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Identification and Characterization of the KlCMD1Gene Encoding Kluyveromyces lactis Calmodulin

TIMOTHY F. RAYNER AND MICHAEL J. R. STARK*

Department of Biochemistry, University of Dundee, Dundee, DD1 4HN, U.K.

Received 16 October 1997; accepted 4 February 1998

The KlCMD1 gene was isolated from a Kluyveromyces lactis genomic library as a suppressor of the Saccharomycescerevisiae temperature-sensitive mutant spc110-124, an allele previously shown to be suppressed by elevated copynumber of the S. cerevisiae calmodulin gene CMD1. The KlCMD1 gene encodes a polypeptide which is 95% identicalto S. cerevisiae calmodulin and 55% identical to calmodulin from Schizosaccharomyces pombe.

Complementation of a S. cerevisiae cmd1 deletion mutant by KlCMD1 demonstrates that this gene encodes afunctional calmodulin homologue. Multiple sequence alignment of calmodulins from yeast and multicellulareukaryotes shows that the K. lactis and S. cerevisiae calmodulins are considerably more closely related to each otherthan to other calmodulins, most of which have four functional Ca2+-binding EF hand domains. Thus like itsS. cerevisiae counterpart Cmd1p, the KlCMD1 product is predicted to form only three Ca2+-binding motifs. TheKlCMD1 sequence has been assigned Accession Number AJ002021 in the EMBL/GenBank database. ? 1998 JohnWiley & Sons, Ltd.

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— calmodulin; CMD1; ALG1; K. lactis; EF hand

*Correspondence to: M. J. R. Stark, Department of Bio-chemistry, University of Dundee, Dundee, DD1 4HN, U.K.Tel.: (+44) 01382 344250; fax: (+44) 01382 322558; e-mail:

INTRODUCTION

Calmodulin (CaM) is a ubiquitous 16 kDa eu-karyotic protein which is known to bind Ca2+ andact as a regulatory molecule in a number ofsystems (Cohen and Klee, 1988). Calmodulinsbind Ca2+ by means of helix-loop-helix domainsknown as EF hands (Gariepy and Hodges, 1983;Kretsinger and Nockolds, 1973), which allow themolecule to act as a Ca2+ sensor protein within thecell. The calmodulins of higher eukaryotes containfour EF hands (Strynadka and James, 1989)whereas the Saccharomyces cerevisiae calmodulin(Cmd1p) has only three such domains (Davis et al.,

m.j.r.stark@dundee.ac.uk

CCC 0749–503X/98/090869–07 $17.50? 1998 John Wiley & Sons, Ltd.

1986; Starovasnik et al., 1993). Although CMD1 isan essential gene, the critical functions of Cmd1pdo not require Ca2+-binding, since cmd1 mutantsin which all three functional EF hand domains aremutationally inactivated for Ca2+-binding arefunctional in vivo (Geiser et al., 1991).

S. cerevisiae calmodulin performs a variety ofessential and non-essential roles in the cell. Pre-viously, it has been shown that the interactionbetween the Cmd1p and the spindle pole body(SPB) component Spc110p is essential for cellviability in S. cerevisiae (Geiser et al., 1993; Stirlinget al., 1994). Allele spc110-124 introduces a singlepoint mutation in the CaM binding site ofSpc110p, disrupting the interaction between thetwo proteins and leading to a temperature-sensitive cell-cycle arrest and loss of viability

during mitosis (Stirling et al., 1996). At the

870 . . . . .

arrest point, spc110-124 cells have replicated theirgenomic DNA but have abnormal microtubuledistributions (Stirling et al., 1996), a pheno-type shared with other similar spc110 mutants(Kilmartin and Goh, 1996; Sundberg et al., 1996).This temperature-sensitive defect can be sup-pressed either by overexpression of S. cerevisiaeCMD1 from the GAL promoter or by a high-copy2 ì vector carrying CMD1 (Kilmartin and Goh,1996; Stirling et al., 1996; Sundberg et al., 1996).This relationship between calmodulin and Spc110penabled isolation of the Kluyveromyces lactishomologue of CMD1 in a genetic suppressorscreen where we sought rescue of the spc110-124Ts" phenotype by clones present in a K. lactisgenomic library.

MATERIALS AND METHODS

General methodsBasic yeast methods and growth media were as

described by Kaiser et al. (1994). General DNAmanipulations were performed as described inSambrook et al. (1989) using either Escherichia coliDH5á or DH10B (Grant et al., 1990). High strin-gency Southern blots were probed and washed at65)C, using 1#SSC, 0·1% SDS as the final wash,while lower stringency Southern blots were probedand washed at 55)C, using 2#SSC, 0·1% SDS asthe final wash.

Isolation of the K. lactis gene for calmodulin(KlCMD1)

Isolation of the KlCMD1 gene was achieved bytransforming S. cerevisiae strain TRY124 (MATaade2-1 his3-11,15 leu2-3,112 trp1-1::spc110-124::TRP1 ura3-1 spc110::LEU2; Stirling et al.,1996) with a YCp50-based K. lactis genomic DNAlibrary (Stark and Milner, 1989), selecting forUra+ transformants with the ability to grow at37)C, a temperature normally restrictive forTRY124. Plasmids were recovered from Ts+ yeasttransformants into E. coli strain DH10B andexamined by Southern blot analysis followingdigestion with EcoRV, using a 605-bp SnaBI-BamHI fragment of pEL1 (Davis and Thorner,1989) carrying the S. cerevisiae CMD1 gene asa hybridization probe under conditions of lowstringency. One plasmid (pTRKL15) containedan insert which hybridized to the probe underthese conditions. Plasmid pTRKLCMD1 wasconstructed by inserting a BstZ17I restriction

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fragment carrying the entire KlCMD1 gene andflanking sequences from pTRKL15 into theEcoRV site of plasmid pRS313 (Sikorski andHieter, 1989). DNA sequencing was performed ondenatured plasmid DNA templates following sub-cloning of regions of the pTRKL15 insert intopRS316 (Sikorski and Hieter, 1989). All presentedsequence was determined on both strands usingthe T7 Sequenase version 2.0 sequencing kit(Amersham International plc) and syntheticoligonucleotide primers.

To demonstrate that KlCMD1 could function-ally complement S. cerevisiae CMD1 loss offunction, strain KWY1 (MATa/MATá ade2-1/ade2-1 his3-11,15/his3-11,15 leu2-3,112/leu2-3,112trp1-1/trp1-1 ura3-1/ura3-1 can1-100/can1-100CMD1/cmd1::URA3) was transformed withpTRKLCMD1 and subjected to sporulation andtetrad analysis.

DNA and protein sequence analysisDNA sequence entry and preliminary analyses

were performed using GeneWorks 2.5 (OxfordMolecular). Homology searches were accom-plished using the TBLASTX program (Altschulet al., 1990) and pairwise alignment of proteinsequences was achieved using the GAP programfrom the GCG software package (Devereux et al.,1984). For multiple protein alignments, the GCGprograms PILEUP and LINEUP were used, whilethe DISTANCES and GROWTREE programswere used for phylogenetic analysis.

RESULTS AND DISCUSSION

Identification of the KlCMD1 geneS. cerevisiae strain TRY124 was transformed

with a K. lactis genomic DNA library and trans-formants were grown at 37)C to screen for cloneswhich suppressed the Ts" phenotype of thespc110-124 mutant allele. Colonies were selectedfor further study on the basis of suppression of thetemperature-sensitive spc110-124 phenotype. Theplasmids from these colonies were recovered andexamined by Southern blot analysis under con-ditions of low stringency using an S. cerevisiaeCMD1 probe. One positive clone was identifiedand the insert sequenced. Homology searchesusing this sequence established that the product ofone of the ORFs encoded by this insert showedhigh sequence similarity to calmodulins. A portionof the 5*-untranslated flanking region (UTR: base

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Figure 1. Sequence of the K. lactis CMD1 gene and its encodedprotein. Possible transcription initiation sites and TATA boxsequences are underlined and double underlined respectively. Aputative MluI motif (see text) is italicized.

pairs "632 to "302 relative to the start codon) ofthe KlCMD1 gene was used to probe a digest of K.lactis genomic DNA at high stringency, confirmingthis organism as the origin of the cloned sequence(data not shown). A similar blot carrying digests ofK. lactis genomic DNA made with each of eightdifferent restriction enzymes was also probedunder the same conditions with a fragment of theoriginal library clone which included the entireKlCMD1 coding region. None of the enzymes usedwas predicted to cleave within the sequence comp-lementary to the probe and each digest yielded asingle hybridizing restriction fragment, demon-strating that KlCMD1 is a unique gene within theK. lactis genome (data not shown).

To verify that KlCMD1 encodes the K. lactiscalmodulin homologue, we first constructedpTRKLCMD1, which carries a minimal fragmentfrom the original library clone in which KlCMD1

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is flanked by 656 bp of the 5*-UTR and 219 bp ofthe 3*-UTR. This plasmid was introduced intoa diploid strain (KWY1) heterozygous for acmd1::URA3 gene deletion and transformantswere induced to sporulate. Dissection of 18 asciestablished that the Ura+ (cmd1Ä) and His+

markers co-segregated in three of the progenyspores, demonstrating complementation ofcmd1::URA3 by the KlCMD1 gene. ThusKlCMD1 can provide the essential functions ofcalmodulin in S. cerevisiae cmd1::URA3 cells. Thelow observed frequency of Ura+ His+ sporesis most likely due to spontaneous loss of theKlCMD1 plasmid during this analysis, since ofthe 36 Ura" spores, only seven inheritedpTRKLCMD1 (i.e. Ura" His+). In contrast, in acontrol experiment where KWY1 was transformedwith pRS313, no viable Ura+ His+ spore progenywere observed (n=13 asci).

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Features of the KlCMD1 geneThe nucleotide sequence of KlCMD1 and its

flanking regions is shown in Figure 1, togetherwith the amino acid sequence of the encodedpolypeptide. The KlCMD1 locus is predicted to en-code a 441-bp ORF (positions 662–1102 in Figure1). The 5*-UTR contains at least three TATA-likesequences (Struhl, 1989), commencing at positions"380, "368 and "287. There are regions fittingboth the TCGA ("211) and Pu-Pu-Py-Pu-Pu("104, "118, "128, "232, "254, "307) con-sensus sequences for transcription initiation sites(Guarente, 1992) downstream of these sites. Thereis also a potential MluI cell cycle box consensussite at "302, a sequence which in S. cerevisiaepromoters can direct elevated expression of genesin late G1 (Andrews and Herskowitz, 1990;Lowndes et al., 1992). The leader region of theKlCMD1 gene is A-rich, and position "3 relativeto the putative initiation codon AUG is also an A,in agreement with observations made by Kozak(1989) concerning yeast translation initiation sites.The protein product of this ORF is deduced toconsist of 147 amino acid residues with a calcu-lated total molecular weight of 16,045 Da. Nosignificant ORFs were found in the region up-

stream of KlCMD1 shown in Figure 1. However,

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downstream of the gene a 175-amino acid ORFreads into the sequence towards the 3* end ofKlCMD1 (not shown). This sequence is proline-rich and shows sequence similarity to the centralregion of the S. cerevisiae protein verprolin(Donnelly et al., 1993).

Figure 2. Multiple sequence alignment of K. lactis calmodulin with other yeast and higher eukaryotecalmodulins. Sequences are from K. lactis (klcam), S. cerevisiae (sccam; Davis et al., 1986), C. albicans (cacam;Saporito and Sypherd, 1991), S. pombe (spcam; Takeda and Yamamoto, 1987), D. discoideum (ddcam; Marshaket al., 1984), D. melanogaster (dmcam; Yamanaka et al., 1987) and H. sapiens (hscam; Sasagawa et al., 1982).Dark shaded residues are identical in more than half the sequences compared; lighter shaded residues indicateconservative amino acid substitutions. The underlined regions labelled I to IV indicate the four EF-hand motifs.The EF-hand consensus sequence is D-X-(DNS)-{ILVFYW}-(DENSTG)-(DNQGHRK)-{GP}-(LIVMC)-(DENQSTAGC)-X-X-(DE)-(LIVMFYW), where ( ) indicates alternative and { } excluded residues. The arrow-head indicates the position of the deletion in EF-hand IV in calmodulin from K. lactis and S. cerevisiae.

Pairwise and multiple sequence comparison ofK. lactis and other calmodulins

K. lactis calmodulin was compared using thelocal sequence alignment program GAP (Devereuxet al., 1984) to a number of previously identifiedcalmodulins from a range of species. The predictedamino acid sequence of K. lactis calmodulin showsstrong similarity to the S. cerevisiae calmodulin(95% identity), with just eight amino acid differ-ences between the two proteins. K. lactis cal-modulin also shows a high level of identity withother calmodulins from Candida albicans (60%),Schizosaccharomyces pombe (55%), Dictyosteliumdiscoideum (61%), Drosophila melanogaster (61%)and Homo sapiens (61%). Results of a multiplesequence alignment of the K. lactis calmodulinwith the various proteins listed above are shown inFigure 2, with a phylogenetic tree based on thisalignment in Figure 3. These data indicate that

the S. cerevisiae and K. lactis calmodulins are

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considerably more similar to each other than toany of the other calmodulins listed, including anyof the other yeast calmodulins. Thus calmodulinis highly conserved between these two yeastsalthough less so than histone H3, which differs bya single conservative replacement between the twospecies (Stark and Milner, 1989).

Of particular note is the observation that bothS. cerevisiae and K. lactis calmodulins are pre-dicted to form only three functional Ca2+-bindingdomains; the C-terminal EF hand motif appears tobe degenerate in each case due to deletion ofposition 1 and mutational alteration of position 12in the EF-hand motif (Figure 2), both of which arekey residues for the co-ordination of the Ca2+ ion.In the case of S. cerevisiae, these deviations fromthe normal EF-hand motif have been shown toprevent high-affinity Ca2+-binding (Starovasniket al., 1993). Thus loss of the fourth EF-handpreceded divergence of S. cerevisiae and K. lactis,an event which occurred approximately 1·5#108

years ago (Wolfe and Shields, 1997). During thattime protein sequences in the two yeasts havediverged such that homologous polypeptides typi-cally show 50% to 70% identity at the protein level(e.g. Khoo et al., 1994; Stark and Milner, 1989).The high degree of identity observed between thecalmodulins from these two species not only re-flects the highly conserved nature of calmodulins

in general but strongly suggests that S. cerevisiae

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Cmd1p is not unique in its structure and propertiescompared with other calmodulins.

In S. cerevisiae, CMD1 (YBR109c) is located onchromosome II between ALG1 (YBR110w) andYBR108w (Cherry et al., 1997), with roughly0·5 kb of non-coding sequence between each genepair. In fact limited sequencing of the regionpreceding that shown in Figure 1 (not shown)revealed that a probable homologue of ALG1(260% amino acid identity over 100 residues) isalso located in the same position and orientationto KlCMD1. By comparison, the downstreamORF discussed above does not appear to be closelyrelated to YBR108w. Following divergence of thetwo yeasts, a genome duplication event occurredfollowed by massive gene deletion such that mostof the duplicated genes were lost (Wolfe andShields, 1997). The association of ALG1 andCMD1 therefore appears to have been preservedduring these events.

Figure 3. Phylogenetic analysis of K. lactis calmodulin. The GCG program DIS-TANCES was used to apply the Kimura protein distance method to an alignment of S.cerevisiae Cdc31p (EMBL accession number X74500: used as an outgroup) to themultiple sequence alignment shown in Figure 2. The GROWTREE program was usedto estimate a gene tree from the resulting distances matrix using the UPGMAalgorithm.

ACKNOWLEDGEMENTS

We thank Katie Welch for constructing strainKWY1, Alastair Murchie for oligonucleotide syn-thesis and Doug Stirling for helpful discussions.This work was funded by a project grant (045689)from the Wellcome Trust. We are also pleased toacknowledge the support of the BBSRC in the

form of a Research Studentship (to T.F.R.).

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