Mutational Studies on Yeast
Calmodulin
- Classical mutagenic studies fail to produce single point
mutation conditional-lethal alleles, or even simple unconditional
lethal alleles.
Ohya, Y. and D. Bostein, Structure-based systematic
isolation of conditional-lethal mutations in the single yeast
calmodulin gene. (1994) Genetics 138.
1041-1054.
- Deletion of the single calmodulin gene is lethal
Davis, T.N., M.S. Urdea, F.R. Masiaz, and J.
Thorner, Isolation of the yeast calmodulin gene: calmodulin is an
essential protein. (1986) Cell 47. 423-431.
Ohya and Bostein used site-directed mutagenesis to systematically
study the role of the phenylalanine residues in yeast calmodulin.
They reasoned that since phe residues are more conserved than any
other amino acid between yeast and vertebrate calmodulin, and since
vertebrate calmodulin can functionally substitute for yeast
calmodulin, mutating phenylalanine residues to alanine might lead to
a conditional-lethal phenotype.
Ohya, Y. and D. Bostein, Structure-based systematic
isolation of conditional-lethal mutations in the single yeast
calmodulin gene. (1994) Genetics 138.
1041-1054.
- Only one of the single site mutations produced a phenotype.
F92A has a
temperature-sensitive phenotype. The phenotype was
dose-dependent: it could be suppressed by over-production of
the mutant calmodulin.
- 24 of the 26 mutliple mutations caused some phenotype:
- five were dose-independent recessive lethal
- F16A + F19A + F92A
- F16A + F19A + F140A
- F68A + F89A
- F68A + F92A
- F92A + F140A
- six were lethal only in synthetic medium
- F16A + F19A + F89A
- F65A + F92A
- F68A + F140A
- F89A + F92A
- F16A + F19A + F65A + F68A
- F12A + F16A + F19A +F65A + F68A
- two were dose-independent temperature sensitive
- F65A + F140A
- F12A + F16A + F19A + F65A
- eleven were dose-suppressible temperature sensitve
- F12A + F16A + F19A
- F12A + F89A
- F12A + F92A
- F12A + F140A
- F16A + F19A + F65A
- F16A + F19A + F68A
- F65A + F68A
- F65A + F89A
- F89A + F140A
- F12A + F65A + F68A
- F12A + F16A + F19A + F68A
- two had no discernable phenotype:
- in most of these mutants, calmodulin is not degraded more
rapidly than wildtype calmodulin is. Three strains contain lower
steady-state levels of calmodulin than wildtype:
- F92A + F140A
- F16A + F19A + F65A + F68A
- F12A + F16A + F19A + F65A + F68A
- In most cases, the mutant phenotypes are suppressed by
high calcium. Two exceptions are only suppressed to a very limited
extent:
- some of the alleles were able to complement each other:
heteroallelic diploids were able to grow at the restrictive
temperature.
- On the basis of the frequencey with which the various
phenylalanine mutations contribute to the more severe phenotypes,
the authors rank the relative
importance of the phenylalanine residues in yeast calmodulin
(note that in these experiments F16 and F19 were mutated together,
and the authors are considering them a single site):
- F92 > F89 = F140 = F16F19 > F68 > F65 > F12
- this corresponds well with the fact that F92 is the only
phenylalanine whose mutation alone produces a temperature sensitive
phenotype
- it appears that in yeast calmodulin, the C-terminal
phenylalanines are more important for function than the N-terminal
phenylalanines.
Roberts et al, developed a synthetic "consensus" calmodulin for
use in mutagenesis studies. This calmodulin contains the conserved
features of plant and animal calmodulins.
Roberts, D.M., R. Crea, M. Malecha, G.
Alvarado=Urbina, R.H. Chiarello, and D.M. Watterson, Chemical
synthesis and expression of a calmodulin gene for site-specific
mutagenesis. (1985) Biochemistry 24.
5090-5098.
Harris, Watterson, and Thorner sunstituted mutants of this gene for
wildtype calmodulin in S. Cerevisiae. The "wildtype"
synthetic gene (which has 60% sequence identity with yeast
calmodulin) was able to substitute for calmodulin, but several of
the mutant synthetic calmodulins caused mutant phenotypes in the
yeast cells.
Harris, E., D.M. Watterson and J. Thorner,
Functional consequences in yeast of single -residue alterations in a
consensus calmodulin
Note: all of the synthetic calmodulins ("wildtype" and
mutant) are under the control of a GAL promoter, and therefore the
proteins are expected to be present at fairly high copy number.
- four mutants of the synthetic calmodulin were unable to
support germination of spores deficient in yeast calmodulin. This
was true even when extra calcium was added to the medium.
- EEE82-84KKK (residues 82-84 are at the end of the region
linking the two domains of calmodulin. Residue 84 is the first
residue of the first helix in the C-terminal domain.)
- EEE82-84KKK + DEE118-120KKK (residues 118-120 are at the
end of linker loop/beginning of helix III in the C-terminal domain
of calmodulin.)
- S101F (S101 is in the first binding loop of the C-terminal
domain. It is one of the residues involved in the short
beta-interaction between binding loops))
- E84K + E120K
- Of these four mutants, only S101F was able to support
continuous growth of haploid cells lacking yeast calmodulin.
However, the ability of this mutant to rescue calmodulin-deficient
cells seems to be dependent on copy-number.
- growth of haploid cells was slower than for the "wildtype" synthetic
calmodulin
- growth of haploid cells is much less consistent when the
S101F mutant is under the control of a different promoter that
results in lower copy-number of the mutant.
- the mutant is unable to sustain growth of diploid cells in
which it was the only copy of calmodulin present. The authors
attribute this to the lower levels of any calmodulin found in
diploid cells relative to levels in haploid cells.
- The same mutation has been carried out in
Paramecium, and is also non-lethal. This mutation is
responsible for altered swimming behavior in the cells, due to a
defect in the calcium/calmodulin-dependent gating of channels for
monovalent ions.
Schaefer, W.H., R.D. Hinrichsen, A.
Burgess-Cassler, C. Kung, I.A. Blair and D.M. Watterson, A mutant
Paramecium with a defective calcium-dependent potassium
conductance has altered has an altered calmodulin: A nonlethal
selective alteration in calmodulinr egulation. (1987) Proc.
Natl. Acad. Sci USA 84. 3931-3935.
Kung, C., R.R. Preston, M.E. Maley, K.Y. Ling, J.A., Kanabrocki,
B.R. Seavey and Y. Saimi, In vivo Paramecium mutants show
that calmodulin orchestrates membrane responses to stimuli. (1992)
Cell Calcium 13. 413-425.
- The single charge-reversal mutant E84K is unable to support
germination or growth, while the similar mutant E140K is.
- K115Y (in the linker loop between helices II and III
in the C-terminal domain of calmodulin.)
- was able to support germination and growth
- caused decreased viability when the cells were grown on
nitrogen-deficient medium
- caused a drastic reduction in sporulation
frequency when it was the only calmodulin in MATa/MAT(alpha)
- steady-state levels of K115Y were only about 25% of that
of all other variants of synthetic calmodulin that were capable of
supporting cell growth.
- R116F, a similar mutant in Schizosaccharomyces pombe
is also present at decreased levels.
Takeda, T., Y. Imai, M. Yamamoto,
Substitution at position 116 in Scizosaccharomyces calmodulin
decreases its stability under nitrogen starvation and results in a
sporulation-deficient phenotype. (1989) Proc. Nat. acad.
Sci. USA 86. 9739-9741.
- the authors postulate that the decreased levels of this
mutant are due to increased proteolysis. In calmodulins in many
different species, K115 is trimethylated, conferring a permanent
positive charge. The authors think that this permanent charge
could be important in determining the half-life of calmodulin in the
cell. In this case, the phenotype during nitrogen starvation could
simply be due to a general increase in proteolysis, as the cells
recover the nitrogen in non-essential proteins.
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