(Received for publication, November 28, 1994; and in revised form, June 19, 1995)
From the
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
H 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.
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
-helix that is flexible in solution(1, 2) . Each
globular domain consists of two EF-hand Ca
-binding
sites that interact via a short
-sheet. Analysis of a
Ca
titration of vertebrate calmodulin (vCaM) (
)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
-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 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.
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/CAM1
INT which encodes S. pombe calmodulin with a T0A mutation. Plasmid pEC/CAM1
allowing isopropyl-
-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/CAM1
INT 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/CAM1
INT 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 PCAM1
creating plasmid pCAM1
.
Plasmid pCAM1
::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 pCAM1
cut with SmaI.
The EcoRI site treated with Klenow polymerase in plasmid
pCAM1 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/CAM1
INT 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.
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.
H chemical
shifts were referenced to a downfield internal standard peak set at
8.04 ppm.
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
4. Because the two
H
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:
represents the peak area of
His-107
;
represents the peak area of
His-107
;
represents the peak area of His-107`. B, comparison of His-107 and Tyr-138 peaks:
represents the peak area of His-107
;
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);
represents the peak area of
His-107`. C, comparison of NH
-terminal and
COOH-terminal peaks:
represents the peak area of
His-107
;
represents the peak area of
Tyr-138
;
represents the peak area of the
H
's from sites 1 and 2.
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.
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.
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
H's that participate in the aromatic box region of the
NH
-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
-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
H
's of the
-sheet, two downfield-shifted H
peaks
give NOEs to the Phe-89 resonances as well as to each other. By analogy
to vertebrate calmodulin, these are the H
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 H and aromatic resonances was observed in NOESY spectra
of Ca
-bound Cam1p. In particular, the two most
downfield-shifted H
peaks in the spectrum give NOEs to each other
and to a phenylalanine ring spin system, identifying the predicted
NH
-terminal pattern. The two H
's should be
derived from Asn-26 and Thr-64, and the aromatic ring protons should be
those of Phe-16. The H
resonances for Asn-26 and Thr-64 are also
the most downfield-shifted H
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 H
spectrum of Cam1p contains a number of well-resolved peaks in the
aromatic and H
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 H
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 H
peaks for
Tyr-99 and Asn-137 from the carboxyl-terminal domain.
Figure 4:
Ca titration of Cam1p by
H 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
H
protons (*) are indicated in the figure. A, downfield
H
and aromatic region; B, expanded spectra, showing the
peaks of His-107 and Tyr-138. Intermediate species are indicated in the
spectrum.
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. (
)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
-terminal domain include the two H
resonances
assigned to Asn-26 and Thr-64, in the amino-terminal
-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
4.0 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-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
-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
-terminal peaks detected in the one-dimensional NMR
experiments display slow exchange behavior in contrast to the fast
exchange behavior displayed by the analogous H
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
-terminal H
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.
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
10
M and for the three lower affinity sites
of 8.0
10
M, 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:
, His-107
;
, 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:
,
Tyr-138`;
, Tyr-138
. C, predicted
populations of species involving Ca
binding to sites
1 and 2. The solid line represents species with only one
NH
-terminal site occupied; the dashed line represents species with both sites 1 and 2 occupied:
,
H
'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
-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.
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
-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.