bacillus subtilis sepf binds to the c-terminus of ftsz枯草芽孢杆菌sepf ftsz糖基结合.pdf

Bacillus subtilis SepF Binds to the C-Terminus of FtsZ ´ 1. 1. 2 1 2 Ewa Krol , Sebastiaan P. van Kessel , Laura S. van Bezouwen , Neeraj Kumar , Egbert J. Boekema , Dirk-Jan Scheffers1* 1 Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands, 2 Department of Electron Microscopy, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands Abstract Bacterial cell division is mediated by a multi-protein machine known as the ‘‘divisome’’, which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z- ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull- down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZDC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that