©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ca Binding to Calmodulin and Its Role in Schizosaccharomyces pombe as Revealed by Mutagenesis and NMR Spectroscopy (*)

(Received for publication, November 28, 1994; and in revised form, June 19, 1995)

Michael J. Moser Sandra Y. Lee Rachel E. Klevit Trisha N. Davis (§)

From the Department of Biochemistry SJ-70, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

As a first step toward identifying the important structural elements of calmodulin from Schizosaccharomyces pombe, we examined the ability of heterologous calmodulins and Ca-binding site mutant S. pombe calmodulins to replace the essential cam1 gene. A cDNA encoding vertebrate calmodulin allows growth of S. pombe. However, calmodulin from Saccharomyces cerevisiae does not support growth even though the protein is produced at high levels. With one exception, all mutant S. pombe calmodulins with one or more intact Ca-binding sites allow growth at 21 °C. A mutant containing only an intact Ca-binding site 3 fails to support growth, as does S. pombe calmodulin with all four Ca-binding sites mutated. Several of the mutant proteins confer a temperature-sensitive phenotype. Analysis of the degree of temperature sensitivity allows the Ca-binding sites to be ranked by their ability to support fission yeast proliferation. Site 2 is more important than site 1, which is more important than site 4, which is more important than site 3. A visual colony color screen based on the fission yeast ade1 gene was developed to perform these genetic analyses. To compare the Ca-binding properties of individual sites to their functional importance for viability, Ca binding to calmodulin from S. pombe was studied by ^1H NMR spectroscopy. NMR analysis indicates a Ca-binding profile that differs from those previously determined for vertebrate and S. cerevisiae calmodulins. Ca-binding site 3 has the highest relative affinity for Ca, while the affinities of sites 1, 2, and 4 are indistinguishable. A combination of an in vivo functional assay and an in vitro physical assay reveals that the relative affinity of a site for Ca does not predict its functional importance.


INTRODUCTION

Calmodulin is a small eukaryotic protein that reversibly binds calcium ions. Crystal and solution structures show that calmodulin is composed of two homologous globular domains connected by a long central alpha-helix that is flexible in solution(1, 2) . Each globular domain consists of two EF-hand Ca-binding sites that interact via a short beta-sheet. Analysis of a Ca titration of vertebrate calmodulin (vCaM) (^1)by nuclear magnetic resonance (NMR) spectroscopy showed that the two COOH-terminal sites have the highest affinity for Ca. Both COOH-terminal sites fill simultaneously, indicating that either both sites have identical affinities or that binding is cooperative (3) . In contrast, S. cerevisiae calmodulin, or Cmd1p, binds only 3 Ca ions. The fourth site lacks key residues required to ligand a calcium ion and does not bind Ca at detectable levels(4, 5) . The three functional sites have similar affinities for Ca, although one site in the NH(2)-terminal domain begins to fill before the other two sites(6) .

The ability to bind Ca and activate target enzymes allows calmodulin to function as an intracellular mediator of the Ca signals induced by extracellular stimuli. In both liver and skeletal muscle, Ca-calmodulin activates phosphorylase kinase, stimulating glycogenolysis(7) . In smooth muscle cells, activation of myosin light chain kinase by Ca-calmodulin leads to muscle contraction(8) . The discovery that immunosuppressive drugs such as cyclosporin function by inhibition of the Ca-calmodulin-dependent protein phosphatase calcineurin suggests a role for calmodulin in T-cell activation(9) .

Study of calmodulin in genetically tractable organisms has yielded many insights into calmodulin functions. Disruption of the unique gene encoding calmodulin in fungi is a lethal mutation(10, 11, 12) . Depletion of Aspergillus nidulans calmodulin causes a failure to progress from a nimT23 cell cycle block at the G(2) to M phase boundary(13) . Many different lines of evidence show that calmodulin performs at least two essential functions in Saccharomyces cerevisiae(14, 15, 16, 17, 18, 19) . An essential mitotic function requires an interaction between calmodulin and a 110-kDa protein component of the spindle pole body, the yeast equivalent of the microtubule organizing center(20, 21) . Calmodulin also plays an essential role in polarized yeast cell growth via an interaction with an unconventional type V myosin, Myo2p(22) .

The wealth of information concerning Ca-dependent functions of calmodulin might suggest the essential roles for calmodulin in S. cerevisiae growth and division are also Ca-dependent. However, mutant S. cerevisiae calmodulins that do not bind detectable levels of Ca support normal rates of budding yeast cell growth and division (23) and localize in a manner identical to wild-type calmodulin throughout the cell cycle(14) . Vertebrate calmodulins with analogous mutations also support normal growth of S. cerevisiae, indicating a Ca-independent function fundamental to the calmodulin molecule(23) .

To explore the universality of a Ca-independent function for calmodulin during cell proliferation, we began an analysis of calmodulin in the fission yeast, Schizosaccharomyces pombe. We report a two-pronged approach involving genetic analysis and Ca-binding measurements by NMR. Combining genetic and physical analyses allows a comparison of the Ca-binding properties of calmodulin in vitro to the functional importance of Ca binding in vivo.


EXPERIMENTAL PROCEDURES

Media

Escherichia coli was grown in LB prepared as described(24) . S. cerevisiae rich medium was YPD and minimal medium was SD (25) supplemented as described(23) . S. pombe rich medium YE and sporulation media SPA and ME were as described(26) . Minimal medium was NBA (27) containing 2% glucose. Media were supplemented with 100 µg/ml adenine, leucine, and uracil as needed. Minimal medium limiting for adenine was NBA containing 5 µg/ml adenine. 5-Fluoro-orotic acid was added to YE at 500 µg/ml. Sensitivity to G418 was tested on YE medium containing 0.1 mg/ml G418.

Strains

The strains of S. pombe were MP18, h, ade1-D25, ade6-M210, leu1-32, ura4-D18, cam1; and MP24-6C, h, ade1-D25, ade6-M210, leu1-32, ura4-D18, cam1Delta::ura4. The 2.5-kilobase AflII fragment containing ade1 was deleted from the S. pombe genome in strain MP18 by two-step gene replacement (28) using plasmid pZA25 linearized with BstEII, creating the ade1-D25 allele. The red/white colony color indicator strain, MP24-6C, was constructed using plasmid pCAM1Delta::URA4 cut with BamHI and HindIII to replace the coding sequences of the cam1 gene with the ura4 gene. S. cerevisiae strain TDY55-5D, MATa, ade2-1oc, ade3Delta-100, can1-100, cmd1Delta::TRP1, his3-11,15, leu2-3,112, lys2Delta::HIS3, trp1-1, ura3-1 was described(15) .

Plasmids

Plasmids are listed in Table 1. A genomic clone pFL20/CAM1-1 containing cam1 was isolated from an S. pombe genomic library (29) in pFL20 (30) by hybridization to a 24-base pair oligonucleotide probe PCAM-348 complementary to base pairs 348-371 of cam1(11) . A SalI/EcoRV fragment from pFL20/CAM1-1 containing cam1 was blunt ligated in both orientations into pBluescript II KS- (Stratagene) digested with SalI and SmaI, creating plasmids pKS-/CAM1 and pKS-/CAM1-2. Plasmid pUTZ/CAM1-1 is an S. pombe shuttle vector containing the ura4 selectable marker and cam1. It was created by ligating the BssHII fragment of pKS-/CAM1 treated with Klenow polymerase into pUTZ4 (gift of Scott Stevens and Jo Ann Wise) cut with BamHI and treated with Klenow polymerase. S. pombe shuttle vectors containing the LEU2 selectable marker and cam1 were created by ligating a BamHI fragment carrying cam1 from pUTZ/CAM1-1 into the BamHI site of pIRT2 (31) in both orientations, creating pIRT2/CAM1-1 and pIRT2/CAM1-2.



Plasmids pZA1 through pZA42 encoding all possible combinations of cam1 mutants with from 1 to 3 E to V Ca-binding site mutations and plasmid pIRT2/CAM1-3 containing all 4 E to V mutations in an S. pombe shuttle vector containing the LEU2 selectable marker were constructed by oligonucleotide directed mutagenesis (32) starting with pIRT2/PCAM1-1 as a template. The presence of each mutation was confirmed by DNA sequence analysis.

The single intron of cam1 was replaced with an NcoI site at the initiating methionine by oligonucleotide directed mutagenesis (32) of pIRT2/CAM1-2 with oligonucleotide DEL-NCO. This created pIRT2/CAM1DeltaINT which encodes S. pombe calmodulin with a T0A mutation. Plasmid pEC/CAM1 allowing isopropyl-beta-D-thiogalactopyranoside inducible expression of S. pombe calmodulin protein from the trc promoter in E. coli was created by ligation of the NcoI/PstI fragment of pIRT2/CAM1DeltaINT containing cam1 into pSB5 (33) cut with NcoI and PstI. Plasmid pSC/PCAM which allows expression of cam1 from the CMD1 promoter in S. cerevisiae was constructed by replacing the NcoI/BamHI fragment of pJG7 (23) with one from pIRT2/CAM1DeltaINT containing cam1.

The cam1 coding sequence of plasmid pKS-/CAM1-2 was precisely deleted and replaced with EcoRI and SmaI restriction sites by oligonucleotide directed mutagenesis (32) using primer PCAM1Delta creating plasmid pCAM1Delta. Plasmid pCAM1Delta::URA4 containing ura4 inserted in place of cam1 was created by ligating the 1.8-kilobase HindIII fragment containing ura4 from pUTZ4 treated with Klenow polymerase into pCAM1Delta cut with SmaI.

The EcoRI site treated with Klenow polymerase in plasmid pCAM1Delta was changed to NcoI by ligation of an NcoI 8-base pair linker (Boehringer Mannheim Biochemicals). This created plasmid pZA13. Plasmid pZA45 containing the cam1 promoter and terminator in an S. pombe shuttle vector carrying the LEU2 selectable marker was created by insertion of the BamHI/SalI fragment of pZA13 into pSP1 (34) cut with BamHI and SalI. In plasmid pZA46, CMD1 was put under control of the cam1 promoter and terminator by ligating the NcoI/SnaBI fragment of pJG7 (23) into pZA45 cut with NcoI and SmaI. Plasmid pZA47 directing expression of vertebrate calmodulin in S. pombe was made by ligating the PstI treated with T4 DNA polymerase and NcoI fragment of pKKCAM (35) into pZA45 cut with NcoI and SmaI.

Two plasmids were required for construction of the colony sectoring assay system used in this study, one containing a calmodulin gene and the S. pombe ade1 gene and another for making a deletion of the ade1 gene from the genome. The ade1 gene was isolated from pPS6 (36) by SphI digestion, treatment with T4 DNA polymerase, and PstI digestion. This fragment was ligated into pBluescript II KS- cut with XbaI treated with Klenow polymerase and cut with PstI yielding pKS-/ADE1. Plasmid pKS-/ADE1 was digested with AflII and then re-ligated, deleting the entire ade1 coding and control sequences creating ade1-D25 in plasmid pZA19. The HindIII fragment of pUTZ4 containing ura4 was ligated into pZA19 cut with HindIII creating pZA25 which can be used to remove ade1 from the S. pombe genome. Plasmid pADH/NPT contains the neomycin resistance gene from pSV2NEO (37) under control of the S. pombe adh1 promoter. It was made by partial digestion of plasmid pARU4IN (gift of Debbie Graves and Jo Ann Wise) with HindIII, then treatment with Klenow polymerase followed by re-ligation to remove the ura4 gene and destroy a HindIII site. Plasmid pNPT/ADE1-3, an S. pombe shuttle vector selectable by G418 and containing ade1 was constructed by ligation of an FspI/SacI fragment from pKS-/ADE1 into pADH/NPT cut with SmaI and SacI. Plasmid pSPVCAM which contains a vertebrate calmodulin cDNA under control of the cam1 promoter and the CMD1 terminator was made by ligation of a NcoI/BamHI fragment of pJG60 (23) into pIRT2/CAM1DeltaINT cut NcoI/BamHI. A fragment containing vCaM was isolated from pSPVCAM by digestion with Asp-718, treatment with Klenow polymerase, and BamHI digestion. This fragment was ligated into pNPT/ADE1 cut with ClaI, treated with T4 DNA polymerase, and then cut with BamHI creating calmodulin indicator plasmid pADE1/VCAM.

Plasmid pMM46, allowing expression from the GAL1 promoter of a gene carried on a BamHI fragment was constructed by deleting the CMD1 coding sequences of pTD52 (38) and replacing them with a BamHI site. Plasmid pTD52 was modified by digestion with SnaBI, ligation of BamHI 8-base pair linkers (Boehringer Mannheim), digestion with BamHI to remove the CMD1 gene and excess linkers, and then re-ligation. A synthetic cam1-E0 cDNA on a BamHI fragment was synthesized by polymerase chain reaction using pIRT2/CAM1-3 as a template. The fragment was digested with BamHI and cloned into pBluescript II KS- cut with BamHI to create pMM23. Nucleotide sequence of pMM23 was determined. Plasmid pMM62 allowing expression in S. cerevisiae of cam1-E0 from the GAL1 promoter and CMD1 terminator was constructed by ligation of the BamHI fragment from pMM23 into pMM46 cut with BamHI.

Red/White Colony Color Assay for S. pombe

The ade6-M210 mutant in adenine biosynthesis causes the accumulation of an adenine intermediate that polymerizes to form a red pigment. The red pigment can be visually detected in a yeast colony grown on solid medium limiting for adenine. An ade1-D25,ade6-M210 double mutant strain forms white colonies because ade1 encodes an enzyme needed for the synthesis of the pigment forming intermediate. Thus, a visual screen for the presence or absence of a plasmid carrying ade1 can be performed using an ade1-D25, ade6-M210 double mutant as an indicator strain. An ade1-D25, ade6-M210, leu1-32, ura4-D18, cam1Delta::ura4 indicator strain MP24-6C can survive and produce red pigment because it contains plasmid pADE1/VCAM, encoding both wild-type vertebrate calmodulin and ade1. The strain is also resistant to G418 due to the neomycin resistant gene encoded by plasmid pADE1/VCAM. A vertebrate calmodulin cDNA was chosen to reduce recombination between plasmids in vivo. A second plasmid encoding a mutant calmodulin and the LEU2 selectable marker is introduced into the assay strain. If the mutant calmodulin can complement the chromosomal deletion of cam1, cells that lose the first plasmid encoding calmodulin and ade1 can survive. Loss of the first plasmid, and thus function of the mutant calmodulin, is scored by the presence of white sectors in red colonies on media limiting for adenine. The white sectors contain yeast cells that are wholly dependent on plasmid-encoded mutant calmodulin for growth. Strains relying solely on a mutant calmodulin and containing no vertebrate calmodulin were isolated by streaking colonies containing white sectors on minimal medium limiting for adenine and choosing solid white colonies. These apparently solid white colonies were then retested on minimal medium limiting for adenine. The loss of plasmid pADE1/VCAM was finally confirmed by demonstrating the strains were sensitive to G418.

Mutations Introduced

The mutations are E31V, E67V, E104V, and E140V if the protein sequence is numbered according to the vertebrate calmodulin sequence. This results in the first residue of S. pombe calmodulin being numbered 0 since Cam1p contains a single amino acid extension at the amino terminus compared with vertebrate calmodulin. To simplify analysis and discussion, mutant cam1 alleles were named according to which Ca-binding sites are intact and contain a glutamate in position 12 of the EF-hand. The strains were given the E designation to indicate glutamate and a number to refer to which sites are not mutated. For example a cam1-E13 mutant is wild-type in site 1 and site 3 and mutant in site 2 and site 4, thus it contains the E67V and E140V amino acid substitutions.

Purification of Cam1p

Calmodulin was partially purified from E. coli strain SB1 containing plasmids pSB6 and pEC/PCAM as described except that cells were lysed in a French pressure cell in the presence of 1 mM phenylmethylsulfonyl fluoride, RNase A, and DNase(23, 39) . Calmodulin was further purified by anion exchange chromatography as described except the buffer was Bis-Tris buffer, pH 6.1, and the calmodulin was eluted with a linear gradient from 80 to 440 mM NaCl(33) . Peak fractions were treated as described to remove residual divalent cations and EGTA(40) . Purified calmodulin was analyzed by electrospray ionization mass spectrometry and the correct molecular weight of 16,745 was confirmed. Protein concentration was determined in duplicate by amino acid analysis following 20 h hydrolysis in 6 N HCl, 0.05% beta-mercaptoethanol, 0.02% phenol at 115 °C. Hydrolysate was analyzed on a 7300 Amino Acid Analyzer with System Gold software (Beckman Instruments). A molar extinction coefficient for Cam1p at 276 nm was determined to be 308 M cm.

Antibody Production and Immunoblot Analysis

Polyclonal antisera to Cam1p were produced in rabbits as described (14) using 250 µg of Cam1p as an immunogen for each injection. Soluble S. pombe protein extracts were prepared by glass bead lysis in 50 mM Tris buffer, pH 7.5, containing 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride. Recombinant Cmd1p was isolated as described(14) . Proteins were transferred to Zeta-Probe membrane as described(23) . Primary antisera were affinity purified anti-Cmd1p immunoglobulin G (14) (1000:1) and anti-Cam1p serum (2000:1). Secondary antibody was blotting grade anti-rabbit immunoglobulin G horseradish peroxidase conjugate (Bio-Rad) (5000:1). Signal was detected using Renaissance luminol reagent (DuPont NEN) and Hyperfilm-MP (Amersham International plc).

Native Gel Analysis and Densitometry of Plasmid Encoded S. pombe Calmodulins

Soluble S. pombe protein extracts were isolated as described above and then protein concentrations were determined in quadruplicate using bicinchoninic acid reagent (Sigma) with bovine serum albumin as a standard. Either 100 or 200 µg of protein extract as well as 200, 400, 600, 800, and 1000 ng of recombinant Cam1p standards were subjected in duplicate to native polyacrylamide gel electrophoresis in the presence of EGTA as described (23) . The amount of Coomassie Brilliant Blue-stained protein present in an extract was quantified by video densitometry using NIH Image (Wayne Rasband, NIH).

NMR Spectroscopy

NMR spectra were acquired on a Bruker AM-500 spectrometer. Typical acquisition parameters were 10,000-Hz spectral width, in 8 K points for one-dimensional spectra and 2 K points for two-dimensional spectra, using a 1.5-s relaxation delay with water presaturation. At each Ca addition, a 64 scan spectrum was collected. Two-dimensional spectra were obtained in the pure-phase absorption mode with time-proportional phase incrementation using standard pulse sequences. A D(2)O NOESY spectrum was collected using a mixing time of 200 ms. A D(2)O TOCSY spectrum was collected using the MLEV-17 mixing sequence with a mixing time of 65 ms(41) .

Data were processed using the software Felix 2.0 (Hare Research, Inc.). Two-dimensional data sets were typically 650 2 K points, processed with sine-bell filters skewed toward t = 0, shifted in both dimensions by 55 °C for the NOESY and by 35 °C for the TOCSY, and zero-filled to produce 1 K 1 K matrices. ^1H chemical shifts were referenced to a downfield internal standard peak set at 8.04 ppm.

CaTitration

The NMR sample for the Ca titrations was prepared by dissolving lyophilized Ca-free Cam1p at a concentration of 2.2 mM in a total volume of 450 µl containing 50 mM imidazole-d(4) and 100 mM KCl in 99.96% D(2)O. The pH was adjusted to a final value of pH 7.6 (meter reading, uncorrected for D(2)O isotope effects). Ca titrations were performed by adding aliquots of a 50 mM stock solution of CaCl(2) in D(2)O.

Since the peaks of interest displayed slow-exchange behavior, data analysis involved measuring the area of each peak of interest. Spectra were plotted on graph paper and the squares under each peak were counted. The values were normalized with respect to the area of a downfield internal standard peak. The area of a given peak is reported as percent of final peak area for that proton, where the final peak area used was the average value taken from spectra collected with Ca equivalents geq4. Because the two Halpha resonances of Asn-26 and Thr-64 showed indistinguishable behaviors, their peak areas were averaged for the plots in Fig. 5C and 6C.


Figure 5: Peak intensities as a function of Ca. Peak areas were quantified, normalized with respect to the area of the downfield internal standard, and plotted as described under ``Experimental Procedures.'' A, His-107 peaks: bullet represents the peak area of His-107; represents the peak area of His-107; up triangle, filled represents the peak area of His-107`. B, comparison of His-107 and Tyr-138 peaks: represents the peak area of His-107; bullet represents the peak area of Tyr-138`; represents the peak area of Tyr-138 (note well: no intensities are plotted for Tyr-138 peaks between the range of 1.5-2.3 Ca equivalents because the two peaks overlap in these spectra and could not be deconvoluted); up triangle, filled represents the peak area of His-107`. C, comparison of NH(2)-terminal and COOH-terminal peaks: represents the peak area of His-107; represents the peak area of Tyr-138; bullet represents the peak area of the Halpha's from sites 1 and 2.



Modeling

The Runge modeling program was used to simulate titration data. The model used consisted of four independent sites; one with higher affinity and three with identical affinity. Since experimentally determined dissociation constants were not available, we adopted the working assumption that the affinity of the strongest site (i.e. site 3) is similar to that measured for vertebrate and for S. cerevisiae calmodulin. Therefore, the K for site 3 was held constant at 3.0 10M while the affinities for the remaining three sites were varied. Each simulation consisted of calculating the populations of each Ca-bound species as a function of [Ca]. The simulated titration curve were compared qualitatively to the NMR titration results.


RESULTS

Genetic Analysis

Heterologous Calmodulins

The structure of calmodulin has been conserved throughout evolution. S. pombe Cam1p is 74% identical to vCaM and 57% identical to Cmd1p of S. cerevisiae at the amino acid level. Functional conservation between these calmodulins was tested by introducing vertebrate and S. cerevisiae calmodulins into S. pombe. A cDNA encoding vCaM supports the growth of S. pombe at all temperatures examined (Table 2). In contrast, Cmd1p cannot support the growth of S. pombe although it is stably produced from a plasmid (Fig. 1, lane 4). The ability of fission yeast calmodulin to support the growth of budding yeast was also tested. A plasmid encoding wild-type Cam1p was introduced into S. cerevisiae. Relying on calmodulin from S. pombe, S. cerevisiae grew at all temperatures tested (Table 2). Thus, vCaM can support the growth of both S. pombe and S. cerevisiae(38) . Cam1p can support the growth of S. cerevisiae; but Cmd1p from S. cerevisiae cannot support the growth of fission yeast.




Figure 1: Immunoblot analysis of heterologous calmodulin expression. 75 ng of calmodulin purified from E. coli or 100 µg of soluble S. pombe protein extract from strains containing plasmids expressing calmodulin were subjected to SDS-polyacrylamide electrophoresis. Resolved proteins were transferred to a membrane and equal halves were probed with either polyclonal antiserum to Cam1p or affinity purified antibody to Cmd1p (14) as described under ``Experimental Procedures.'' Lane 1, purified recombinant Cam1p; Lane 2, purified recombinant Cmd1p; Lane 3, extract from strain with cam1 plasmid; Lane 4, extract from strain with vCaM plasmid + CMD1 plasmid; Lane 5, extract from strain with vCaM plasmid. The ▸ indicates the mobility of Cam1p and the ◂ indicates the mobility of Cmd1p.



Ca-binding Mutants

The observation that S. cerevisiae calmodulin was not functional in S. pombe suggested that S. pombe has different structural requirements for calmodulin than S. cerevisiae. An unusual attribute of calmodulin in S. cerevisiae is that Ca binding is not required. The importance of Ca binding to calmodulin function in S. pombe was tested by mutagenesis and genetic analysis. Substitution of a side chain that lacks Ca ligands for the conserved glutamic acid in position 12 of an EF-hand dramatically reduces the affinity of the mutated site for Ca(23, 40, 42) . A mutant fission yeast calmodulin gene, cam1-E0, was created encoding valine in place of this glutamate in all 4 Ca-binding sites (E31V, E67V, E104V, E140V) of S. pombe calmodulin. The mutant protein encoded by cam1-E0, Cam1-E0p, does not support the growth of S. pombe. Native gel analysis of fission yeast containing the cam1-E0 plasmid shows that the protein is stably produced (Fig. 2, lane 6). Thus, unlike S. cerevisiae, S. pombe requires the Ca binding activity of calmodulin for cellular proliferation.


Figure 2: Native gel analysis of mutant calmodulin expression. Soluble protein extracts from S. pombe strains containing plasmids expressing different calmodulins (as stated below and see Table 1) were subjected to native gel electrophoresis as described(23) . Lanes 2 and 3 contained 100 µg of total soluble S. pombe protein and Lanes 4-6 contained 200 µg of total soluble S. pombe protein. The gel was stained with Coomassie Brilliant Blue. Lane 1, 1 µg of purified recombinant Cam1p; Lane 2, Cam1-E13p; Lane 3, Cam1-E1p; Lane 4, Cam1-E3p + vCaM; Lane 5, Cam1p; Lane 6, Cam1-E0 + vCaM. The } indicates the mobilities of Cam1p and the ◂ indicates the mobility of vCaM.



We tested the ability of the Cam1-E0p mutant protein to function in S. cerevisiae to distinguish if the inability of Cam1-E0p to support growth of S. pombe was due to a deficiency in the mutant protein or a specific requirement in S. pombe for Ca binding. A plasmid encoding the cam1-E0 mutant under control of the S. cerevisiae GAL1 promoter was introduced into budding yeast. Mutant Cam1-E0p is able to support the growth of S. cerevisiae on medium containing galactose as the sole carbon source. Thus, Cam1-E0p functions as calmodulin in S. cerevisiae although it fails to support the growth of S. pombe, confirming the requirement of S. pombe for Ca binding to calmodulin.

The contribution of each Ca-binding site to viability was determined by characterizing the growth phenotypes conferred by mutant fission yeast calmodulins with all possible combinations of one, two, or three Ca-binding site mutations (Table 3). Remarkably, almost all combinations of mutations allow growth. The only mutant that fails to support growth contains Cam1-E3p in which only Ca-binding site 3 is intact. Therefore, Ca-binding sites 1, 2, or 4 are sufficient to allow for a functional calmodulin protein.



The degree of temperature sensitivity conferred by mutant calmodulins containing one intact Ca-binding site indicates that the different sites are not identical in their ability to support growth (Table 3). Cam1-E2p with an intact Ca-binding site 2 allows wild-type growth up to the highest temperature tested, 37 °C. Cam1-E1p with an intact site 1 supports growth up to 30 °C, while Cam1-E4p with an intact Ca-binding site 4 only supports growth up to 25 °C. Mutants containing two intact sites confirm that the effects of mutating Ca-binding sites are not equivalent for all sites. Only Cam1-E34p and Cam1-E13p confer a temperature-sensitive phenotype (Table 3). All other mutants with two or three intact sites support growth at all temperatures tested.

Mutant Calmodulin Protein Expression Levels

The mutant phenotypes could be caused by either inadequate levels of protein in the cells or a deficiency in the protein to perform an essential function. We therefore tested whether the mutant calmodulins were produced at levels sufficient to support growth. Calmodulin was resolved as a single band from other proteins in a crude yeast extract by native polyacrylamide gel electrophoresis and was quantified by densitometry (Fig. 2). Wild-type calmodulin comprised 0.25 ± 0.03% of the total soluble protein in cells containing a plasmid encoding cam1. Calmodulin from cells not carrying a plasmid was below the level of detection. Most mutant proteins were produced at levels greater than that of wild-type (Table 4). In particular, calmodulins that confer a temperature-sensitive growth defect are not underproduced. The two mutant proteins that fail to support growth, Cam1-E3p and Cam1-E0p, were found at slightly reduced levels. However, even this reduced amount of mutant protein is far greater than the amount of wild-type calmodulin normally in a cell, which has no plasmids. Thus, the observed temperature sensitivity does not result from decreased cellular protein levels.



NMR Spectroscopy

Resonance Assignments

The in vivo results suggest that the Ca-binding sites of S. pombe Cam1p are not equivalent. To assess the relative Ca affinities of the sites and their relationship to the functional ability of that particular site to maintain viability, we monitored Ca binding of Cam1p by one-dimensional ^1H NMR. Spectra were collected on Ca-free Cam1p and then following each addition of Ca. To interpret NMR data in detail, resonances in the spectrum were first assigned to specific residues in S. pombe calmodulin. Given the 74% amino acid sequence identity between calmodulin from S. pombe and vertebrates, the overall folding topology of these two proteins is likely to be similar, especially with respect to their EF-hand topology(11) . Therefore, we utilized an assignment protocol that has proven to be successful in spectra of several EF-hand containing proteins(3, 6, 43, 44) . NMR spectra from various calmodulins contain specific patterns indicative of the canonical EF-hand topology(6) . The best characterized of these patterns involves a group of aromatic resonances and Halpha resonances, which display a series of interconnected NOEs observed in calmodulin from S. cerevisiae to that from vertebrates, illustrated in Fig. 3(6, 45, 46) . A conserved phenylalanine (Phe-89 in Fig. 3) gives NOEs to another aromatic ring in the neighboring EF-hand due to its involvement in the ``aromatic box,'' a structural motif found in all calmodulin structures and in other EF-hand domain structures. The same highly conserved phenylalanine gives NOEs to two downfield-shifted Halpha resonances. These Halpha's belong to a two-stranded beta-sheet between two neighboring Ca-binding sites and are located beneath the conserved phenylalanine. This pattern is present twice in the spectrum of all calmodulins studied to date. One set of correlated peaks derives from the two NH(2)-terminal Ca-binding sites, and the other set of peaks derives from the COOH-terminal sites(6, 45, 46) .


Figure 3: Aromatic box region from the COOH-terminal domain of vCaM. Depiction of the residues involved in the COOH-terminal aromatic box region from the crystal structure of vCaM (PDB ID# 1CLL)(55) . The residues involved in the assignment process of Tyr-138 are shown and labeled. The dotted lines between Phe-89 and Asn-137, Tyr-99, and Tyr-138 indicate NOEs conserved in the NMR spectra of other calmodulins and troponins. The analogous Halpha's that participate in the aromatic box region of the NH(2)-terminal domain of S. pombe are those of Asn-26 and Thr-64 while the aromatic rings would be Phe-16 and Phe-65.



Two-dimensional TOCSY and NOESY spectra of Ca-bound Cam1p were analyzed. As expected, two sets of correlations analogous to those previously observed are evident in Cam1p spectra. One set involves a tyrosine and a phenylalanine and therefore must be due to the COOH-terminal domain pattern since there are no tyrosines in the NH(2)-terminal domain. The correlations involving these aromatic resonances yield assignments for Tyr-138 and Phe-89. First, NOESY spectra reveal a tyrosine ring spin system (as established in the TOCSY spectrum) that is close in space to a phenylalanine side chain. An identical pattern is observed in vertebrate calmodulin between Tyr-138 and Phe-89. Second, consistent with the NOE pattern between the conserved phenylalanine and the two Halpha's of the beta-sheet, two downfield-shifted Halpha peaks give NOEs to the Phe-89 resonances as well as to each other. By analogy to vertebrate calmodulin, these are the Halpha resonances from Tyr-99 and Asn-137. The peaks also exhibit strong NOEs to a spin system identified in a TOCSY spectrum as a tyrosine, but different from Tyr-138. Since Tyr-99 is the only other tyrosine in Cam1p, these observations yield a set of self-consistent assignments. The peak assigned to Tyr-138 is the most upfield aromatic resonance at 6.38 ppm, similar to all other assigned calmodulin spectra(3, 47, 48) .

A second set of correlated Halpha and aromatic resonances was observed in NOESY spectra of Ca-bound Cam1p. In particular, the two most downfield-shifted Halpha peaks in the spectrum give NOEs to each other and to a phenylalanine ring spin system, identifying the predicted NH(2)-terminal pattern. The two Halpha's should be derived from Asn-26 and Thr-64, and the aromatic ring protons should be those of Phe-16. The Halpha resonances for Asn-26 and Thr-64 are also the most downfield-shifted Halpha peaks in spectra of other calmodulins, again lending support to this assignment approach based on the expected strong similarities among calmodulin spectra(3, 6, 48) .

Finally, the C2H resonance of the unique histidine, His-107, was easily identified from its position downfield of the main aromatic envelope and from a TOCSY cross-peak that correlates it with another singlet (i.e. C4H) consistent with the pattern of a His side chain.

In summary, as illustrated in Fig. 4, the one-dimensional ^1H spectrum of Cam1p contains a number of well-resolved peaks in the aromatic and Halpha regions that can be followed throughout a Ca titration. These have been assigned by analogy to other well characterized variants of calmodulin and include: the Halpha peaks for Asn-26 and Thr-64 from the amino-terminal domain, aromatic peaks for Tyr-138, the C2H peak of His-107, and the Halpha peaks for Tyr-99 and Asn-137 from the carboxyl-terminal domain.


Figure 4: Ca titration of Cam1p by ^1H NMR. The amount of Ca present in a given sample is expressed as moles of Ca added per mole of protein. Titrations were performed as described under ``Experimental Procedures.'' His-107, Tyr-138, and the two Halpha protons (*) are indicated in the figure. A, downfield Halpha and aromatic region; B, expanded spectra, showing the peaks of His-107 and Tyr-138. Intermediate species are indicated in the spectrum.



Relative Order of CaBinding to Cam1p

Spectra collected throughout a Ca titration of Cam1p revealed some general features in common with vertebrate calmodulin. First, all detectable spectral changes are complete by the addition of 4.0-4.5 equivalents of Ca, consistent with the 4 Ca-binding sites predicted (Fig. 4A). Second, resonances associated with the carboxyl-terminal domain, in particular His-107, are the first to be perturbed in the titration.

Interestingly, although the Ca-induced perturbations in the spectra of Cam1p resemble the changes previously observed for other calmodulin spectra, they are more complicated than those observed for other calmodulins. The His-107 C2H peak is the first resonance to be perturbed detectably by Ca. Intensity at its original position, labeled His-107, disappears with a concomitant appearance of intensity at the resonance position assigned to His-107 in the fully Ca-bound form labeled His-107 (Fig. 4B). In addition, a third His-107 peak labeled His-107`, appears just downfield of His-107. The intensities of the three observed His-107 resonances are plotted as a function of added Ca in Fig. 5A. His-107 loses 50% of its original intensity at 1.0 equivalent Ca. His-107 reaches half its final intensity at 1.5 equivalents Ca and appears at full intensity in spectra collected following addition of >3.0 equivalents Ca. His-107` is first detected after addition of 0.75 Ca equivalents, reaches a maximal intensity that corresponds to 20% of full His-107 intensity, and then disappears gradually from the spectrum by 3.5 Ca equivalents. Such behavior indicates that when less than 2.0 Ca equivalents are present, Cam1p exists in at least three states that are detectable and distinguishable by NMR.

Tyr-138 provides additional information concerning the Ca-binding behavior of the COOH-terminal sites (Fig. 4B). The resonance for Tyr-138 is not resolved in the spectrum of apo-Cam1p and therefore that intensity cannot be quantitated. A new Tyr-138 peak appears early in the titration (labeled Tyr-138`) whose resonance position is slightly upfield of the Tyr-138 position in the fully Ca-bound spectrum. In the presence of 2-3 Ca equivalents, the Tyr-138 peak splits into two peaks, with the new peak resonating at the final Tyr-138 position (labeled Tyr-138). Tyr-138 continues to increase with subsequent Ca additions, with concomitant decrease in Tyr-138`, which ultimately disappears from the spectrum. Hence, similar to His-107, the intensities of at least three Tyr-138 resonances change as a function of added Ca, confirming that more than two species of the COOH-terminal domain are present during the filling of Cam1p.

The intensities of all detectable His-107 and Tyr-138 peaks are plotted in Fig. 5, A and B. The shape of the curves suggests the following interpretation. First, we note that His-107 is in Ca-site 3 and Tyr-138 is in Ca-site 4. Since His-107 and Tyr-138` initially increase in parallel (Fig. 5B), they must correspond to the same state. However, His-107 appears in the fully Ca-bound Cam1p spectrum, whereas Tyr-138` does not. The simplest explanation for this is that these peaks represent a state in which site 3 is filled with Ca. Once site 3 is filled, His-107 is not perturbed further by binding at site 4 and hence appears at its final resonance position, His-107. On the other hand, Tyr-138 is affected by binding at site 3, yielding Tyr-138`, but is further affected by binding to its own site, site 4, to give the Tyr-138 peak. Unfortunately, it is impossible to follow Tyr-138 during the early part of the titration as it is obscured by Tyr-138` until it has reached 40% of its full final intensity. Nevertheless, it does not reach this intensity until well after the His-107 peak has done so. Therefore, the appearance of Tyr-138 must correspond to the binding of Ca at sites 3 and 4. Finally, the appearance and disappearance of His-107` most likely corresponds to the state in which site 4 is filled and site 3 is empty. The chemical shift of His-107` is only slightly different from the chemical shift of His-107, indicating that this site 3 residue is only mildly affected by binding to site 4. No such singly occupied species has been detected for vertebrate calmodulin. (^2)The fact that His-107` and Tyr-138` are observed in the same spectra indicate that two species of Cam1p coexist, one species with site 3 filled and site 4 empty and the other with site 3 empty and site 4 filled.

Resonances that reflect the Ca-binding behavior of the NH(2)-terminal domain include the two Halpha resonances assigned to Asn-26 and Thr-64, in the amino-terminal beta-sheet between sites 1 and 2. The peaks assigned to these two protons grow in slow exchange as Ca is added and exhibit biphasic behavior (Fig. 5C). Both peaks are first detectable in the spectrum at 0.75 Ca equivalents and remain as broad, low intensity peaks in spectra up to 2.0 Ca equivalents. At 2.0 Ca equivalents, the two peaks coalesce into one very broad peak which then splits into two peaks of equal intensity as more Ca is added. These two peaks continue to grow in parallel, reaching half their final intensities by 2.5 Ca equivalents, and are not at full intensity until geq4.0 equivalents Ca. The parallel behavior of these two peaks indicates that they titrate together due to the same event, the filling of sites 1 and 2 with Ca.

The behaviors just described for COOH- and NH(2)-terminal resonances are compared in Fig. 5C. This presentation reveals that sites 1, 2, and 4 have affinities that are indistinguishable from each other. Fig. 5B shows that site 3 has the highest affinity for Ca. These Ca-binding properties are in contrast to those of vertebrate calmodulin, where binding to the NH(2)-terminal and COOH-terminal sites are completely separable events as detected by NMR(3, 49) . The differences between the affinities of high and low affinity sites in Cam1p are less than in vertebrate calmodulin. This difference could be achieved either if the high affinity site is weaker or if the low affinity sites are stronger. Although the high protein concentrations required for NMR measurements do not allow for the determination of absolute binding constants for Ca binding to Cam1p, the NMR data offer support for the latter of the two possibilities. The NH(2)-terminal peaks detected in the one-dimensional NMR experiments display slow exchange behavior in contrast to the fast exchange behavior displayed by the analogous Halpha resonances in vertebrate calmodulin(3, 47) . Therefore the low affinity sites in Cam1p have higher Ca affinities than the vertebrate low affinity sites. Slow exchange behavior was also observed for NH(2)-terminal Halpha resonances in Cmd1p, where Ca affinities were measured directly by flow dialysis (6) . The low affinity and high affinity binding constants in vertebrate calmodulin differ by 10-fold, whereas in S. cerevisiae they differ only by 2-fold. The relative difference between the high and low affinity sites in Cam1p is somewhere between these two values and may lie closer to the S. cerevisiae value.

Modeling

Modeling of Ca binding to four sites was performed to corroborate our interpretation of the NMR spectra as indicating the coexistence of singly occupied species. The populations of singly and doubly occupied species were predicted using a model that consists of four independent binding sites, with one high affinity site and three lower affinity sites. The predicted populations are plotted in Fig. 6with the relevant measured intensities overlaid. Qualitatively, the plots predict the behavior observed by following individual NMR resonances and support the assignments of the peaks to the Ca-bound species described above. In particular, the species with only site 4 occupied is predicted to exist at a low level throughout much of a Ca titration, similar to the behavior of the His-107` peak (Fig. 6A). Species with only site 1 or site 2 occupied have the same behavior as the species with only site 4 occupied. Thus, His-107` could be due to any singly occupied species. The species with only site 3 occupied is predicted to reach almost 50% at 2 Ca equivalents and then to disappear at higher Ca additions (Fig. 6B), mimicking the behavior of Tyr-138`. In addition, the modeling suggests an explanation for the somewhat unusual behavior observed for the two NH(2)-terminal peaks in which there was a small but measurable intensity during the first half of the Ca titration followed by a steep increase in intensity in the latter half of the titration (Fig. 6C). The intensity observed at low Ca additions could correspond to species in which a single NH(2)-terminal site is occupied (solid line, Fig. 6C), while the intensity observed at Ca additions higher than 2.0 equivalents corresponds to the species in which both NH(2)-terminal sites are occupied.


Figure 6: Ca titration behavior predicted from a model of four independent binding sites. Modeling was performed as described under ``Experimental Procedures'' with a model representing four independent sites: one high affinity site and three lower affinity sites. The behavior shown was calculated by using a dissociation constant for the high affinity site of 3.0 10M and for the three lower affinity sites of 8.0 10M, based on conclusion from the NMR data that the difference between the high and low affinity sites is between 2- and 10-fold. In all panels, lines represent the behavior predicted by the modeling and the symbols show the measured data (same as in Fig. 5). A, predicted populations of species involving Ca binding to sites 3 and 4 detected by His-107. The dotted line represents species with sites 3 and 4 unoccupied; the solid line represents species with site 4 occupied; the dashed line represents species with site 3 occupied plus those with site 3 and 4 occupied: bullet, His-107; up triangle, filled, His-107`; , His-107. B, predicted populations of species involving Ca binding to sites 3 and 4 detected by Tyr-138. The solid line represents species with site 3 occupied; the dashed line represents species with site 4 occupied plus those species with site 3 and 4 occupied: bullet, Tyr-138`; Delta, Tyr-138. C, predicted populations of species involving Ca binding to sites 1 and 2. The solid line represents species with only one NH(2)-terminal site occupied; the dashed line represents species with both sites 1 and 2 occupied: bullet, Halpha's from sites 1 and 2.



However, the observed behavior differs from that predicted by the model involving independent sites. Regardless of the values chosen for the high and low affinity binding constants, the behavior predicted for the His-107 peak is hyperbolic in shape, whereas the observed behavior is sigmoidal. Also, the observed behavior for NH(2)-terminal peaks is steeper than that predicted from the model. These differences between the predicted and observed behavior indicate that some of the sites in Cam1p interact with each other. The data available from NMR peak intensities are not accurate enough to allow for a more quantitative assessment of the binding model. Nevertheless, the modeling supports the interpretation of the NMR data that Cam1p contains one high and three lower affinity binding sites.


DISCUSSION

In contrast to S. cerevisiae, an essential calmodulin function in S. pombe requires at least one intact Ca-binding site. Substitution of a valine for the conserved glutamic acid in position 12 of all four of the EF-hand Ca-binding sites of S. pombe calmodulin yields a protein that fails to support proliferation. The same mutant S. pombe protein allows the growth of S. cerevisiae. The importance of Ca binding may reflect essential Ca-dependent calmodulin functions in S. pombe not present in S. cerevisiae. Alternatively, essential functions that are Ca independent in S. cerevisiae may be Ca dependent in S. pombe. Since the essential functions of calmodulin in S. pombe have not been identified, it is not yet possible to distinguish between these alternatives. None of the 4 Ca-dependent calmodulin-binding proteins from S. cerevisiae nor the Ca-calmodulin-dependent protein phosphatase from fission yeast is essential(50, 51, 52, 53) . Note that unlike the unicellular yeasts, the filamentous fungus, A. nidulans, requires the Ca-calmodulin-dependent protein phosphatase for proliferation(54) .

The fact that S. pombe does require some Ca binding by calmodulin was exploited to assess the importance of each Ca-binding site in vivo. Examination of the temperature sensitivity of strains containing cam1 with Ca-binding site mutations suggests that the four sites are not equivalent and that they can be ordered in terms of their importance to fission yeast cell viability. A mutant containing only site 2 intact supports growth at all temperatures tested, site 1 allows growth up to 32 °C, and site 4 allows viability at 25 °C. Alone, site 3 fails to support growth at any temperature tested. These observations lead to the following ranking of functional importance: site 2 > site 1 > site 4 > site 3.

The ranking based on single intact site mutants is consistent with the growth phenotypes of the mutants containing two intact sites. All combinations of two intact sites containing site 2 confer wild-type growth at elevated temperatures. The cam1-E13 mutant allows growth up to 32 °C, while cam1-E34 allows growth up to 30 °C. Thus, an intact site 1 is more important than an intact site 4 even when combined with the poor site 3. The cam1-E14 mutant grows at all temperatures tested, confirming that sites 1 and 4 are more critical than site 3.

Analyzing the effects of mutations to the Ca-binding sites in terms of the structural organization of calmodulin yields two further conclusions. First, the more COOH-terminal Ca-binding site of each two-site domain appears to be of greater importance to the viability of S. pombe than does the first site of a domain. Second, the NH(2)-terminal globular domain containing sites 1 and 2 is more important to fission yeast cell viability than the COOH-terminal domain.

The four Ca-binding sites of S. pombe Cam1p differ in their contributions to the maintenance of cell viability. Our initial hypothesis was that the fundamental difference in the importance of the sites was a reflection of their different affinities for Ca, with the least important sites having the lowest affinities for Ca. Surprisingly, the Ca-binding characteristics of Cam1p revealed by the NMR experiments contradict the model. Site 3, predicted to have the lowest affinity for Ca, actually has the highest relative affinity and binds Ca before the other three sites. Furthermore, the relative affinities of the other 3 sites cannot be distinguished by NMR even though their contributions to viability are not equal. Therefore, importance in vivo does not increase with increasing affinity in vitro. More likely, contributions of an individual site to interactions with target proteins determines its importance to cell viability.

Genetic analysis indicates that the ability of heterologous calmodulins to function in vivo differs between S. pombe and S. cerevisiae. Both Cam1p and vCaM can substitute for Cmd1p in budding yeast. Only vCaM can substitute for Cam1p in fission yeast, Cmd1p cannot. Thus, calmodulin may have additional essential functions in S. pombe that are not present in S. cerevisiae. Alternatively, all the essential functions for calmodulin may be the same in both organisms, but calmodulin and its target proteins in each organism have sufficiently diverged that Cmd1p fails to form a productive interaction with an essential fission yeast target. Further analysis of the essential functions of calmodulin in fission yeast will help distinguish between these two alternatives.

An analysis of the physical properties shared by Cam1p and vCaM but not Cmd1p could identify features of calmodulin required by fission yeast. The most obvious feature is that both Cam1p and vCaM bind four calcium ions while wild-type Cmd1p lacks a functional site 4 and only binds three calcium ions. However, our mutational analysis indicates that neither an ability to bind 4 calcium ions nor a functional site 4 is a critical feature in S. pombe calmodulin.

An examination of more subtle features in the Ca-binding properties of the three proteins also does not explain the heterologous calmodulin substitution data. NMR experiments indicate that each calmodulin possesses its own unique Ca binding characteristics. We found that S. pombe calmodulin binds Ca first at the single high affinity site 3, and singly occupied intermediates of Cam1p can be observed. In contrast, the two high affinity sites of vCaM, sites 3 and 4, bind Ca with positive cooperativity, thus no singly occupied species of vCaM can be detected by NMR. In addition, mutation to site 4 of calmodulin from Drosophila melanogaster, which differs from vCaM at only 3 residues, reduces the Ca binding affinities of both site 3 and site 4, and vice versa(40, 42) . These observations are consistent with the strong coupling observed between the two sites in the vertebrate protein. Finally, site 4 in Cmd1p does not bind Ca at detectable levels although site 3 in the S. cerevisiae Cmd1p retains its high affinity behavior(6) . These functionally similar proteins appear to contain three different types of Ca-binding sites in their COOH-terminal domains. Vertebrate calmodulin has two cooperative sites, Cmd1p has a single high affinity site, while Cam1p has two independent sites with different affinities.

In conclusion, we assessed the abilities of both mutant and heterologous calmodulins to support proliferation of fission yeast. We then analyzed the Ca-binding properties of Cam1p by NMR. Surprisingly, our results indicate that the relative affinity of each site for Ca does not parallel the functional importance of that site. Furthermore, despite the observed differences in the Ca-binding properties of Cam1p and vCaM, calmodulin from vertebrate sources can substitute for that of S. pombe. Our dual approach reveals the limitations of each single approach and emphasizes the importance of caution when interpreting either in vivo or in vitro results alone. Future studies of the interactions between Ca-binding site calmodulin mutants as well as heterologous calmodulins with essential target proteins will be important in gaining further insights into the relationship between the Ca affinity of calmodulin and its functional significance.


FOOTNOTES

*
This work was supported by Grant GM-40506 from the National Institutes of Health (to T. N. D.), by Training Grant T32 GM-08437 from the National Institutes of Health (to M. J. M.), by an American Heart Association Established Investigatorship and Grant DK-35187 from the National Institutes of Health (to R. E. K.), and by Training Grant T32 GM-08437 from the National Institutes of Health (to S. Y. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 206-685-1792; tdavis{at}u.washington.edu.

(^1)
The abbreviations used are: vCaM, vertebrate calmodulin; Cam1p, Schizosaccharomyces pombe calmodulin; Cmd1p, Saccharomyces cerevisiae calmodulin; NOE, nuclear Overhauser effect; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.

(^2)
It is also a formal possibility that His-107` reflects binding to a site in the NH(2)-terminal domain.


ACKNOWLEDGEMENTS

We thank Dr. David Teller and Dr. Peter Brzovic for their help and insights on the modeling of Ca binding. We thank Dr. Jo Ann Wise for plasmids and helpful suggestions in working with S. pombe. We thank Dr. Eric Muller for critical reading of the manuscript. We thank Lowell Ericsson for assistance with mass spectrometry and amino acid analysis.


REFERENCES

  1. Babu, Y. S., Sack, J. S., Greenhough, T. J., Bugg, C. E., Means, A. R., and Cook, W. J. (1985) Nature 315,37-40 [Medline] [Order article via Infotrieve]
  2. Ikura, M., Spera, S., Barbato, G., Kay, L. E., Krinks, M., and Bax, A. (1991) Biochemistry 30,9216-9228 [Medline] [Order article via Infotrieve]
  3. Klevit, R. E., Dalgarno, D. C., Levine, B. A., and Williams, R. J. P. (1984) Eur. J. Biochem. 139,109-114 [Abstract]
  4. Matsuura, I., Ishihara, K., Nakai, Y., Yazawa, M., Toda, H., and Yagi, K. (1991) J. Biochem. (Tokyo) 109,190-197 [Abstract]
  5. Luan, Y., Matsuura, I., Yazawa, M., Nakamura, T., and Yagi, K. (1987) J. Biochem. (Tokyo) 102,1531-1537 [Abstract]
  6. Starovasnik, M. A., Davis, T. N., and Klevit, R. E. (1993) Biochemistry 32,3261-3270 [Medline] [Order article via Infotrieve]
  7. Cohen, P. (1988) in Calmodulin (Cohen, P., and Klee, C. B., eds) pp. 123-144, Elsevier, Amsterdam
  8. Stull, J. T. (1988) in Calmodulin (Cohen, P., and Klee, C. B., eds) pp. 91-122, Elsevier Science Publishers, Amsterdam
  9. Liu, J., Farmer, J. D., Jr., Lane, W. S., Friedman, J., Weissman, I., and Schreiber, S. L. (1991) Cell 66,807-815 [Medline] [Order article via Infotrieve]
  10. Davis, T. N., Urdea, M. S., Masiarz, F. R., and Thorner, J. (1986) Cell 47,423-431 [Medline] [Order article via Infotrieve]
  11. Takeda, T., and Yamamoto, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,3580-3584 [Abstract]
  12. Rasmussen, C. D., Means, R. L., Lu, K. P., May, G. S., and Means, A. R. (1990) J. Biol. Chem. 265,13767-13775 [Abstract/Free Full Text]
  13. Lu, K. P., Osmani, S. A., Osmani, A. H., and Means, A. R. (1993) J. Cell Biol. 121,621-630 [Abstract]
  14. Brockerhoff, S. E., and Davis, T. N. (1992) J. Cell Biol. 118,619-629 [Abstract]
  15. Davis, T. N. (1992) J. Cell Biol. 118,607-617 [Abstract]
  16. Sun, G. H., Hirata, A., Ohya, Y., and Anraku, Y. (1992) J. Cell Biol. 119,1625-1639 [Abstract]
  17. Sun, G. H., Ohya, Y., and Anraku, Y. (1992) Protoplasma 166,1625-1639
  18. Ohya, Y., and Botstein, D. (1994) Science 263,963-966 [Medline] [Order article via Infotrieve]
  19. Ohya, Y., and Botstein, D. (1994) Genetics 138,1041-1054 [Abstract/Free Full Text]
  20. Geiser, J. R., Sundberg, H. A., Chang, B. H., Muller, E. G., and Davis, T. N. (1993) Mol. Cell. Biol. 13,7913-7924 [Abstract]
  21. Stirling, D. A., Welch, K. A., and Stark, M. J. (1994) EMBO J. 13,4329-4342 [Abstract]
  22. Brockerhoff, S. E., Stevens, R. C., and Davis, T. N. (1994) J. Cell Biol. 124,315-323 [Abstract]
  23. Geiser, J. R., van Tuinen, D., Brockerhoff, S. E., Neff, M. M., and Davis, T. N. (1991) Cell 65,949-959 [Medline] [Order article via Infotrieve]
  24. Miller, J. H. (1972) Experiments in Molecular Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  25. Sherman, F., Fink, G. R., and Hicks, J. B. (1986) Methods in Yeast Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  26. Gutz, H., Heslot, H., Leupold, U., and Loprieno, N. (1972) in Handbook of Genetics (King, R. C., ed) Vol. 1, p. 395, Plenum, New York
  27. Ponticelli, A. S., and Smith, G. R. (1989) Genetics 123,45-54 [Abstract/Free Full Text]
  28. Boeke, J. D., Truehart, J., Natsoulis, G., and Fink, G. R. (1987) Methods Enzymol. 154,164-175 [Medline] [Order article via Infotrieve]
  29. Elliott, S., Chang, C., Schweingruber, M. E., Schaller, J., Rickli, E. E., and Carbon, J. (1986) J. Biol. Chem. 261,2936-2941 [Abstract/Free Full Text]
  30. Losson, R., and Lacroute, F. (1983) Cell 32,371-377 [Medline] [Order article via Infotrieve]
  31. Hindley, J., Phear, G., Stein, M., and Beach, D. (1987) Mol. Cell. Biol. 7,504-511 [Medline] [Order article via Infotrieve]
  32. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) in Methods Enzymol. 154,367-382 [Medline] [Order article via Infotrieve]
  33. Brockerhoff, S. E., Edmonds, C. G., and Davis, T. N. (1992) Protein Sci. 1,504-516 [Abstract/Free Full Text]
  34. Cottarel, G., Beach, D., and Deuschle, U. (1993) Curr. Genet. 23,547-548 [Medline] [Order article via Infotrieve]
  35. Persechini, A., Blumenthal, D. K., Jarrett, H. W., Klee, C. B., Hardy, D. O., and Kretsinger, R. H. (1989) J. Biol. Chem. 264,8052-8058 [Abstract/Free Full Text]
  36. McKenzie, R., Schuchert, P., and Kilbey, B. (1987) Curr. Genet. 12,591-597 [Medline] [Order article via Infotrieve]
  37. Southern, P. J., and Berg, P. (1982) J. Mol. Appl. Genet. 1,327-341 [Medline] [Order article via Infotrieve]
  38. Davis, T. N., and Thorner, J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,7909-7913 [Abstract]
  39. Putkey, J. A., Slaughter, G. R., and Means, A. R. (1985) J. Biol. Chem. 260,4704-4712 [Abstract]
  40. Starovasnik, M. A., Su, D. R., Beckingham, K., and Klevit, R. E. (1992) Protein Sci. 1,245-253 [Abstract/Free Full Text]
  41. Bax, A., and Davis, D. G. (1985) J. Magn. Reson. 65,355-360
  42. Maune, J. F., Klee, C. B., and Beckingham, K. (1992) J. Biol. Chem. 267,5286-5295 [Abstract/Free Full Text]
  43. Dalgarno, D. C., Levine, B. A., Williams, R. J. P., Fullmer, C. S., and Wasserman, R. H. (1983) Eur. J. Biochem. 137,523-529 [Abstract]
  44. Brito, R. M., Putkey, J. A., Strynadka, N. C., James, M. N., and Rosevear, P. R. (1991) Biochemistry 30,10236-10245 [Medline] [Order article via Infotrieve]
  45. Dalgarno, D. C., Klevit, R. E., Levine, B. A., Williams, R. J. P., Dobrowolski, A., and Drabikowski, W. (1984) Biochim. Biophys. Acta 791,164-172 [Medline] [Order article via Infotrieve]
  46. Ikura, M., Minowa, O., and Hikichi, K. (1985) Biochemistry 24,4264-4269 [Medline] [Order article via Infotrieve]
  47. Ikura, M., Hiraoki, T., Hikichi, K., Minowa, O., Yamaguchi, H., Yazawa, M., and Yagi, K. (1984) Biochemistry 23,3124-3128
  48. Seeholzer, S. H., and Wand, A. J. (1989) Biochemistry 28,4011-4020 [Medline] [Order article via Infotrieve]
  49. Ikura, M., Hiraoki, T., Hikichi, K., Mikuni, T., Yazawa, M., and Yagi, K. (1983) Biochemistry 22,2573-2579 [Medline] [Order article via Infotrieve]
  50. Cyert, M. S., Kunisawa, R., Kaim, D., and Thorner, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,7376-7380 [Abstract]
  51. Pausch, M. H., Kaim, D., Kunisawa, R., Admon, A., and Thorner, J. (1991) EMBO J. 10,1511-1522 [Abstract]
  52. Liu, Y., Ishii, S., Tokai, M., Tsutsumi, H., Ohki, O., Akada, R., Tanaka, K., Tsuchiya, E., Fukui, S., and Miyakawa, T. (1991) Mol. & Gen. Genet. 227,52-59
  53. Yoshida, T., Toda, T., and Yanagida, M. (1994) J. Cell Sci. 107,1725-1735 [Abstract/Free Full Text]
  54. Rasmussen, C., Garen, C., Brining, S., Kincaid, R. L., Means, R. L., and Means, A. R. (1994) EMBO J. 13,3917-3924 [Abstract]
  55. Chattopadhyaya, R., Meador, W. E., Means, A. R., and Quiocho, F. A. (1992) J. Mol. Biol. 228,1177-1192 [Medline] [Order article via Infotrieve]

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