Direct Interaction between the Two Z Ring Membrane Anchors FtsA and ZipA

Abstract

The initiation of Escherichia coli cell division requires three proteins, FtsZ, FtsA, and ZipA, which assemble in a dynamic ring-like structure at midcell. Along with the transmembrane protein ZipA, the actin-like FtsA helps to tether treadmilling polymers of tubulin-like FtsZ to the membrane. In addition to forming homo-oligomers, FtsA and ZipA interact directly with the C-terminal conserved domain of FtsZ. Gain-of-function mutants of FtsA are deficient in forming oligomers and can bypass the need for ZipA, suggesting that ZipA may normally function to disrupt FtsA oligomers, although no direct interaction between FtsA and ZipA has been reported. Here, we use in vivo cross-linking to show that FtsA and ZipA indeed interact directly. We identify the exposed surface of FtsA helix 7, which also participates in binding to ATP through its internal surface, as a key interface needed for the interaction with ZipA. This interaction suggests that FtsZ's membrane tethers may regulate each other's activities.IMPORTANCE To divide, most bacteria first construct a protein machine at the plane of division and then recruit the machinery that will synthesize the division septum. In Escherichia coli, this first stage involves the assembly of FtsZ polymers at midcell, which directly bind to membrane-associated proteins FtsA and ZipA to form a discontinuous ring structure. Although FtsZ directly binds both FtsA and ZipA, it is unclear why FtsZ requires two separate membrane tethers. Here, we uncover a new direct interaction between the tethers, which involves a helix within FtsA that is adjacent to its ATP binding pocket. Our findings imply that in addition to their known roles as FtsZ membrane anchors, FtsA and ZipA may regulate each other's structure and function.

Keywords: Escherichia coli; cell division; cross-linking; ftsA; ftsZ; zipA.

FIG 1

FtsA-R260-BpA specifically cross-links with ZipA. (A and B) Strains expressing either FLAG-FtsA or FLAG-FtsA with an amber codon replacing R260 (R260X) were grown with the antibiotics specified, with or without BpA, and either subjected to UV cross-linking or not. Protein extracts were separated by SDS-PAGE, followed by Western blotting using anti-FLAG to detect the FLAG-FtsA (A) or anti-ZipA (B). Full-length FtsA or ZipA bands are indicated by arrowheads, as are the most prominent cross-linked species (CLS). The band representing FtsA protein terminated at codon 260 is indicated by an unlabeled arrowhead. (C to E) Strains expressing FtsA or FtsAR260X, carrying ftsA12 (WM1115), ΔzipA ftsA* (WM1657) or WM1657 with ZipA-Strep-Tag produced from plasmid pPK9 (selection with spectinomycin), were grown with BpA and either UV cross-linked or not, and their proteins were separated by SDS-PAGE. Western blots were probed with anti-Strep-Tag to detect ZipA-Strep-Tag (C), anti-ZipA (D), or anti-FtsA (E). Bands representing CLS are indicated by arrowheads. Molecular weight markers in kilodaltons are shown to the left of all blots.

FIG 2

Residues on the outer face of helix 7 of FtsA preferentially cross-link with ZipA. (A) WM1115 strains expressing FtsA amber substitutions at all the residues within helix 7 were grown in the presence of BpA and tested for their ability to complement ftsA12 at 42°C. After growth at 30°C, all strains shown were serially diluted 10-fold, spotted on plates containing different concentrations of IPTG to induce expression of the ftsA derivatives at different levels plus arabinose to induce pEVOL and BpA, and incubated either at 30°C or 42°C for 24 h. To save space, the “Mutants” row shows one of the mutants that grew at all IPTG levels at 30°C, because all of the other mutants behaved similarly. (B and C) The same strains in panel A were UV cross-linked, and protein extracts were separated by SDS-PAGE. Western blots were probed with either anti-FtsA (B) or anti-ZipA (C), and arrowheads indicate the general position of CLS. Molecular weight markers in kilodaltons are shown to the left. (D) Shown at the left is the position of each residue in FtsA helix 7 (including P250, which is outside the helix) and the relative ability of BpA at each position to cross-link with ZipA. The protein sequence of ZipA from E. coli was used to generate a model PDB file with the Phyre2 server and subsequently analyzed in UCSF Chimera. Summarized in the table at the right is the ability of each amber substitution under the conditions of the experiments in panels A and C to cross-link with ZipA and to complement at 42°C, along with the relative conservation of each substituted residue. The scoring system for CLS reflects the intensity of CLS bands, from ++++ (strongest) to − (undetectable). Scoring for complementation reflects the ability to grow at no IPTG like WT FtsA (++++), weak at no IPTG but strong growth with 10 µM IPTG (+++), weak with 10 µM IPTG but stronger growth at higher IPTG levels (++), or little growth at any induction level (+). Scoring for conservation depends on the degree of conservation across the species aligned in Fig. S5 in the supplemental material: ++++, identity across all species; +++, identity across most species; ++, identity in at least 4 other species in the list; +, some identity or similar characteristics in other species; −, no obvious conservation.

FIG 3

Disruption of FtsA helix 7 reduces FtsA-ZipA interaction. (A and B) Various FtsA constructs with residue changes in helix 7 were expressed in ftsA12(Ts) cells (WM1115) and tested for viability at different concentrations of inducer under either permissive (A) or nonpermissive (B) conditions. (C) A subset of these residue changes were converted into BpA-containing derivatives and tested for their ability to cross-link with ZipA. Protein extracts from cultures supplemented with BpA and cross-linked with UV were separated by SDS-PAGE and probed on Western blots with anti-FtsA or anti-ZipA. FtsA, ZipA, and cross-linked species (CLS) are highlighted. Blots contain spliced lanes from the same gel. Molecular weight markers in kilodaltons are shown at the left.

FIG 4

FtsA* forms CLS in the absence of ZipA and cross-links as well as FtsA. Cross-linking experiments with FtsAR260-Bpa or FtsA containing both R286W (FtsA* allele) and R260-BpA tested whether the presence of the R286W allele (ftsA*), which decreases FtsA oligomerization, influences the relative levels of cross-linking to ZipA or another FtsA. The zipA⁺ strain WM1115 or the ftsA* ΔzipA strain WM1657, with or without pPK9-ZipA to overproduce ZipA, was used to express either FtsAR260-BpA (lanes 1 and 2) or FtsA*R260-BpA (lanes 3 to 8) in the presence of BpA, UV cross-linked or not, followed by separation of proteins by SDS-PAGE and probing Western blots with anti-FtsA (A) or anti-ZipA (B). Un-cross-linked FtsA and ZipA, as well as major and minor CLS bands, are denoted with arrowheads. Molecular weight markers are shown at the left. Lanes 1 and 2 and lanes 3 to 8 were from separate gels.

FIG 5

Bacterial two-hybrid assay for ZipA-FtsA interactions. BTH101 cells containing pUT18c derivatives carrying full-length FtsA (WT) or small deletions within helix 7 plus pKNT25 or pKT25 derivatives fused to FtsA, FtsN, FtsZ, or ZipA (or empty vector) were spotted onto LB X-Gal plates with antibiotics and 50 µM IPTG, incubated for 24 h at 30°C, and photographed. All six replicates for each pair had identical phenotypes, and so only one example of each pair is shown.