Bacillus subtilis SepF binds to the C-terminus of FtsZ.
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 FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.
Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
Ewa
E
Cendrowicz
Sebastiaan P
SP
van Kessel
Laura S
LS
van Bezouwen
Neeraj
N
Kumar
Egbert J
EJ
Boekema
Dirk-Jan
DJ
Scheffers
D001412:Bacillus subtilis / Q000166:cytology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology*; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D008854:Microscopy, Electron; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics
D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D017931:DNA Primers; C121014:EzrA protein, Bacillus subtilis; C071525:FtsZ protein, Bacteria