A non predicted amphipathic transmembrane segment is responsible for perceiving changes in membrane lipid fluidity.

CYBULSKI, L.; MARTIN, M.; MARIA CECILIA MANSILLA; ALBANESI, D.; DE MENDOZA, D

Rosario

Congreso; XXXV Reunión Anual Sociedad Argentina de Biofísica; 2006

SAB

Biophysics of proteins and nucleic acids A non predicted amphipathic transmembrane segment is responsible for perceiving changes in membrane lipid fluidity. Larisa E. Cybulski, Mariana Martín, María C. Mansilla, Daniela Albanesi and Diego de Mendoza Department of Microbiology, National University of Rosario. IBR- CONICET. Suipacha 531, 2000 Rosario, Argentina e-mail: cybulski@ibr.gov.ar Bacillus subtilis, a gram-positive bacterium, frequently encounters stress conditions in its natural environment, the soil. In order to detect and respond to these stressful and variable conditions, it employs two-component signal transduction systems. Changes in nutrients or in the growth temperature alter the membrane fluidity. The B. subtilis Des pathway has been identified as the first system that senses changes in membrane lipid fluidity and responds accordingly. It is composed of a membrane histidin-kinase, DesK, a soluble response regulator, DesR, and the effector enzyme, D5-desaturase (1, 2). Under conditions of restricted membrane fluidity, the Des pathway is induced. DesK phosphorylates DesR, enabling its binding to the des promoter as a tetramer. A tetramer of phosphorylated DesR positioned on Pdes recruits the RNA Polymerase to increase the transcription of the acyl-lipid desaturase (3). This enzyme introduces double bonds into the acyl chains of membrane lipids. The newly synthesized unsaturated fatty acids decrease the phase transition temperature of the phospholipids, favoring the phosphatase activity of DesK, what results in the interruption of des transcription (1). Several membrane topology prediction programs applied to the protein sequence of DesK predict four primary transmembrane segments (TMS) and one putative secondary TMS. In this study we analyzed the topology of DesK by single cysteine introduction and labeling of these Cys residues with the membrane non-permeable reactive MBP. We found that DesK has in fact five TMS instead of four. Sequential deletion of different TMS led us to the conclusion that i) TMS are responsible not only for anchoring the cytoplasmic kinase/phosphatase domain of  DesK to the membrane, but also to perceive the signal, and ii) the first amphipathic helix of DesK is a TMS that is crucial for the detection and signaling of the membrane fluidity in B. subtilis, since its remotion leads to constitutive kinase activity regardless of the growth temperature. The behavior of the other forms of DesK lacking different TMS, as well as single mutants in TMS1 is discussed. References: [1] Aguilar PS, Hernandez-Arriaga AM, Cybulski LE, Erazo A, and de Mendoza D The EMBO Journal 2001. 20: 1681-1691. [2] Cybulski LE, Albanesi D, Mansilla MC, Altabe S, Aguilar P, and de Mendoza D Mol. Microbiol. 2002. 45: 1379-1388. [3] Cybulski LE, del Solar G, Craig P, Espinosa M, and de Mendoza D J. Biol. Chem . 2004, 279: 39340-39347. We acknowledge Ariel Fernandez for helpful discussions.