Evaluation of the kinetic properties of the sporulation protein SpoIIE of Bacillus subtilis by inclusion in a model membrane.
Starvation induces Bacillus subtilis to initiate a developmental process (sporulation) that includes asymmetric cell division to form the prespore and the mother cell. The integral membrane protein SpoIIE is essential for the prespore-specific activation of the transcription factor sigmaF, and it also has a morphogenic activity required for asymmetric division. An increase in the local concentration of SpoIIE at the polar septum of B. subtilis precedes dephosphorylation of the anti-anti-sigma factor SpoIIAA in the prespore. After closure and invagination of the asymmetric septum, phosphatase activity of SpoIIE increases severalfold, but the reason for this dramatic change in activity has not been determined. The central domain of SpoIIE has been seen to self-associate (I. Lucet et al., EMBO J. 19:1467-1475, 2000), suggesting that activation of the C-terminal PP2C-like phosphatase domain might be due to conformational changes brought about by the increased local concentration of SpoIIE in the sporulating septum. Here we report the inclusion of purified SpoIIE protein into a model membrane as a method for studying the effect of local concentration in a lipid bilayer on activity. In vitro assays indicate that the membrane-bound enzyme maintains dephosphorylation rates similar to the highly active micellar state at all molar ratios of protein to lipid. Atomic force microscopy images indicate that increased local concentration does not lead to self-association.
Searls
Tim
T
Microbiology Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
Chen
Xingyong
X
Allen
Stephanie
S
Yudkin
Michael D
MD
D001412:Bacillus subtilis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D018625:Microscopy, Atomic Force
D001426:Bacterial Proteins; D008051:Lipid Bilayers; C030069:spore-specific proteins, Bacillus