Research Article: Proteolysis-Dependent Remodeling of the Tubulin Homolog FtsZ at the Division Septum in Escherichia coli

Date Published: January 23, 2017

Publisher: Public Library of Science

Author(s): Marissa G. Viola, Christopher J. LaBreck, Joseph Conti, Jodi L. Camberg, Eric Cascales.


During bacterial cell division a dynamic protein structure called the Z-ring assembles at the septum. The major protein in the Z-ring in Escherichia coli is FtsZ, a tubulin homolog that polymerizes with GTP. FtsZ is degraded by the two-component ATP-dependent protease ClpXP. Two regions of FtsZ, located outside of the polymerization domain in the unstructured linker and at the C-terminus, are important for specific recognition and degradation by ClpXP. We engineered a synthetic substrate containing green fluorescent protein (Gfp) fused to an extended FtsZ C-terminal tail (residues 317–383), including the unstructured linker and the C-terminal conserved region, but not the polymerization domain, and showed that it is sufficient to target a non-native substrate for degradation in vitro. To determine if FtsZ degradation regulates Z-ring assembly during division, we expressed a full length Gfp-FtsZ fusion protein in wild type and clp deficient strains and monitored fluorescent Z-rings. In cells deleted for clpX or clpP, or cells expressing protease-defective mutant protein ClpP(S97A), Z-rings appear normal; however, after photobleaching a region of the Z-ring, fluorescence recovers ~70% more slowly in cells without functional ClpXP than in wild type cells. Gfp-FtsZ(R379E), which is defective for degradation by ClpXP, also assembles into Z-rings that recover fluorescence ~2-fold more slowly than Z-rings containing Gfp-FtsZ. In vitro, ClpXP cooperatively degrades and disassembles FtsZ polymers. These results demonstrate that ClpXP is a regulator of Z-ring dynamics and that the regulation is proteolysis-dependent. Our results further show that FtsZ-interacting proteins in E. coli fine-tune Z-ring dynamics.

Partial Text

Cell division in bacteria is a conserved and highly coordinated dynamic process involving many cellular proteins that function together to divide a single cell into two daughter cells [1]. During cell division the Z-ring assembles at midcell, the site of septation. The Z-ring contains the essential cell division protein FtsZ and many other division proteins, which are recruited to the septum. FtsZ is a GTPase that is structurally homologous to eukaryotic tubulin and forms large, dynamic polymers [2]. Each FtsZ monomer contains a compact, globular N-terminal polymerization domain, a flexible unstructured linker region, and a conserved region near the C-terminus that is important for protein interactions [2,3]. Several proteins in E. coli bind to FtsZ and have been shown to modulate the polymerization state of FtsZ in vitro, including MinC, SlmA and Z-ring associated proteins (ZAPs) [4]. Many of these protein-protein interactions occur near the FtsZ C-terminus.

Using direct biochemical degradation assays, we demonstrate that an extended FtsZ C-terminal region (317–383), which includes two sites previously identified as important for ClpX recognition, is sufficient to target the non-native substrate Gfp for degradation (Fig 1B). We also show that full length FtsZ fused to Gfp at the N-terminus assembles into polymers with GTP and is also degraded by ClpXP in vitro (Fig 1C and 1D). As expected, degradation of Gfp-FtsZ occurs more rapidly in the presence of GTP, which promotes assembly of FtsZ into polymers. Titrating the FtsZ concentration above the critical concentration for polymer assembly shows a cooperative increase in the rate of degradation by ClpXP, which is consistent with multimerization enhancing recognition through concentration of FtsZ C-terminal binding sites (Fig 6B). In vivo Gfp-FtsZ localizes to the Z-ring, consistent with a previous report using a similarly constructed N-terminal Gfp-FtsZ fusion protein [58]. The polymerization domain is critical for FtsZ incorporation into the Z-ring because Gfp-ZC67 does not localize to a ring (data not shown), even though the protein interaction site near the C-terminus is intact.




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