Contribution of proline residues of DesK in the sensing and transmission of cold stimulus.

PORRINI, L; MANSILLA, MC; DE MENDOZA, D

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2012

SAIB

Bacillus subtilis regulates the expression of the des gene coding for the Δ5 acyl lipid desaturase by a two-component system composed of a membrane-associated kinase, DesK, and a soluble transcriptional regulator, DesR, which stringently controls transcription of the des gene. This pathway is activated in response to a decrease in membrane fluidity provoked by a temperature downshift. However, 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. DesK has five proline residues in its TMS which are conserved in several membrane-bound thermosensors. To investigate whether these residues play an essential role in the function of DesK, we mutated each proline individually to alanine. All DesKPA mutants were unable to activate des transcription upon a temperature downshift, whereas phosphatase activity was retained. By error prone PCR mutagenesis on desKPA alleles we obtained additional mutations that restored the wild type behavior. These results strongly suggest that proline residues of DesK TMS play an important role in sensing and transmission of cold stimulus by kinking the helices in order to bring cytoplasmic domains into optimal positions for catalysis.