Biochimica et Biophysica Acta, 1093 (1991) 169-177
© 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 016748899100197L
Cell cycle control by calcium and calmodulin in Saccharomyces cerevisiae Y a s u h i r o A n r a k u 1,2, Y o s h i k a z u O h y a 1 a n d H i d e t o s h i Iida 2 t Department of Biology, Faculty of Science, University of Tokyo, Tokyo (Japan) and 2 Division of Cell Proliferation, National Institute for Basic Biology, Okazaki (Japan) (Received 3 April 1991)
Key words: Cell cycle; Calcium ion; Calmodulin; Calcium ion concentration, transient; Cytosolic free calcium concentration; DNA replication; Mitosis; Nuclear division
Contents I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. Calcium and cell cycle control in yeast cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Is Ca 2+ essential for cell proliferation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B. Measurement of [Ca2+l i in single yeast cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The vacuole as an intracellular Ca 2+ store . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Transient increase in [Ca2+]i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
170 170 170 171 171
llI.Calmodulin and cell cycle control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Calmodulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Calmodulin is required for cell proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Several checkpoints of calmodulin in cell cycle progression of mammalian cells . . . . . . . . . . . . . D. Terminal phenotype of calmodulin deficiency in a yeast mutant . . . . . . . . . . . . . . . . . . . . . . . . E. Roles of calmodulin in mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Is yeast calmodulin required only for nuclear division? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
172 172 172 172 173 173 174
IV. Calcium and calmodulin: toward a search for their targeting enzymes and cellular machineries . . . .
V. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The cell cycle is a complex, temporally programmed event, and involves cellular processes associated with not only a number of coordinated enzymatic reactions including DNA and membrane syntheses but also cy-
Abbreviations: [Ca2+]i, cytosolic free Ca 2+ concentration; EGTA, glycoetherdianine.N,N,N',N'-tetraacetic acid. Correspondence: Y. Anraku, Department of Biology, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan.
tomechanodynamics including nuclear division and cell separation [1-3]. The cell cycle is divided into four phases: the gap between mitosis and DNA replication (G~), the DNA synthetic phase (S), the gap between DNA replication and mitosis (G 2) and the mitotic phase (M). Genetic and molecular biological research in the last decade has greatly increased our understanding of the structure and functional complexity of the cell cycle as well as the regulatory cascades involved in its progression. Ca2+ is present ubiquitously in eukaryotic cells and is known to be a growth-regulating divalent cation during the cell cycle. By direct cell proliferation stud-
170 ies, Ca 2+ has been shown to be an essential element for events in G~ during the cell cycle of cultured mammalian cells [4-6]. In addition, cell biological studies in the last decade have clearly shown that the actions of growth factors elicit a transient increase in the cytosolic free Ca 2+ concentration ([Ca2+]i) by inducing Ca 2+ release from internal stores or Ca 2+ influx through the plasma membrane [7-9]. However, little is known about how free Ca 2+ in the cytosol regulates cell proliferation at the molecular level in mammalian cells. The main reason for this is that it is difficult to apply ~enetic and molecular biological approaches to mammalian cells. The yeast Saccharomyces cerevisiae is one of the best eukaryotes for such approaches. We have chosen the yeast for study with the hope that this organism will help us to unravel the molecular mechanisms of the function of Ca 2+ during the cell cycle, although there have been few studies on even the physiological role of Ca 2+ in this organism. Most Ca2+-mediated cellular functions are generated in one of two ways: direct activation of enzymes by Ca 2+  or modulation of enzyme activities by Ca'+.binding proteins such as calmodulin . For Ca 2+ to act as an activator or a modulator, eukaryotic cells have acquired a variety of sophisticated mechanisms by which [Ca-~+]i is maintained homeostatically and fluctuates temporally. Temporal and spatial changes in [Ca2+]| in response to hormone- and ~rowth factor-induced stimuli must be sensed by most :.inds of cells as a Ca 2+ signal or a second messenger, which directs the regulation of Ca2+-mediated cellular processes [7-9]. In this review, we shall discuss recent advances in studies on cell cycle control by Ca 2+ and calmodulin, especially focusing on the importance of calcium physiology in single yeast cells and the genetic manipulation of Ca'÷-regulatory processes. Comprehensive reviews of studies on cell cycle control by Ca '++ [12,13] and calmodulin  in mammalian cells have appeared.
plete medium (SD) containing 681 ~ M Ca 2+ were used as standard media for analysis of growth kinetics. They found that yeast cells could grow indefinitely in SD-Ca with a mean doubling time of 4.3 h at 22°C as in SD (mean doubling time, 3.5 h). This observation was consistent with previous results of Kov/l~ . However, cell growth in SD-Ca at 22°C was completely inhibited by simultaneous additions of 10 ~M A23187 (a Ca 2+ ionophore) and 10 mM EGTA (a Ca 2+ chelator). No growth inhibition was observed when either one of these two reagents was added singly, and the inhibition occurred in parallel with a decrease in the intracellular Ca 2+ content, lida et al. also confirmed that the inhibition was brought about by deficiency of Ca 2+ by showing that readdition of Ca 2+ reversed the inhibition, whereas additions of Mg 2+, Cu 2+ and Mn 2+ did not . Cytochemical analysis by flow cytometry revealed that this growth inhibition results in transient G I arrest followed by block, mostly at G2/M. Interestingly, after the transient G~ arrest, DNA synthesis is reinitiated in most cells, whereas bud emergence is rescued in only half the population. Most cells in which DNA synthesis is reinitiated are finally arrested in G2/M. These observations suggest that Ca '+ is required mainly in G~ and G2/M  and that the checkpoint in G~ can be eventually overcome. Iida et al.  also demonstrated that after addition of A23187 and EGTA the intracellular cAMP level decreases within 10 rain. No significant transient G t arrest was observed in wild-type cells incubated with 8-Br-cAMP, in RAS2 vau9 mutant cells, which produce a high level of cAMP, or in Abcyl mutant cells, which have constitutively activated cAMP-dependent protein kinase. From these results, it can be concluded that Ca 2+ is essential for cell cycle progression in yeast cells and that G t events may be mediated by cross-talk between Ca 2+ and cAMP pathways.
11. Calcium and cell cycle control in yeast cells
The fact that deprivation of extracellular Ca 2+ arrests yeast cells in G~ suggests that a break of [Ca2+]i homeostasis may seriously affect cAMP metabolism and its regulatory cascade. Iida et ai.  established a sensitive epifluorescence microscopic method for measuring [Ca2+] i in individual yeast cells using fura-2 as a Ca2+-specific probe in conjunction with digital image processing. In this procedure, yeast cells are loaded with fura-2 by electroporation and the loaded cells are observed under the objective of a Nikon Microphot-FX microscope with an integral fluorimeter system. Images of fura-2 fluorescence at excitation wavelengths of 340 nm and 380 nm are acquired with a SIT camera and relayed into both a TV monitor and an ARGUS-100 image processor (Hamamatsu Photonics). The ratio
11-,4. Is Ca 2 + essential for cell proliferation? Until recently, the answer to the basic question of whether Ca 2+ is essential for cell proliferation of yeast cells was uncertain , although in mammalian cells, there is increasing evidence that deprivation of extracellular Ca 2+ causes growth arrest in G l of the cell cycle [16-18]. lida et al.  established a suitable experimental system for study of cell cycle regulation by Ca" + in the yeast S. cerevisiae. For investigation of the effect of extraceilular Ca 2+ on cell growth, a Ca 2+deficient synthetic dextrose medium (SD-C~), containing Ca 2+ at a concentration of 0.24 ~M, and a com-
II-B. Measurement o f [Ca2+] i in single yeast cells
171 (340/380 nm) is converted to [Ca2+]i, using the equation described by Grynkiewicz et al.  and displayed essentially as described by Poenie et al. . This procedure permits the measurement of steady state levels of [Ca2+]i of 10 nM to 1400 nM in single yeast cells. Using this method, lida et al.  determined that the average [Ca2+]i in single cells of a mating type growing exponentially was 116+ 90 nM in SD, but 136 + 124 nM in SD-Ca. This indicates that the yeast cells were able to maintain a homeostatic level of [Ca2+]i of about 100 nM, despite the large difference in extracellular Ca 2+ concentrations in SD and SD-Ca. This [Ca2+] i level is essentially the same as the basal level in animal cells . Studies by atomic absorption spectrophotometry have shown that the total Ca 2+ content of yeast cells in SD-Ca is 49 pmoi/106 cells or 49 pmol/31 # m a x 10 6, which is equivalent to 1.6 mM Ca 2+ in yeast cells if all of this Ca 2+ is in solution . This means that the cytosolic free Ca 2+ in yeast cells is only 0.006% of the total, and that most of the Ca 2 + in the cells is stored in the vacuole, probably as bound forms [24,25]. These findings answer the question of why yeast cells can grow indefinitely in 'Ca2+-free ~ media. Even when incubated in a medium completely free from Ca 2 ~, yeast cells should be "able to grow for 14 generations until this Ca 2+ content reaches the basal [Ca2+]i level, i.e., about 100 nM. The bound form of Ca 2+ may well be released to generate the cytosolic free Ca 2÷. Since 'Ca2+-free ' media contain considerable amounts of contaminating Ca 2÷ salts, yeast cells can continue to proliferate for further generations using extracellular and intracellular Ca 2+ salts. It is noteworthy that mammalian cells do not proliferate in Ca2+-deficient medium, although the total calcium content and [Ca 2+ ]~ in the cells are determined to be the same order, i.e., abou,: 1 mM and 100 nM, respectively. The reason for this ~itriking contrast is unknown. II-C. The vacuole as an intracellu!ar Ca 2+ store Vacuoles in yeast cells are a major storage site of Ca 2+ [25,26] and Ca 2+ homeostasis is thought to be maintained via the function of the vacuolar Ca2+/nH + antipo: system [24,27], which is driven by a proton motive f,~ree generated by the vacuolar membrane H÷-ATPase [2~,]. Several lines of evidence show that the vacuolar capr_~.~ity for Ca 2+ storage is large: (1) Wild-type yeast ceiis can grow normally in a nutrient medium supplemented with !00 mM CaCI 2 ~ and (2) Ca 2+ taken up 1:/the ceils is mostly sequestered into vacuoles and stol~d stably as forms of Ca-polyphosphates . These fa:ts indicate that the vacuole functions as a physiological Ca 2+ reservoir in this organism.
The epifluorescence microscopic method was alse used to measure the apparent free Ca 2+ concentration in the vacuolar lumen, which was determined to be approx. 900 nM 2. Thus, the vacuolar H ÷ pump and Ca 2÷ antiporter provide the cell with an internal regulatory system for maintenance of [Ca2+]i homeostasis under conditions with w~:dely different extracellular Ca 2+ concentrations. Recently, Ohya et al. demonstrated that Ca2+-sensi tive mutants (cls7, cls8, cls9, c/sl0 and clsll)  have defects in vacuolar membrane H+-ATPase activity and that vacuoles prepared from these mutants are also all defective in ATP-dependent Ca2+-uptake activity . Consistent with these observations, [Ca2+]i in the individual mutant cells is increased to a level of 900 + 100 nM, which is 6-times that of wild-type cells. This finding indicates that loss of [Ca2+]i homeostasis, e.g., about 6-fold increase in [Ca2+] i, is due to the defect in the vacuolar membrane H+-ATPase activity and triggers generation of serious metabolic lesions with a Ca2+-sensitive phenotype . Recently, TFPI, whose dominant mutation confers cells with trifluoperazine resistance , was isolated and found to be identical to CLS8 , a gene encoding subunit a of the vacuolar membrane iI+-ATPase . Therefore, one target protein of trifluoperazine in yeast cells may be subunit a of vacuolar membrane H +-ATPase. II-D. Transient increase in [Ca 2 +]i Biochemical and enzymological studies have shown that a number of Ca 2+ -activated enzymes and Ca 2+ -binding proteins have dissociation constants for Ca 2+ in the order of 10-6-10 -4 M [33,34]. Obviously, the observed ranges of Ca2+-dependent enzyme activation or modulation are much higher than the basal level of [Ca2+]i . Studies in the past few years have highlighted the abilities of animal cells, both in excitable and non-excitable cells, to generate periodic [Ca2+]i increase or calcium oscillation in response to extracellular and intracellular stimuli [8,9,35,36]. The transient increase in [Ca2+]i is induced in target cells either by opening Ca 2+ channels in the plasma membrane to allow Ca 2+ influx from the external medium or by activating cell surface receptors that trigger hydrolysis of membrane phosphoinositide lipids to generate inositol 1,4,5-trisphosphate, which in turn releases free Ca 2+ from internal stores [8,9]. Measurements of the transient increase in [Ca2+]i have mainly been made with aequorin as a Ca2+-dependent luminescence probe, and so crucial determination of the increase in [Ca2+]i in terms of a concentration difference has been difficult to assess. Nevertheless, the increase in [Ca2+]i is thought to be sensed by cell systems, such as Ca 2+ signals, and to direct a variety of cellular functions including hormone secre-
172 tion, neurotransmitter release, muscle contraction, fertilization and lymphocyte activation [8,9]. A Ca e+luminescence method using aequorin can also be applied to small cells such as those of S. cerevisiae, if the cells produce apoaequorin in coelenterazine-loaded conditions and maintain the aequorin complex stably in a sufficient amount. By use of recombinant DNA techniques in yeast cells, Nakajima-Shimada et al. established a novel Ca2+-luminescence method and detected a transient rise in [Ca2+]i upon addition of glucose to yeast cells arrested in G0/G I of the cell cycle by glucose starvation .
111. Calmodulin and cell cycle control
IliA. Calmodulin Caimodulin is a ubiquitous Ca2+-binding protein found in all eukaryotes thus far examined . Calmodulin was discovered independently by Kakiuchi and Yamazaki  and Cheung  in 1970 and named after its 'Ca2+-modulating' function . In 1980, Watterson et al.  determined the amino acid sequence of bovine calmodulin. Since then calmodulins from more than twenty species have been purified and their molecular properties and gene structures have been determined . Vertebrate calmodulins show complete identity in amino acid sequence except that the electric eel contains two species of calmodulin. Vertebrate calmodulins contain 148 amino acid residues and are dumbbell-shaped Ca'+-binding proteins . They have four conserved EF-hand structures to which Ca 2+ binds with a dissociation constant in the micromolar range. Calmodulins from invertebrates and plants show over 90% homology with bovine calmodulin, whereas those from fungi show 60-80% homology. Calmodulin from S. cerevisiae, which is the most distantly related to mammalian calmodulin, displays 59% sequence identry. The protein contains 147 amino acid residues  and three Ca2+-binding sites . Although calmodulin of S. cerevisiae has a unique structure, it has similar properties to other calmodulins in Ca2+-dependent in vitro reactions . Moreover, for its essential function in cell proliferation, calmodulin of S. cerevisiae can be replaced by either chicken  or Xenopus  calmodulin.
lll.B. Calmodulin is required for cell proliferation Biochemical and molecular biological research in the last decade has greatly increased our appreciation of the roles of caimodulin as a regulator of cell proliferation. The calmodulin level has been shown to increase in mammalian cells that have been transformed by either an oncogenic virus or a chemical carcinogen [50,51]. Studies have suggested that calmodulin levels
are not critical in maintaining the transformed state [52,53], but that an increased level of calmodulin-binding proteins may play a key role . Tanaka et al.  found that a rise in calmodulin level in CHO cells is required for transit from the G O to G I / S phase. During normal growth of CHO cells, the intracellular concentration of calmodulin doubles at the Ga/S boundary . Means and collaborators  demonstrated that the sharp rise in calmodulin content associated with transition from the G O stage to the S phase is regulated at the mRNA level. These observations suggest that calmodulin levels are controlled by some cellular mechanisms, which are responsible for promoting cell proliferation. Genetic analyses have also proved useful for studying the roles of calmodulin in cell proliferation. The study with a bovine papilloma virus-based expression vector for calmodulin mini gene showed that over-expression of the protein leads to a shortened G~ period . In addition, the rate of G I progression was positively correlated with calmodulin concentration, suggesting that calmodulin has a regulatory effect on the rate of cell cycle progression . Calmodulin genes have been isolated from two lower eukaryotes, S. cerevisiae  and Schizosaccharomyces pombe . In both organisms, haploid cells containing a disrupted calmodulin gene are unable to grow from spores, clearly indicating that calmodulin is an essential protein for cell proliferation [45,58,59].
III-C. Several checkpoints of calmodulin in cell cycle progression of mammalian cells Various events regulating the cell cycle may take place dependent on ordered pathways and coordinate accurate progress through the whole cell cycle. Hartwell and Weinert  originally named the control mechanisms that enforce order-dependency in the cell cycle checkpoints. Order-dependent relationships have been well analyzed genetically using a number of cdc mutants of the yeasts S. cerevisiae and S. pombe. The observations described in the previous section that the level of calmodulin doubles at the GI//S boundary  and that over-expression of the protein decreases the duration of the G, period  suggest that calmodulin is involved in a control mechanism(s) initiating a n d / o r carrying out cell division cycle. Pharmacological studies with anti-calmodulin drugs have also indicated various possible roles of calmodulin in cell cycle progression. Hidaka and colleagues have explored a great number of anti-calmodulin drugs including derivatives of phenothiazine, thioxanthene and naphthalenesulfonamide . When CHO cells were treated with the anti-calmodulin drug W13, their growth was arrested at both the G1/S boundary and in the S phase : at a relatively high concentration, this
173 drug was found to inhibit cell growth in the M phase . Rasmussen and Means found that expression of anti-sense calmodulin mRNA in C127 cells, which resuited in a decreased level of calmodulin synthesis, caused growth arrest in both the G~ and M phases . This study indicates that calmodulin is required for the progression of the Gt and M phases. In addition, a recent study by Baitinger et al.  showed that oligopeptides that correspond structurally to the inhibitory domain of calmodulin-dependent multifunctional protein kinase II, inhibit nuclear envelope breakdown in dividing sea urchin eggs, and so proposed that this kinase is involved in the control of entry into mitosis. These observations together with the pharmacological data strongly suggest that calmodulin functions at many stages of the mammalin cell cycle including the G~, S, G2/M and M stages. III-D. Terminal phenotype o f calmodulin deficiency in a yeast mutant
Studies on the molecular biology of yeast have already been shown to be potentially advantageous for defining several checkpoints in the cell cycle [60,66]. To determine the key step requiring calmodulin in the mitotic process of yeast cells, Ohya and Anraku  constructed a conditional-lethal mutant in which expression of calmodulin is controlled by the galactoseinducible G A L l promoter. The G A L l promoter is turned on in galactose medium and turned off in glucose medium. The mutant diploid strain YOG18 carries a disrupted chromosomal calmodulin gene (cmdl : : URA3 / c m d l : : URA3) and harbors the pGCAM104 plasmid, in which expression of calmodulin is under the control of the G A L l promoter. The mutant cells were found to grow normally in galactose medium, but to stop growing after 12-15 h in glucose medium. This growth arrest was associated with a decrease in the intracellular calmodulin level: cells cultured in galaetose medium contained 2.3 /~g calmodulin/mg protein. After 8 h incubation in glucose medium their calmodulin content had decreased to about 0.1/zg/mg protein, which was the same level as that found in growing wild-type cells. Moreover, after 13 h in glucose medium, no calmodulin ( < 0.01 p.g/mg protein) could be detected in the mutant cells by Western blot analysis . Cytoehemical analyses of the terminal phenotype showed that the mutant cells subjected to growth arrest by deprivation of calmodulin have a bud, a nucleus after the S phase and a short mitotic spindle. Thus, the defect was mainly in nuclear divisior, . It was also shown that 27% of the cells stopped growing with a small bud, indicating that bud growth was partially inhibited in the mutant cells. These observations to-
gether with the fact that the yeast calmodulin level increases in parallel with the budding process~ reaching 2-fold the initial level before nuclear division , suggest that nuclear division is the step in the cell cycle requiring most calmodulin for its progression. The fidelity of mitotic chromosome transmission in S. cerevisiae is known to be high and the spontaneous rate of loss of chromosome V has been estimated as 8.3" 10 -6 events per cell division . Since several yeast cdc mutants deficient in nuclear division show increased rates of chromosome loss, this fidelity is due to precise chromosome segregation as well as complete chromosome replication and condensation . Ohya and Anraku  examined the stabilities of chromosomes III, IV, and V in conditional calmodulin-depleted mutant cells grown in galactose and glucose media and found that calmodulin deficiency increased the rates of chromosome loss, as the result of a defect in precise chromosomal segregation. A further interesting finding was that expression of chick calmodulin under the control of the GALl promoter in the yeast calmodulin-disrupted mutant was able to support full progression of the cell cycle of the mutant cells in galactose medium, but in glucose medium growth was eventually arrested by the block of nuclear division . These findings indicate that a key role of calmodulin in nuclear division is conserved in eukaryotic cell systems. III-E. Roles o f calmodulin in mitosis
Positive involvement of [Ca2+]~ oscillation in mitosis have been suggested [22,69-73]. Tombes and Borisy  have shown that anaphase in mammalian fibroblasts and epithelial cells is a calcium-modulated event, usually associated with sustained elevation of [Ca2+]i to above 50 riM. Using a Ca2+-pulse method, Kao et al.  demonstrated that local Ca 2+ movement may be involved in nuclear envelope breakdown. In addition, mitosis was inhibited by microinjection of a specific antibody against Ca2+-sequester proteins in the membrane of Ca 2+ stores [74,75]. Several lines of biochemical and immunocytological evidence also indicate that calmodulin can interact with cytoskeletai components and modulate their specific functions ir~ cytomechanodynamic processes . Calmodulin is known to be localized in the mitotic apparatus, especially in the kinetochore spindle in mammalian  and plant  cells, and thought to have a key role in the local depolymerization of kinetocore microtubules during anaphase . Calmodulin-dependent phosphorylation is thought to play important roles in entry a n d / o r progression of mitosis. For instance, calmodulin-dependent phosphorylation of non-histone proteins has been observed during premature chromosome condensation in a tern-
174 perature-sensitiv¢ baby hamster kidney cell line . Inhibitory oligopeptidcs for calmodulin-dependent multifunctional protein kinase II inhibit nuclear envelope breakdown , suggesting that this kinase is involved in the control of entry into mitosis. Recently, Dinsmore and Sloboda [84,85] reported that a substrate of a calmodulin-dependent protein kinase, the 62 kDa mitotic apparatus protein, is phosphorylated maximally at the metaphase-anaphase transition, and that phosphorylation of this protein by a calmodulindependent protein kinase results in microtubule disassembly in vitro. Functional involvement of the 62 kDa protein in mitosis is further supported by the finding that microinjection of its specific antibodies into dividing sea urchin embryos arrests mitosis . The results described above suggest the positive involvement of Ca 2+ and calmodulin in the entry and progression of mitosis in mammalian cells, which is quite consistent with the results in yeast. Deprivation of Ca 2+ in yeast cells brings about growth arrest finally at G2/M , possibly because break of [Ca2+]i homeostasis seriously affects a regulatory cascade of calmodulin. It has not yet been proved that local Ca 2+ movement occurs during mitosis in yeast, all available evidence so far suggests that Ca 2+ and calmodulin are essential for regulation of yeast nuclear division [19,59,67].
III-F. Is yeast calmodulin required only for nuclear division? Mammalian cells apparently have several checkpoints during the cell cycle where calmodulin is required. In contrast, the yeast calmodulin-deficient mutant has a main defect in nuclear division : although the mutation was associated with partial inhibition of bud growth, no obvious defects in the G I and GI/S stage appeared. One possible explanation is that the mechanism(s) for control of G~ events by calmodulin is different between yeast and mammalian ceils. In fact, over-expression of calmodulin reduces the duration of the G~ period in mammalian cells , whereas overexpression of yeast calmodulin  or vertebrate calmodulin [48,49] has little affect on the growth rate of yeast ceils. Another explanation may be that the defect in nuclear division observed in the yeast mutant is due to the nature of a conditional-lethal mutant. As discussed already, this terminal phenotype of the yeast mutant is associated with a decrease in the intracellular calmodulin level after prolonged incubation in glucose medium . Therefore, other types of conditional-lethal mutant (Ts or Cs) in which calmodulin immediately loses its function at a non-permissive temperature may reveal another terminal phenotype. Recently, temperature-sensitive calmodulin mutants were obtained during the domain analysis of yeast
calmodulin required for cell prolifearation: Sun et al.  constructed plasmid.'~ e.xpressing a series of N- and C-terminal halves of the calmodulin under the control of the GALl promoter and introduced them into a CMDl-disrupted strain. The plasmids expressing the N-terminal half (Serl-Lcu76) and the C-terminal half (LeuSS), which each maintain two complete EF-hand structures, complemented the growth defect of the calmodulin null mutation . This is the first evidence showing that each half of the calmodulin is not only a structural unit but also a functional unit in vivo. Interestingly, all the mutant cells depending solely on half calmodulins show a temperature-sensitive growth phenotype . These temperature-sensitive calmodulin mutants may be useful for further analysis of the checkpoints of calmodulin in cell cycle control. IV. Calcium and calmodulin." toward a search for their targeting enzymes and cellular machineries As mentioned above, growth factors are known to induce an increase of [Ca2+] i, which may trigger a variety of biochemical events finally leading to initiation and/or progression of the cell cycle. However, little is known about the targets of the mobilized Ca 2+ that are responsible for the regulation of cell proliferation in animal cells. One target may be protein kinase C (PKC). On increasing in [Ca2+]i, cytosolic PKC is converted to membrane-bound PKC, whose activity can be elevated by a higher concentration of either Ca 2+ or diacylglycerol [87,88]. Protein kinase C may initiate the cell cycle by inducing expression of cellular oncogenes such as c-los and c-myc . Another possible target is calmodulin. Ca2+/cal modulin may facilitate cell proliferation by moduiating the activities of many enzymes and by changing the organization of the cytoskeleton. Growth factors such as epidermal growth factor and insulin have been shown to induce glycolysis depending up~a Ca ~+ in the medium . Multimeric skeletal phosphorylase ki nase, which is responsible for the regulation of glycolysis, is composed of calmodulin (8 subuait) ~nd calmodulin-dependent prote:a kinase (1' subunit) . Therefore, one role of calmodulin in cell proliferation may be in regulating glycolysis. Ca2+/calmodulin can also modulate the activities of adenylate cyclase, cyclic nucleotide phosphodiesterase, several protein kinases and protein phosphatase 2B in vitro . These enzymes all have a potential to regulate cell proliferation by changing the phosphorylation levels of their target proteins directly or indirectly. DNA polymerase may also be regulated by calmodulin, as this enzyme is associated with some calmodulin-binding proteins . Calmodulin may regulate entry into the S phase by changing the stability of microtubules. Evidence for this is that microtubule-depolymerizing drugs, such as colchicine
175 induce initiation of DNA synthesis in quiescent chick and mouse cells [94,95]. Carafoli and colleagues [96,97] found that calmodulin and a number of calmodulinbinding proteins are located in liver nuclei. After partial hepatectomy, the amount of nuclear calmodulin increases, and redistribution of calmodulin within the nuclei takes place during the replicative period, which occurs by a Ca2+-dependent regulatory mechanism . Possible targets of calmodulin during mitosis were discussed in the previous section. In addition to these observations on animal cells by biochemical and cell biological approaches, studies on yeast cells by genetic and molecular biological approaches have contributed to an understanding of the regulatory cascade triggered by Ca 2+ mobilization in the cytosol. Besides calmodulin, other novel, putative Ca2+-binding proteins and also a putative protein kinase C have been found in S. cerevisiae. Ohya et al. isolated many Ca2+-sensitive mutants and classified them into 18 complementation groups . In Ca2+-rich medium, one of them, the cls4 mutant, shows a defect in bud emergence and its reproductive organization. The cls4 mutation is allelic to the cdc24 mutation , and the CLS4/CDC24 gene encodes a putative Ca 2+binding protein . Recently, Pringle and colleagues identified a gene family whose products may interact with the CLS4/CDC24 gene product [100,101]. Their genetic studies of multicopy suppressors of the cdc24 mutation demonstrated the existence . f four new genes, two of which, CDC42 and RSR1, have significant homology with ras oncogenes and appear to encode GTP-binding proteins. By molecular cloning of CDC genes, another putative Ca2+-binding protein encoded by the CDC31 gene was also identified . A cdc31 mutation resulted in a defect in duplication of the spindle pole body, the yeast microtubule organization center on the nuclear envelope. Thus, Ca 2+ is postulated to regulate bud formation and spindle pole body duplication through interplays with the CLS4/CDC24 and CDC31 gene products, respectively. Protein kinase C has been purified from S. cerevisiae cells  and its putative structural gene (PKC1) has been isolated and characterized . The PKC1 gene is essential and in PKCI-depleted cells the cell cycle is arrested at a stage with a small bud and G2/M DNA . Interestingly, a Cae+-dependent mutant call-l, whose terminal phenotype is very similar to that of PKCl-depleted cells, has already been isolated by Ohya et al. . Genetic studies on CALl and PKC1 should be useful for determining whether the two gene products function in a common, Ca2+-dependent regulatory pathway. Several target proteins for calmodulin have been identified in yeast cells. A number of uncharacterized yeast calmodulin-binding proteins were identified using 125I-labeled calmodulin  and most of these were
found to be concentrated in the nucleus. There are two reports of calmodulin-dependent protein kinase activity in yeast cell extracts [107,108], and Londesborough  reported purification of a yeast calmodulin-dependent protein kinase to near homogeneity. Recently, CMK1 and CMK2 genes of S. cerevisiae, encoding calmodulin-dependent multifunctional protein kinases II, were isolated and characterized : the CMK1 gene encodes the yeast calmodulin-dependent protein kinase that was purified by Londesborough , and the CMK2 gene was cloned by screening at a low stringency with a CMK1 fragment as a probe. Since disruption of both kinase genes is not lethal, another gene product may substitute for in vivo function of the CMK1 and CMK2 kinases . Further molecular cloning and analysis of genes for calmodulin-dependent enzymes in yeast cells will aid a more thorough understanding of the regulatory mechanism(s) of calmodulin in the cell cycle progression. V. Conclusion Evidence that Ca2+functions as a growth-regulating substance in eukaryotes from yeast to mammalian cells is now convincing. The strongest evidence for this concept is that deprivation of Ca 2+ in either mammalian or yeast cells causes cell cycle arrest. The precise roles of Ca 2+ in cell cycle control in mammalian and yeast cells have not been compared throughly, but genetic and molecular biological studies have revealed similar functions of calmodulin in the regulation of mitosis in these two types of cell. Studies on the molecular biology of yeast have a potential advantage for defining the checkpoints ~n the cell cycle and identifying the regulatory components involved in Ca2+-dependent a n d / o r Ca2+-signal transferring systems. Little is yet known, however, about how local and temporal changes of [Ca2+]i are sensed by, or sense, other ~ignai-transferring networks directed by cAMP, inositol 1,4,5-trisphosphate and diacylglycerol as potential signal transducers. Moreover, no functional interaction between the cdc2 +/CDC28 regulatory gene and genes affecting Ca2+-regulatory pathways in yeast has yet been reported. In this regard, the recent finding of linkage between Ca 2+ regulatory processes and the cdc2+/CDC28 homologue in mammalian cells [13,111] is very interesting. Future efforts to unravel the molecular mechanisms underlying the cross-talk among these second messengers will provide new msight into the roles of Ca 2÷ in cell biology. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan
176 a n d a grant f r o m t h e U e h a r a M e m o r i a l F o u n d a t i o n , J a p a n . This w o r k is a contribution to t h e N I B B Prog r a m for B i o m e m b r a n e R e s e a r c h .
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