IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Contribution of proline residues of DesK in the sensing and transmission of cold stimulus.
Autor/es:
PORRINI, L; MANSILLA, MC; DE MENDOZA, D
Reunión:
Congreso; IX Congreso Argentino de Microbiología General. Rosario; 2013
Resumen:
Bacillus subtilis responds to a sudden decrease in
temperature by transiently inducing the expression of the des gene
encoding
for a lipid desaturase, Δ5-Des,
which introduces a double bond into the acyl chain of preexisting membrane
phospholipids. This
Δ5-Des-mediated membrane remodeling is
controlled by the cold-sensor DesK. The molecular detail of how the input
signal is
sensed by the transmembrane segments (TMS)
of DesK and transmitted to the cytoplasmic catalytic domain is completely
unknown. In order to answer this
fundamental issue, we decided to identify residues critical for cold sensing.
We found five
proline residues in TMS of DesK which are
conserved in several membrane-bound thermosensors. To investigate whether these
prolines play an essential role in the
sensing and transmission of cold stimulus, we mutated each residue individually
to alanine.
We first assayed the effect of Pro to Ala
substitution on expression of the des gene using strain DAK3. This
strain carries a
DesK null mutation, expresses desR from
Pxyl promoter and contains a Pdes-lacZ transcriptional fusion.
This strain was
transformed individually with plasmids
expressing DesKPAs alleles. The b-galactosidase activity of these strains
showed that all
DesKPA mutants were unable to activate des
transcription upon a temperature downshift. This could be due either to
DesKPA
mutants were locked in a
phosphatase-dominant state or the mutations producing an inactive enzyme in
both kinase and
phosphatase activities. In order to answer
this question we assayed in vivo phosphatase activity of DesKPA
variants. Our
findings show that DesK mutants retain the
phosphatase activity, indicating that Pro to Ala replacement does not
completely
inactivate DesK but brings it to adopt a
phosphatase dominant state.
In an attempt to elucidate the mechanistic
basis of this unexpected role of prolines on the signaling state of DesK we
plan to
isolate mutants of DesKPA that revert its
phenotype deficient in kinase activity. The search for new mutants, now able to
detect
the order of membrane lipids, was
performed by generating random mutations within the gene sequence coding for
the TMSs
(desKPAtms)
by error prone PCR mutagenesis. Surprisingly we isolated two clones that
incorporate a leucine to proline
substitutions at position 174, which
restored wild type phenotype in desKPA alleles. These new DesKPA-L174P mutants
are
able to response to changes in temperature
and membrane composition. These results suggest that the helix distortion
generated by Pro174 would be favoring the
kinase conformation lost in DesKPA variants.
Our findings show that the prolines in
DesK TMS are critical for conformational changes that lead DesK to adopt a
kinase state
and so these residues could play an
important role in signal transduction probably inducing regions of dynamic
flexibility (hinges)
in the helices in such a way as to bring
cytoplasmic domains into optimal positions for catalysis.