Mutational analysis of calmodulin in Saccharomyces cerevisiae T.N. DAVIS

Department of Biochemistry, University of Washington, Seattle, Washfngton, USA recaptor in Abstract - Galmoduiin is well characterized as an intracellular c$’ nonprolifmiting tlssues such as muscle and brain. Several observations indkate that caimodulin Is also required for cellular growth and division. Deietlon of the calmodulln gene Is a lethal mutation In Saccharomyces car&s&e [l], Schkcsaccharomyces pom&e [2] and AspergiMfs nidulans [3]. Expression of calmodullnantisense RNA in mouse Gl27 cells causes a transient arrest at 01 and metaphase [4]. Although these results indicate caimodulln plays a crltlcal function during proliferation,they do not reveal tha function. S. cewisiae ofkrs an excellent system for ldenttfylng calmodulin functions. BeoaUSe calmodulln mutants can be readily constructedby gene replacementthe conaequencea of mutations in calmoduiin can be dlrectiy examined in vivo without lnterfemnce from wild-type calmodulin. The avaliable weaith of informattonconcerning all aspects of the yeast life cycle provides a large framework for intarpretation of new results. The recent dissection of ceil cycle regulation is just the iatest exampie of the important InsIghts provided by analyzing basic cellular processes in yeast Whether studles of calmoduiin in yeast wlli reveal a universal function Is unknown. One encouraging result Is that yeast cells relying on vertebrate caimoduiin as their only source of oalmoduilnsurvive and grow well, even if the amount of vertebrate calmodulln is equivalent to the normal steady state levels of yeast calmoduiln [5]. This review discussesthe varied techniques wa are using to identify the functions of caimodulln in yeast As part of the analysis, we are deftnlng the essential elements of caimoduiinstructure.

for Ca2+is not uquired for cahxiuliu tu perfonu its essentialfunction [a]. Yeast calmbids 3 Mutants defective in C2’-binding calcium ions with high affinity [7] (TN. Davis, unpublishedresults). We characterized3 mutautsiu Since calm&h is best understoodas an iutra- which all three active Ca2+-binding sites are cellular Ca2+-receptor,a likely hpthesis for its inactivated. Iu the mutaut 3D+A, the cuuserved fuuctionisthatittxausmitsaCa2 signalxequired aspartateat the flmtpositionof each loup is changed for cell pdiferatiou. However,analysisof a set of to an ala&e. Iu the mutant3EdV, the conserved Ca2+-bindingmutants suggests that a high affiuity glutamateat the twelfth position of each loop is 435 Mutational analysis of calmudulin

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changed to a valine. The most severe mutant, the

sextuplet, has all six mutations 3D+A and 3E+V. As expected from our understanding of EF hands [8], the mutan~~roteins have a dramatically reduced affiiity for Ca . The sextu let calmodulin does not bind detectable levels of 49 Ca2’ either during gel filtration or when bound to a solid support Despite their defects, all three mutant proteins can satisfy the cellular re@ement for calmodulin. Yeast strains containing the mutant proteins in place of wild-type cahnoduliu grow at a rate that is indistinguishable tiom wild type. The simplest ex lauation of our results is that a high affinity for CaR is not required for calmodulin to perfom its essential function. At the time that the original work was published, two other possibilities were entertained [6]. One was that the structures of the mutant proteins mimic the conformation of (Ca2t)s-caImodulin, and thus they constitutively activate the target proteius. The other was that the ability of the mutant proteins to bind Ca2’ and respond to Ca2’ signals is restored by the presence of target proteins. The identification of a yeast Ca2t-calmodulin dependent protein phosphatase similar to calcineurin [9, 101 and a yeast Ca2’calmodulin dependent protein kinase [ll] allowed us to test these possibilities. Neither the phosphatase [9, 101 nor the kiuase [12, 131 am required for cell growth, and thus activation of these enzymes does not represent the essential calmodulin function. However, yeast calcineurin is required in the mating pheromone pathway. Yeast cells deleted for both genes encoding catalytic subunits do not recover from arrest with a-factor [9]. In contrast, wild-type cells only arrest transiently in the same assa calmodulin mutants defective in binding Ca% E; do not recover from arrest with &factor (J.R. Geiser and T.N. Davis, unpublished results). The severity of the defect is very similar in the calmodulin mutauts as in the calcineuriu mutants suggesting the mutant calmodulins cannot activate calcineuriu. Therefore in vivo, their structure apparently does not mimic the conformation of (Ca2+)3-calmodulin nor is their ability to bind Ca2’ and activate targets restored by calcineuriu. A similar set of experiments with the Ca2’calmodulin dependent kinase is not possible because characterization of the strains lacking the kinase has

cELLczALauM

not revealed a phenotype [12, 131. However, the 3E+V mutant protein was purified and assayed for its ability to activate the purified kinase (gift of M. Pausch and J. Thomer) in vitro. The kinase is activated 20-fold by wild-type (Ca2t)3-calm~. In contrast, the activity of the kinase in the presence of the 3E+V mutant protein was the same as in the absence of calmodulin (D. van Tuinen and T.N. Davis, unpublished result). Thus, in vitro, the structure of 3E+V mutant calmodulin does not mimic the conformation of (Ca2+)3-calmodulin nor is its ability to bind Ca2’ and activate targets mstored by the kinase. In conclusion, mutant calmodulius have been constructed that have a dramatically reduced affiiity for calcium ions and cannot activate either of two yeast Ca2t-calmodulin dependent enzymes. Nevertheless, the mutant proteins support the growth of yeast cells. Taken together, our results argue that the essential role of calmodulin is to perform an as yet unidentified Ca2t-independent function. Means and coworkers [14] constructed a mutant Aspergillus cahodulin analogous to the 3E+V yeast mutant in which the glutamate at the twelfth position of each Ca2+-binding loop is changed to valine. When expressed under the control of the Aspergillus calmodulin promoter, the mutant cDNA cannot support the growth of AspergiUus. This result seems to suggest that the essential function of calmodulin in Aspergillusrequires a high affinity for Ca2’. However, the levels of the mutaut protein produced by this construct have not been measured, and thus the mutant protein may be present at too low a level to support growth. Similar mutations in yeast calmodulin decrease steady state levels of protein to 45% wild-type levels [6]. Unlike S. cerevisiae, Aspergillus is sensitive to even small changes in calmodulin levels [14]. It cannot yet be determined if the mutant protein is inactive because it does not bind Ca2’ or because it is unstable. Temperature-sensitiveand null mutationsin calmodulin

One purpose of the mutational analysis of yeast calmoduliu is to identify structural elements required for calmoduliu function. The surprising result that mutant calmodulins defective in binding Ca2’

h4UTATIONALANALYSIS OF CALMODULIN IN !i. CEREWSXAE

support growth indicates that we do not know enough about cahnodulin function to design defective mutants by site-directed mutagenesis. Instead, we use a plasmid shuffling technique to screen a set of random mutations in the yeast gene encoding cahnodulin (CMDI) for those that inactivate or destabilize calmodulin. A plasmid carrying CMDl is randomly mutagen&d and transformed into an indicator yeast strain. The strain was constructed such that colonies carrying a plasmid in which the CMDl gene is not functional are solid red. Colonies carrying a plasmid in which the CMDl gene is functional are white or have white sectors. Colonies carrying plasmids with temperaturesensitive mutations in CMDl sector white at 25’C but remain a solid red at 37°C. (See [6] and [15] for a mom thorough discussion of this technique.) Calmodulin is surprisingly resilient. In the initial screen of 35 000 colonies, only 15 contained mutations that inactivated calmodulin [16]. All were either nonsense mutations, frameshift mutations or a missense mutation in the initial ATG and thus merely prevented translation of an intact protein. A single missense mutation that resulted in production of an intact but inactive protein was not identified. One temperature-sensitive mutant was isolated, but it carried two mutations, IlOON and E104V. ‘Ihe CMDI gene has 441 nucleotides. From the rate of occurrence of the nonsense mutations, I calculate that 198 of the 1323 possible mutations were screened, but only 8 base changes conferred a growth phenotype. Thus 96% of the mutations were silent, Unlike most enzymes, small changes in calmodulin do not alter function. Since single mutations have little effect on calmodulin function, we repeated the plasmid shuffling screen starting with a plasmid that aheady carried one mutation in CMDI. We then screened for mutations that in combination with the one mutation would confer a phenotype. Thirkcn new temperatum-sensitive mutants were isolated and, as expected from the design of the screen, all contain at least 2 mutations (D. van Tuinen and T.N. Davis, unpublished msults). Surprisingly, 8 contain at least 3 mutations. In one, two-thirds of the second EF hand is deleted. These results am completely consistent with the idea that calmodulin is not readily inactivated.

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using sitemlltagti or tk plasmid shuffling tech@ue, we isolated 57 mutants carrying various comlk&ions of 29 mknsenlut&msaud a deletion (D. van Tuinen, B. Chang, JR. Geiser, and T.N. Davis, unpublished results). Thme mntants, including the one carrying a mutation in the initial ATG, am not viable and 20 am tempaaturesensitive. The other 34 mutants do not display a growth phenotype. Fifteen of the mutauts carry missense mutations in 1, 2 or all 3 Ca2+-binding loops [6] (J.R. Geiser and T.N. Davis, unpublished results). All of the mutants defective in binding Ca2+ can substitute for wild-type calmoduk. Although inactivating all three loops de&&&es cahnodulin and decreases the steady state levels [6], only the most severe mutant containing 2 mutations in each loop shows a tempera~sensitive phenotype. The mutant in which most of the second EF hand is deleted only displays a growth phenotype at the highest temperature that our wild-type strain of yeast can grow, 38°C. These results strongly argue that the interaction between cahnodulin and the essential targets is substantially diffemnt from well described Ca dependent interactions. A change of a hydrophobic amino acid residue toapolarorchargedresiduehasamoredramatic effect on the stability of calmodulin than changes in the charged residues. Eighteen of the nineteen temperature-sensitive mutants contain at least one such mutation. Although, we have heavily mutagenized the second EF hand of &noduEn, none of the temperature-sensitive mutants isolated in the random screen carry mutations in residues that farm ligands to the calcium ion, whereas, changes in the hydrophobic residues were isolated frequently. The most severe single mutant is IlOON. Cells carrying the IlOON mutation alone can grow at 37’C. but form smaller colonies than a wild-type strain. A mutant carrying IlOON and the corn- parable mutation in the N-terminal domain, 127N, is not viable. In vertebrate cahnodulin 1100 is part of the B-sheet between Ca2’-binding loops 3 and 4, 127 is part of the bsheet between loops 1 and 2 [17]. . Analysis of NMR spectra of yeast c&nod&l suggests that the fkheets may also be present in yeast cahnod&n &I. Starovasnik, TN. Davis, and R.E. Klevit, manuscript in preparation). Gur results suggest that the interaction between the loops is

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important for stability. Sun and coworkers I181 have shown that when overexpressed, the N-terminal or the C-terminal half of calmodulin can substitute for the full length protein. We have confirmed this result @ Stevens and T.N. Davis, unpublished results) using strains expressing either residues l-76 (the N-terminal half) or residues 75-146 (the C-terminal half). In our constructs, the N-terminal half is expressed under control of the GAU promoter. Expression of full length CMDl under control of GAL1 leads to 180-fold overexpression of cahnodulin. However, expression of the N-terminal half under control of GAL1 only results in a 3-fold increase relative to the steady state levels of full length protein. Thus, either the message encoding the N-terminal half or the protein itself is very unstable. In agreement with previous results [18], strains expressing the C-terminal half grow well at temperatures up to 32°C but are dead at 37°C. In our hands, strains expressing the N-terminal half grow poorly and accumulate suppressors unless the medium is supplemented with 50 mM CaCk, in which case, the cells grow well at all temperatures. A high concentration of Ca” in the medium also suppresses the temperature-sensitive phenotype of the strains expressing the C-terminal half [18]. The extra Ca” may stabilize the half-cahnodulins. That both the N-terminal and the C-temunal half contain the essential structural information of cahuodulin explains why single mutations do not abolish the function of the protein unless they prevent translation. Structural studies of yeast calmodulln Although neither a solution structure nor a crystal structure is available for yeast calmodulin, several lines of evidence suggest that yeast calmodulin adopts a structure with similarities to vertebrate calmodulin [17, 191. Yeast and vertebrate calmodulin sham 60% identity in primary structure [ll. The secondary structure of yeast calmodulin as predicted by a Chou and Fasman algorithm is very similar to that of vertebrate calmodulin [20]. As measured by circular dichroism, the amount of a-helix in yeast apo-calmodulin [21] is nearly the

CELLCA-

same as in vertebrate [221 or scallop calmodulin [211. The general features of the NMR spectra am reminiscent of the spectra of vertebrate calmodulin (M. Starovasnik, T.N. Davis, and R.E. Klevit, unpublished ObSeNatiOnS). Most significantly, vertebrate calmodulin can perform all the calmodulin functions required for yeast cell growth [5,23, 241. Thus, the structural elements required for yeast growth are conserved in vertebrate cahnodulin. Structural differences between yeast and vertebrate cahnodulin are reflected in the high concentrations of yeast calmodulin mquired to activate vertebrate enzymes [7, 231. One obvious difference is that yeast cahnodulin binds 3 mole calcium ions per mole of protein [7] (TN. Davis, unpublished results), whereas all other cahnodulins bind four 1251. Half-maximal binding occurs at a similar concentration of Ca2’ in yeast calmodulin as in other cahnodulins [7, 251 (T.N. Davis, unpublished results). Yeast calmodulin contains four EF hand homologs, but since it only binds three calcium ions, one of the homologs must be inactive. In EF hands 1 and 3, 15 out of the 16 residues that defme an EF hand are conserved [8]. In EF hands 2 and 4, 14 of the 16 residues am conserved. The sixth position of the Ca2+-binding loop of the second EF hand is histidine instead of glycine, but aU the residues that ligand the calcium ion are conserved and there are no deletions. EF hand 4 is more severely altea one residue is deleted from the Ca2’-binding loop and glutamine replaces glutamate at the twelfth position of the loop. On the basis of the sequence analysis, the fourth loop is predicted not to have a high affinity for Ca2’. As an initial structural analysis of yeast cahuodulin, we identified the products of limited proteolysis with trypsin [26]. A similar study of vertebrate calmodulin provided early evidence that the protein has two globular domains connected by a flexible tether 127-291. The tryptic fragments generated in the presence of EGTA or Ca2’ are remarkably conserved in yeast calmodulin [261. Both yeast and vertebrate apo-cahnodulin are prefer- entiall cleaved at residue 106. In the presence of Ca21, both yeast and vertebrate cahnodulin contain a relatively stable N-terminal domain and am cleaved at Lys-77 and Lys-115 (if it is not lrimethylated). In general, the susceptibilities of the basic residues in

MUTATIONAL ANALYSIS OF CALMODULIN IN S. CEREWSL4E

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S phase until G2, when the size of the bud is only slightly smaller than the mother cell. ‘Ihe nucleus then migrates to the neck and mitosis begins. At this stage a spindle can be readily observed by immunofluomscent staining of tubulin 1311. The spindle elongates and the chromosomes am segregated into mother and daughter cells. Cytokinesis followed by cell separation produces two unbudded

yeast cahnodulin am similar to those in vertebrate calmodulin. The exception is Arg-126, which is rapidly cleaved in the yeast protein but stable in vertebrate calmodulin. In vertebrate calmodulin, Arg-126 is stable to proteolysis presumably because it is part of the fast a-helix of the fourth EF hand. The unusual accessibility of Arg-126 in yeast (Ca2+)3-cahnodulinsuggests that the structure of the surrounding region is irregular and is not folded into an EF hand. This provides the first direct evidence that the fourth loop does not bind Ca*‘. In agreement, Matsuura and coworkers [21] recently showed that a mutant yeast calmodulin in which the fourth loop is deleted still binds three calcium ions as does the intact protein. An inactive fourth Ca*‘-binding loop may be the primary cause of the decreased affinity of yeast calmodulin for mammalian targets.

Calmoduh~is essential for proliferation of yeast cells [l]. We have taken two approaches to understand what aspects of proliferation require calmodulin. One has been to characterixe a temperature-sensitive cahnodulin mutant 1161 and the other has been to localixe calmodulin by immunofluorescence [37]. Interestingly, the two approaches highlight different functions so I will discuss them separately.

The fimctlons of cahnodnlln in yeast

Characterizationof a temperature-sensitive calmadulinmutant

77reyeast cell cycle S. cerevisiae proliferates by a mitotic cell cycle during which the daughter cell is produced by budding. The budding process can be divided into four steps, selection of a bud site, assembly of the bud site, organization of the cytoskeleton and polarized growth (for review, see [30]). Several proteins including actin [31] and the components of the lo-nm filaments [32, 331 accumulate at the nascent bud site. Then a ring of chitin is formed [34] and new cell wall growth is locaked to the region bounded by the chitin ring 1351. The result is the selective growth of the bud. The ability to establish polarity within a cell is not unique to budding yeast but is performed in numefous other cell types including neurons, epithelial cells and fibroblasts. The fact that the size of the bud correlates with the stage of the cell cycle simplifies analysis of the cell cycle in S. cerevisiae. Growing unbudded cells are in the Gl phase. In most strains, the initiation of DNA synthesis closely correlates with bud emergence, but analysis of mutants defective in one or the other reveals that the two events are independent 1361. The bud continues to grow throughout

CdlS.

The mutant allele c&l-l, which carries two mutations IlOONand E104V. was integrated at the Ch4Dl locus thereby replacing the wild-type gene with a temperature-sensitive allele. The resulting strain grows well at 20°C. poorly at 3o’C and dies at temperatures of 34°C and above. An isogenic wildtype strain grows well up to 38°C. When shifted to the nonpermissive temperature, the temperaturesensitive calmodulin mutant dies with a complex phenotype [16]. RNA synthesis and DNA synthesis continue in the calmodulin mutant and the cells grow large at the nonpeimissive temperature. Thus, calmodulin is not required for general macromolecular synthesis. The cmdl-1 sbain does not display a uniform terminal morphology. After 4 hours at 37°C. cultures accumulate 8596 budded cells, but the size of the bud varies from a third the size to the same size as the mother cell. Time-lapse photomicroscopy reveals that, as judged by bud morphology, 27% of the cells in an asynchronous culture can complete one bud and start another before arresting growth (T.N. Davis, unpublished results). Thus, arrested asynchronous ndl-I cultums contain a mixture of cells, some that stopped growth in the first cell cycle after the temperature shift and others produced during

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CELLCALCIUM

subsequent cell cycles. The heterogeneous arrest nonpermissive temperature will continue through indicates either that the response to lack of cytokinesis aud some will bud again before caImodulin is heterogeneous or that the temperature- arresting, In contrast, nearly all lose viability during sensitive mutation is leaky. The two possibilities the first mitosis. Thus, the calm&lin mutant cannot be distinguished by analysis of asynchronous shows a heterogeneous terminal bud morphology cultures. Similarly, characterization of the hetero- but uniformly loses viability coincidentally with geneous population does not allow the distinction mitosis. Therefore, the temperature-sensitive mubetween primary and secondary effects of tation is not leaky, but the response to the miscalmodulin inactivation. Instead, to examine the segregation of the chromosomes is heterogeneous. roles of calmoduliu at each stage of the cell cycle, A gradual depletion of calmodulin from S. cultures synchronized at the permissive temperature cerevisiue cells also leads to an accumulation of are monitored after a shift to the nonpermissive cells with buds of different sizes and with a GYM temperature. It should be noted that the phenotype content of DNA [38]. These results confirm that of the calmodulin mutant is distinct from cdc calmodulin is required for efficient growth of a bud mutants, which arrest during the first cell cycle with but not for DNA synthesis. However, the spindle approximately 90% of the cells with the same sized does not elongate in the calmodulin-depleted cells bud. This feature of cdc mutants dramatically suggesting there is a different response to the slow simplifies their characterizationby allowing analysis depletion of wild-type calmodulin (14 h) as opposed of asynchronous cultures. to the inactivation of a temperature-sensitive Synchronous populations of unbudded mutant calmodulin. Ohya and Anraku [38] presented cells in Gl progress through S phase at the non- preliminary results that suggested an increase in permissive temperatnm [16]. As compared to the chromosome loss but the rate of loss was not initiation of DNA synthesis, bud emergence is determined. slightly delayed in the mutant culture and bud The phenotype of the temperature-sensitive growth is slowed. Although the cells show defects calmodulin mutant at the nonpermissive temperature in bud growth, they can recover if mtumed to the is very similar to that of mutants defective in permissive temperature before mitosis. In contrast, chromosome disjunction. In the fiis mutants of S. as mitosis begins, the mutant cells perform an pombe, the spindle elongates but the chromosomes irreversibly lethal step. Mutant cells synchronized do not separate. ‘Ihe result is a random arrangement in GZM lose viability immediately upon the shift to of the chromosomes along the spindle [39]. The the nonpetissive temperature. Thus, them is a dis2+ gene encodes a type 1 protein phosphatase strict requirement for calmodulin to successfully [39]. Temperature-sensitive mutants in topoisocomplete mitosis. In the absence of cahnodulin, the merase II in S. cerevisiue [40] or S. pombe [41] die spindle elongates, but the duplicated DNA is at the nonpermissive temperature as they attempt to dispersed along the spindle rather than closely separate sister chromatids that are still entwined. associated with the spindle pole bodies. Analysis of The similarity between the top2 mutants in S. the DNA content of the cells after the first mitosis cerevisiue and the cmdl-1 mutant is especially reveals that many am aneuploid containing either striking. Like the calmodulin mutant, mntants in mom or less than the normal content of DNA. top2 do not arrest with a uniform terminal Fewer than 5% am viable. At a semi-permissive morphology, and synchronized cultures die aa they temperature, the calmodulin mutant shows approx- proceed through mitosis 1401. In both, the DNA imately a lo-fold increase in the rate of chromo- appears to be stretched along the spindle, although some loss compared to a wild-type strain. The this phenotype is more pronounced in the calmitotic phenotypes of the temperature-sensitive modulin mutant Both calmodulin and topoisomutant strongly suggest that calmodulin is required merase II [42] am required immediately after a for proper segregation of the chromosomes. release from a G2JA4arrest and lose viability with More than half the mutant cells from a culture nearly identical kinetics. synchronized in G2/M and then shifted to the Not all mutants that die as they proceed through

MUTATIONAL ANALYSIS OF CALMODULIN LNS. CEREViSL4E

mitosis have a phenotype similar to the calmodulin mutant Mutants that produce multiple spindle pole bodies, [43, 441 show a triangular spindle very different from the spindle observed in the calmodulin mutant at the nonpermissive temperature. Mutants in a novel mitotic motor with similarity to kinesin are unable to separate the spindle pole bodies resulting in a distinctive V-shaped spindle [45] not seen in the calmodulin mutant. Some cells produced by the calmodulin mutant are aploid suggesting that there may be a defect in the connection between the spindle and the DNA or in the formation of the spindle pole bodies. However, the phenotype of the calmodulin mutant is distinct from that of mutants defective in spindle pole body function or morphogenesis. A mutant that has a complete defect in attachment of the DNA to one of the spindle pole bodies (n&l-I) does not die as it proceeds through mitosis, but instead goes through an asymmetric cell division in which one daughter cell doubles in ploidy and the other inherits no chromosomes [46]. Two mutants that form monopolar spindles do not proceed through mitosis and instead arrest as large budded cells and remain viable [47, 481. One mutant with a monopolar spindle proceeds through mitosis and dies [48], but the DNA remains compact and not elongated as in the calmodulin mutant Results from higher eukaryotic cells also strongly suggest calmodulin is involved in chromosome segregation although its exact role is unknown. Depletion of calmodulin in mouse cells leads to a delay at u-sztaphasesuggesting the chromosomes can line up but not separate [4]. Calmodulin colocalizes with the kinetochore microtubules in plant endospem [49] and rat kangaroo PtK2 cells [SO]. The localization seems different in yeast cells in that calmodulin is not detected on the mitotic spindle [37] (see below). However, in yeast there is only one kinetochom microtubule per chromosome [51], as opposed to many in higher eukaryotic cells. Thus, even if calmodulin had a similar mitotic distribution in yeast, it could be below our level of detection. In yeast cells, the mitotic function can be performed by mutant calmodulins that do not bind detectable levels of Ca2’ [6]. The essential mitotic function does not involve Ca2’-binding. In agree-

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ment with this conclusion in yeapt, the ability of calmctdulintobindtothemitoticappamtusinFtKr cells is not dependent on the presence of Ca2+. Even a modified &uodubn unable to activate cyclic nucleotide phosphodiesterase can stabike the kinetochore microtubules [52]. Thus, a possible Ca2t-independent function is stabikration of the kinetochore microtubules. Determining how this could translate into a defect in chromosome disjunction requires mom information abont the dynamics of kinetochom microtubules during chromosome segregation in yeast. Immunolocalization

Antibodies against yeast calmodulin were raised in rabbits, affiity-purified and carefully chamckkzed for specificity 1371. The latter is especially important for antibodies raised against yeast antigens because the preimmune sera of rabbits often recognize a variety of yeast proteins [53]. Merely a comparison of preimmune sera to immune sera is not an adequate test of the speciticity of an autibody because the titer of a nonspecific antibody can be increased by immunization with an apparently unrelated antigen 1531. Ideally, the specitkity of the antibody should be determined by a comparison of the immunofluorescent signal in wild-type cells to the signal in cells depleted for the antigen. Although yeast cells deleted for the calmodnlin gene (cmdlA) am not viable, we were able to test the specificity of the antibody by capitalizing on the ability of vertebrate calmodulin to rescue cmdlA cells. The affinity-purified antibody raised against yeast &noduhn does not recognize vertebrate calmodulin. We compared the staining pattern of wild-type yeast cells to the staining pattern of control cmdlA yeast cells relying on a vertebrate calmodulin cDNA. No staining over background was seen in the control, but a distinctive bright staining pattern was observed in the wild-type yeast Thus, the aflinity-purified anti-yeast1371. calmodulin specifically localizes yeast calmodulin. Consistent with the bud growth defect displayed by the temperatum-sensitive mutant, the distribution of calmodulin in yeast cells suggests that it plays a roleinthepolarizedgmwthrequiredtomakeabud [37]. In unbudded cells, calmodulin coacentrates in

CBLLc-

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a patch 10-15 min before bud emergence shortly after actin concentrates there. The timing of patch formation and the fact that actin also concentrates in this region suggest that calmodulin is accumulating at the nascent bud site. Calmodulin concentrates throughout the bud in small budded cells and remains in the tip as the bud grows. During mitosis, calmodulin is dispersed throughout the cell and bud and then moves to the neck during cytokinesis. Interestingly, the mutant calmodulins 3D+A and 3E+V, in which the Ca2+-bindingsites have been inactivated, distribute very similarly to wild-type calmodulin. Thus a high affinity for Ca2’ is not required for a polarized distribution of calmodulin. A functional actin cytoskeleton is required to organize surface growth. Temperature-sensitive actin mutants arrest as unbudded cells and enlarge uniformly when shifted to the nonpermissive temThe polarized distribution of perature [54]. calmodulin is disrupted in these cells [37]. In wildtype cells, actin cables am aligned towards the bud in the mother cell and actin cortical patches accumulate in the bud [31]. In the calmodulin mutant at the nonpermissive temperature, the actin cytoskeleton is disorganized [37]. Thus, the polarized distributions of calmodulin and actin are interdependent. Actin has a similar relationship with two actin-binding proteins Abplp and fmbrin [55, 561. However, since calmodulin and actin distrib utions overlap but am not identical, we believe that the interaction between cahnodulin and actin is indirect. Another site of cell growth is the septum that forms at the neck between the mother and daughter at cytokinesis. Actin and other proteins involved in cell growth localize at the neck during cytokinesis [31, 571. Calmodulin also localizes here consistent with it playing a role in cytokinesis [37]. A culture of the temperature-sensitive cahnodulin mutant synchronized at G2 and then released at the nonpermissive temperature shows a mild defect in cytokinesis [161. However, since the calmodulin mutant displays a severe defect in mitosis, it is difficult to distinguish a primary defect in cytokinesis from a secondary effect of the mitotic defect. Interestingly, if synchronized at Gl and released at the nonpermissive temperature, the mutant shows a severe defect in cytokinesis. A

possible explanation of this result is that calmodmin is involved in organizing the 10 nm filaments, which form during Gl and are required for cytokinesis [32]. A possible target for calmodulin action during polarized growth is an unconventional myosin recently identified in a screen for mutants defective in bud formation [58]. The product of the My02 gene has six putative cahnodulin-binding sites. Since mutant yeast calmodulins defective in Ca2’binding localize similarly to wild-type calmodulin, the interaction ms nsible for this localization is expected to be CaYt-independent. Interestingly, a related myosin from bovine brain Plgo, has been shown to bind calmodulin in a Ca2’ independent manner [58, 591. One model is that calmodulin binds directly to Myo2p and facilitates bud growth and cytokinesis.

Future studies Characterization of the calmodulin mutants have identified many functions for calmodulin in yeast cells including recovery from arrest with cl-factor, polarized bud growth, cytokinesis, and chromosome disjunction. Calmodulin very likely acts through calcineurin during response to mating pheromones, but its other targets ate elusive. Future studies will establish whether calmodulin is a component of unconventional myosins in yeast as in other organisms and will characterize the role of calmodulin in facilitating cell growth. Identifying the interacting proteins involved in chromosome segregation may be more difficult. The two proteins already known to be required for chromosome disjunction, topoisomemse II and protein phosphat- ase type I seem unlikely targets because there is little or no pmcedence for their regulation by calmodulin. We will use both genetic and bio- chemical methods to discover the protein-protein interactions essential for calmodulin action.

Acknowledgements I thank Dr HC3.D. Mdler. S.E. Brokerhoff.

J.R. Geiicr. and M. Moserfor criticalnading of the manuscript.

MUTATIONAL ANALYSIS OF CALMODULIN IN S. CEREI?J&%l?

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Mutational analysis of calmodulin in Saccharomyces cerevisiae.

Calmodulin is well characterized as an intracellular Ca2+ receptor in nonproliferating tissues such as muscle and brain. Several observations indicate...
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