isduplicated, but not separated, and the cell is committed torecombination. This arrest requires the DNA damagecheckpoint Rad17p, Rad24p, Ddc1p, and Mec1p proteins inbudding yeast, ensuring the order of meiotic events by pre-venting chromosome segregation when recombination is

Table 7.9 Spo proteins in meiosis and sporulation.

Protein Function

Spo1 prospore protein; required for meiotic SPB duplication and separationSpo2 involved in nuclear membrane integrity at meiosis I and IISpo3 controls meiotic nuclear divisionSpo4 required for spore wall elongation, closure, and maturationSpo5 required for spore wall elongation, closure, and maturationSpo7 regulatory subunit of Nem1–Spo7 phosphatase; controls phospholipid biosynthesis, premeiotic replicationSpo8 mRNA N6-A methyltransferase; required for entry in meiosisSpo9 required for premeiotic DNA synthesis, SPB duplication, and sporesSpo10 required for SPB duplication, meiosis I and II, and sporesSpo11 initiates meiotic recombination by inducing DSBs in DNASpo12 regulator of mitotic exit; required for release of Cdc14p from nucleolusSpo13 required for sister chromatid cohesion during meiosis; proper attachment of kinetochoresSpo14 phospholipase DSpo15 dynamin-like GTPase involved in vacuolar sorting, cytoskeleton organization, endocytosisSpo16 required for synaptonemal complex assemblySpo17 required for premeiotic DNA synthesis; amyloglucosidas activity in sporulationSpo19 bending force for forespore membrane assemblySpo20 subunit of meiosis-specific t-SNARE complex; required for prospore formationSpo21 component of SPB, modifying outer plaque for prospore formationSpo22 essential for chromosome synapsisSpo23 associate of Spo1Spo50 required for premeiotic DNA synthesisSpo51 required for sporulationSpo53 required for sporulationSpo69 component of meiosis-specific cohesin complexSpo70 activator of meiotic APC/C complex; member of Cdc20 family; required for spore wall assembly and for degradation of Clb1

during meiosisSpo71 involved in spore wall formationSpo72 membrane protein busy in vesicle formation during autophagy and in Cvt pathwaySpo73 involved in spore wall formationSpo74 component of SPB, modifying outer plaque for prospore formationSpo75 involved in spore wall formationSpo77 involved in spore wall formation

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incomplete or the synaptonemal complex is defective. Insuch a case, Pch2p (San-Segundo and Roeder, 1999), anessential nucleolar component of the pachytene checkpoint,prevents chromosome segregation. Nucleolar localization ofPch2p depends on the silencing factors Sir2p and Dot1p,mutation of each of which disrupts the pachytene check-point. Pch2p also functions to repress interhomologrecombination in the rDNA during meiosis by excludingthe meiosis-specific DNA-binding protein Hop1p, which isrequired for homologous chromosome synapsis and the for-mation of chiasmata.

The meiosis-specific Red1p and Mek1p proteins are alsorequired for pachytene arrest. Mek1p is a protein kinase andassociates with Red1p (Rockmill and Roeder, 1988) at synap-tonemal complexes. Such complexes are found at synapsesbetween homologous chromosomes during meiosis, andform when sister chromatids condense upon axial elements,whereby it is important that Red1p, Hop1p, and Mek1p are

kept in an appropriate stoichiometry to reach effectivechromosome segregation and to serve the meioticrecombination checkpoint. Red1p, Hop1p, and Mek1p arealso responsible for keeping normal levels of DSB forma-tion (in localizing Dmc1p to DSBs), but also to ensure thatcross-overs occur between homologous chromosomes andnot between sister chromatids. Red1p is a multifunctionalprotein that controls maximum activity of Mek1p, sisterchromatid cohesion, and proper timing of the first meioticdivision. The RED1 gene is induced early in meiosis, itremains present on chromosomes during pachytene afterHop1p has left, and begins to dissociate from chromo-somes in late pachytene or early diplotene. Condensin isrequired for the proper chromosome localization ofRed1p. The protein is phosphorylated, which appears todepend on meiotic recombination, but not by Mek1p;dephosphorylation is effected by Glc7p (Yu and Koshland,2003; Eichinger and Jentsch, 2010).

Summary

� Cell growth and propagation are two sides of the samecoin. When yeast cells have reached a critical size duringvegetative growth under appropriate environmental condi-tions, they are prepared to divide and generate progeny bybudding. The single steps of this process are subject tocontrol by the mitotic cell cycle, which invokes periodicevents to induce and regulate chromosome duplication,carefully avoiding multiple duplications as well as check-ing possible DNA damage and taking measures for DNArepair. Concomitantly, all morphogenetic changes that areconnected to proliferation have to be organized in anorderly programmed interplay. All of these important pro-cesses have been intensely investigated in S. cerevisiae. Infact, research in budding yeast growth and cell cycleopened perspectives on many processes that turned out tobe basically the same in higher eukaryotes. Admittedly,our present knowledge on decisive aspects of the cell cyclewould not have come true without important complemen-tary findings in other systems.

� Our discourse starts with a description of the buddingprocess, including cell polarity and bud site selection, alongwith their regulation, as well as dynamics of morphogenicstructures such as the bud neck, the spindle, and the SPB.Briefly, morphogenic differences toward mating, the sexual

mode of yeast reproduction, filamentous growth, and celldeath are discussed.

� A larger part of this chapter is devoted to dynamicsand regulation of the cell cycle, introducing the cyclinsand the CDK activities, and focusing on their regulationand interplay. Since most aspects of DNA replicationduring the S phase have been presented in Chapter 5,we concentrate here on the pronounced events occurringduring mitosis: cohesion of sister chromatids, structureand assembly of the spindle, and the significance of thespindle assembly checkpoint, steps during chromosomesegregation, and exit from mitosis.

� A final part briefly touches on chromosome behavior inmeiosis, determining the life cycle of sexual reproduction,which in yeast follows the conjugation of partners at mat-ing and ends in the production of (normally) four haploidspores. In meiosis there is one round of chromosomeduplication, but in contrast to mitosis, there are two suc-cessive nuclear divisions. To allow for exchange of geneticmaterial between the two partners, cross-overs of theparental chromosomes are invoked before meiosis I. Wedescribe the regulation of the meiotic events, stressingthe relevance of checkpoints.

Further Reading

Botstein, D., Amberg, D., Mulholland, J. et al. (1997) The yeastcytoskeleton, in The Molecular and Cellular Biology of theYeast Saccharomyces: Cell Cycle and Cell Biology (eds J.R.Pringle, J.R. Broach, and E.W. Jones), Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY, pp. 1–90.

Bouck, D.C., Joglekar, A.P., and Bloom, K.S. (2008) Designfeatures of a mitotic spindle: balancing tension andcompression at a single microtubule kinetochoreinterface in budding yeast. Annual Review of Genetics, 42,335–359.


Carmona-Gutierrez, D., Eisenberg, T., Buttner, S., Meisinger,C., Kroemer, G., and Madeo, F. (2010) Apoptosis in yeast:triggers, pathways, subroutines. Cell Death and Differentia-tion, 17, 763–773.

Douglas, L.M., Alvarez, F.J., McCreary, C., and Konopka, J.B.(2005) Septin function in yeast model systems and patho-genic fungi. Eukaryotic Cell, 4, 1503–1512 (review).

Enserink, J.M. and Kolodner, R.D. (2010) An overviewof Cdk1-controlled targets and processes. Cell Division, 5,11–53.

Fuller, B.G. and Stukenberg, P.T. (2009) Cell division: rightingthe check. Current Biology, 19, R550–R553.

Hardwick, K.G. (1998) The spindle checkpoint. Trends inGenetics, 14, 1–4.

Hoi, J.W.S. and Dumas, B. (2010) Ste12 and Ste12-like pro-teins, fungal transcription factors regulating developmentand pathogenicity. Eukaryotic Cell, 9, 480–485.

Hollingsworth, N.M. (2008) Deconstructing meiosis onekinase at a time: polo pushes past pachytene. Genes andDevelopment, 22, 2596–2600.

Kassir, Y., Adir, N., Boger-Nadjar, E. et al. (2003) Transcrip-tional regulation of meiosis in budding yeast. InternationalReview of Cytology, 224, 111–171.

Marston, A.L. and Amon, A. (2004) Meiosis: cell-cycle controlsshuffle and deal. Nature Reviews Molecular Cell Biology, 5,983–997.

Moreno-Borchart, A.C. and Knop, M. (2003) Prospore mem-brane formation: how budding yeast gets shaped in meio-sis.Microbiological Research, 158, 83–90.

Primig, M., Williams, R.M., Winzeler, E.A. et al. (2000) Thecore meiotic transcriptome in budding yeasts. Nature Genet-ics, 26, 415–423.

Queralt, E. and Uhlmann, F. (2008b) Cdk-counteracting phos-phatases unlock mitotic exit. Current Opinion in Cell Biology,20, 661–668.

Ruchaud, S., Carmena, M., and Earnshaw, W.C. (2007) Chro-mosomal passengers: conducting cell division. NatureReviews Molecular Cell Biology, 8, 798–812.

Shimoda, C. (2004) Forespore membrane assembly in yeast:coordinating SPBs and membrane trafficking. Journal ofCell Science, 17, 389–395.

Sclafani, R.A. and Holzen, T.M. (2007) Cell Cycle Regulationof DNA Replication. Annual Review of Genetics, 41, 237–280.

Slaughter, B.D., Smith, S.E., and Li, R. (2009) Symmetrybreaking in the life cycle of the budding yeast. Cold SpringHarbor Perspectives in Biology, 1, a003384.

Tolliday, N., Bouquin, N., and Li, R. (2001) Assembly and regu-lation of the cytokinetic apparatus in budding yeast. CurrentOpinion in Microbiology, 4, 690–695.

Vershon, A.K. and Pierce, M. (2000) Transcriptional regulationof meiosis in yeast. Current Opinion in Cell Biology, 12,334–339.

Zich, J. and Hardwick, K.G. (2010) Getting down to the phos-phorylated “nuts and bolts” of spindle checkpoint signal-ling. Trends in Biochemical Sciences, 35, 18–27.

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