A specific role for the ZipA protein in cell division: stabilization of the FtsZ protein

Abstract

In Escherichia coli, the cell division protein FtsZ is anchored to the cytoplasmic membrane by the action of the bitopic membrane protein ZipA and the cytoplasmic protein FtsA. Although the presence of both ZipA and FtsA is strictly indispensable for cell division, an FtsA gain-of-function mutant FtsA* (R286W) can bypass the ZipA requirement for cell division. This observation casts doubts on the role of ZipA and its need for cell division. Maxicells are nucleoid-free bacterial cells used as a whole cell in vitro system to probe protein-protein interactions without the need of protein purification. We show that ZipA protects FtsZ from the ClpXP-directed degradation observed in E. coli maxicells and that ZipA-stabilized FtsZ forms membrane-attached spiral-like structures in the bacterial cytoplasm. The overproduction of the FtsZ-binding ZipA domain is sufficient to protect FtsZ from degradation, whereas other C-terminal ZipA partial deletions lacking it are not. Individual overproduction of the proto-ring component FtsA or its gain-of-function mutant FtsA* does not result in FtsZ protection. Overproduction of FtsA or FtsA* together with ZipA does not interfere with the FtsZ protection. Moreover, neither FtsA nor FtsA* protects FtsZ when overproduced together with ZipA mutants lacking the FZB domain. We propose that ZipA protects FtsZ from degradation by ClpP by making the FtsZ site of interaction unavailable to the ClpX moiety of the ClpXP protease. This role cannot be replaced by either FtsA or FtsA*, suggesting a unique function for ZipA in proto-ring stability.

FIGURE 1.

Levels of proto-ring in maxicells. Samples from cultures of E. coli CSR603, VIP978 (CSR603 clpP::kan), and VIP979 (CSR603 clpX::kan) were withdrawn at different stages along the maxicell production procedure. No UV, non–UV-irradiated cells; UV, 3 h after irradiation; and MX, 16 h after d-cycloserine addition. The amounts of FtsZ, FtsA, and ZipA were measured by Western blotting using antibodies raised against each protein (see “Experimental Procedures”). The three proto-ring proteins were analyzed in the case of CSR603, whereas only the FtsZ levels were measured in VIP978 and VIP979.

FIGURE 2.

Protection of FtsZ from degradation by overproduction of ZipA in maxicells. FtsZ protein level during the maxicell production procedure of E. coli CSR603 cells bearing empty plasmid (control) and overproducing His-ZipA (pPZV23), His-ZipA3 (pPZV24), FtsA (pPNV40), or FtsA* (pPZV33) is shown. MX+IPTG, 16 h after addition of d-cycloserine and IPTG; other abbreviations are as in the legend to Fig. 1. Error bars indicate the S.D. in each case.

FIGURE 3.

FtsZ-protective properties of different ZipA mutants. A, ZipA structural domains (H6, hexahistidine tag; TM, transmembrane domain; ±, charged domain) and the different mutants used in the present work. B, FtsZ protein levels during the maxicell production procedure of E. coli CSR603 cells overproducing the different ZipA mutants. Abbreviations are as in the legend to Fig. 2. Error bars indicates the S.D. in each case.

FIGURE 4.

FtsZ protection under ZipA and FtsA or FtsA* overproducing conditions. A, FtsZ protein levels during the maxicell production procedure of E. coli CSR603 cells overproducing ZipA (pPZV128), FtsA (pPNV40), or FtsA* (pPZV33) or a combination of ZipA with FtsA or with FtsA*. Error bars, S.D. B, levels of ZipA, FtsA, and FtsA* on maxicells overproducing separately ZipA, FtsA⁺, or FtsA*, or pairwise combinations of ZipA⁺ together with FtsA⁺ or with FtsA*. MX+Ara, MX+IPTG, and MX+Ara+IPTG, 16 h after addition of arabinose, IPTG or both, respectively, and d-cycloserine. Other abbreviations are as in the legend to Fig. 1.

FIGURE 5.

FtsA or FtsA* cannot substitute or interfere with FZB in FtsZ protection. FtsZ levels on maxicells overproducing His-ZipA1 (pPZV29), His-ZipA4 (pPZV30) and FZB (pPZV38) alone (top panel) or in combination with FtsA (pPZV131) (middle panel) or FtsA* (pPZV132) (bottom panel) are shown. Abbreviations are as in the legend to Fig. 4. Error bars indicate the S.D. in each case.

FIGURE 6.

FtsZ helical-like structures formed by ZipA. Snapshots show a three-dimensional reconstruction of deconvoluted immunofluorescent image stacks of FtsZ and ZipA in CSR603/pPZV23 (His-ZipA) 16 h after addition of d-cycloserine and IPTG.

FIGURE 7.

Structure of the ZipA and FtsA regions involved in the binding to the FtsZ C terminus. A, left, E. coli ZipA (green) and FtsZ C terminus (red; adapted from PDB 1F47) (8). Right, T. maritima FtsA (cyan) and FtsZ C terminus (yellow; adapted from PDB 4A2A) (13). B, structures shown in A oriented to maintain the overlap of the two conformations of the FtsZ C terminus as published by Szwedziak et al. (13) (colors as in A).

FIGURE 8.

The C terminus of FtsZ as a central hub to integrate signals that modulate divisome assembly in E. coli. The C terminus of FtsZ is represented as a cylinder protruding from the bulk of the protein on which different modulators can exert their action. FtsA and ZipA would promote septation whereas ClpX and MinC would prevent it.