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The cytokinetic Z ring is required for bacterial cell division. It consists of polymers of FtsZ, the bacterial ancestor of eukaryotic tubulin, linked to the cytoplasmic membrane. Formation of a Z ring in Escherichia coli occurs as long as one of two proteins, ZipA or FtsA, is present. Both of these proteins bind FtsZ suggesting that they might function to tether FtsZ filaments to the membrane. Although ZipA has a transmembrane domain and therefore can function as a membrane anchor, interaction of FtsA with the membrane has not been explored. In this study we demonstrate that FtsA, which is structurally related to eukaryotic actin, has a conserved C-terminal amphipathic helix that is essential for FtsA function. It is required to target FtsA to the membrane and subsequently to the Z ring. As FtsA is much more widely conserved in bacteria than ZipA, it is likely that FtsA serves as the principal membrane anchor for the Z ring.
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Bacterial cell division requires the cytokinetic Z ring consisting of polymers of FtsZ tethered to the cytoplasmic membrane at the division site (Bi and Lutkenhaus, 1991; Lutkenhaus and Addinall, 1997; Errington et al., 2003; Romberg and Levin, 2003). The ring is positioned at the division site through the activity of two negative regulatory systems, Min and Noc, which prevent its formation elsewhere in the cell (de Boer et al., 1989; Yu and Margolin, 1999; Wu and Errington, 2004). It is a highly dynamic structure with FtsZ in the ring turning over with a half-life on the order of 10 s (Stricker et al., 2002). The Z ring functions to recruit additional cell division proteins, of which more than a dozen have been identified in Escherichia coli. These proteins are recruited in an ordered fashion resulting in a complex (septal ring) capable of carrying out cytokinesis (Buddelmeijer and Beckwith, 2002). Two of these proteins, FtsA and ZipA, interact directly with FtsZ and appear to localize simultaneously with FtsZ at the Z ring (Addinall and Lutkenhaus, 1996; Wang et al., 1997; Hale and de Boer, 1999). A third protein ZapA also interacts directly with FtsZ but is not essential for cell division (Gueiros-Filho and Losick, 2002; Johnson et al., 2004). Only after FtsA and ZipA are present at the Z ring, are additional downstream cell division proteins recruited resulting in the formation of a septal ring capable of carrying out cytokinesis (Weiss et al., 1999; Bernhardt and de Boer, 2003; Schmidt et al., 2004).
In addition to functioning as a scaffold for the assembly of the septal ring, the Z ring may have a more direct role in providing the force for constriction. Models have been proposed in which energy from depolymerization of FtsZ filaments is used to power the invagination of the septum (Erickson et al., 1996; Bramhill, 1997). However, there has been no direct evidence to date for this model.
One of the main questions in bacterial cell division is how the Z ring is linked to the cytoplasmic membrane. In E. coli either one of two proteins, FtsA or ZipA, is capable of supporting formation of a Z ring (Addinall and Lutkenhaus, 1996; Hale and de Boer, 1999; Pichoff and Lutkenhaus, 2002). Both proteins bind to a short conserved sequence located at the extreme carboxy end of FtsZ (Ma and Margolin, 1999; Mosyak et al., 2000; Haney et al., 2001; Pichoff and Lutkenhaus, 2002). As long as one of these two proteins is present a Z ring forms, however, a complete septal ring is not formed as additional downstream proteins are not recruited (Pichoff and Lutkenhaus, 2002). Whether additional proteins are also required at this early stage of assembly is not clear. However, when both FtsA and ZipA are inactivated Z rings do not form (Pichoff and Lutkenhaus, 2002). Also, FtsZ mutants, unable to bind FtsA or ZipA because of substitutions or deletions of its conserved C-terminal motif, are unable to assemble into Z rings in the absence of wild-type FtsZ (Ma and Margolin, 1999; Pichoff and Lutkenhaus, 2002).
It is clear that ZipA could link FtsZ polymers to the membrane. The FtsZ interacting domain of ZipA is located in a C-terminal domain that is linked to an amino terminal transmembrane domain by an extended linker region (Hale and de Boer, 1997). This transmembrane domain of ZipA is required for ZipA to be functional so it likely serves a membrane-anchoring function. However, ZipA is unlikely to serve as the primary link to the membrane because it is only found in some Gram-negative bacteria (Hale and de Boer, 1997).
FtsA is structurally homologous to actin and is more conserved than ZipA (van den Ent and Lowe, 2000). It is present in most bacteria suggesting it may have a more important role in Z-ring formation than ZipA (Rothfield et al., 1999). The ftsA and ftsZ genes form one of the more highly conserved pair of tandemly linked genes in bacteria. Significantly, a gain of function mutation in the ftsA gene of E. coli bypasses the requirement for ZipA (Geissler et al., 2003). This ftsA allele allows all downstream division proteins to be recruited to the Z ring in the absence of ZipA, suggesting that FtsA plays a more direct and important role in Z ring assembly and in the recruitment of downstream proteins.
Since FtsA is a key protein interacting with FtsZ, how does it link FtsZ polymers to the membrane? One possibility is that FtsA interacts with some as yet unidentified membrane protein. Another is that FtsA interacts directly with the membrane even though no sequence interacting with the membrane has been identified. Cell fractionation studies revealed about 30% of FtsA is in the membrane fraction (Pla et al., 1990). Also, FtsA–GFP fusions show some tendency to be at the membrane and deletion analysis suggests that an internal domain is responsible (Ma et al., 1996). These results raise the possibility that FtsA is a peripheral membrane protein but do not differentiate whether FtsA binds directly to the membrane or to a protein in the membrane.
In this study, we explored the possibility that FtsA binds directly to the membrane. Previous deletion analysis of the C-terminal region of FtsA revealed that four residues could be deleted without affecting FtsA function; however, further deletion resulted in inactivation and failure of the truncated FtsA to localize to the septum (Yim et al., 2000). Our results indicate that a conserved C-terminal motif in FtsA functions as a membrane targeting sequence (MTS) by forming an amphipathic helix that anchors FtsA, and therefore, the Z ring to the membrane.
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The first event readily detected in bacterial cytokinesis is the formation of a Z ring at the division site just beneath the cytoplasmic membrane. In E. coli, FtsZ interacts with FtsA and ZipA and either of these proteins is sufficient to promote Z-ring formation suggesting both are able to tether the Z ring to the membrane. For ZipA, which has a transmembrane domain, the mechanism appears straight forward; however, for FtsA, which did not appear to contain a specific sequence suggesting a membrane interaction it was less clear. In this report, we present clear and convincing evidence that FtsA is a membrane binding protein and that this interaction occurs through a conserved C-terminal amphiphatic helix that serves as an MTS. We also report that the interaction of FtsA with the membrane is essential for its function in cell division and that it regulates the interaction of FtsA with FtsZ. As the FtsZ–FtsA pair is well conserved in evolution, we suggest that FtsA tethers Z rings to the membrane in most bacteria.
FtsA is targeted to the membrane through a conserved C-terminal amphipathic helix
The conserved motif at the extreme C-terminus of FtsA is not present in the crystal structure indicating that it is not organized although computer predictions indicate that this motif is a potential amphiphatic helix (van den Ent and Lowe, 2000; Lowe and van den Ent, 2001). When GFP–FtsA was overexpressed in cells, the fluorescence was clearly located at the membrane and this was in sharp contrast to the cytoplasmic localization of the truncated version, GFP–FtsAΔ15, which lacks the conserved C-terminal motif (MTS). Importantly, this MTS was sufficient to target GFP to the membrane and when added to MinDΔ10 was as effective as the MinD MTS as assessed by MinC activation and membrane localization.
Similar to results with MinD (Zhou and Lutkenhaus, 2003), we found that substitution of the large internal hydrophobic residues within FtsA's putative amphipathic helix with a negatively charged amino acid led to loss of function. GFP fusions of the substituted proteins were unable to localize at the membrane and had a cytoplasmic localization just like the C-terminally truncated FtsA. Taken together these results demonstrate that the FtsA conserved C-terminal motif forms an amphipathic helix that serves as an MTS responsible for tethering FtsA to the membrane. Consistent with this motif functioning as a membrane targeting sequence, it could be functionally replaced by the well characterized membrane targeting helices of MinD.
Binding of FtsA to the membrane is essential for its function and regulates its interaction with FtsZ
Deletion of the MTS from FtsA or mutagenesis of the large hydrophobic residues to charged residues abolished the ability of FtsA to localize to the membrane and complement the ftsA12 (Ts) mutation. In contrast, FtsA-hybrid proteins containing amphiphatic helices from the MinD proteins of E. coli or B. subtilis complemented ftsA12 (Ts) as long as these replacement helices were a functional MTS. These results strongly suggest that targeting of FtsA to the membrane by an amphiphatic helix is essential for its function but that the actual sequence or length of the helix, as long as it is sufficient to promote membrane binding, is not important.
All FtsA constructs with ability to bind to the membrane localize to Z rings and cause filamentation when overexpressed indicating that they disturbed the normal cell division process. In contrast, overexpression of GFP–FtsAΔ15, which lacks the MTS, or FtsA mutants in which hydrophobic residues within the MTS were replaced by charged residues did not localize efficiently to Z rings or cause filamentation but produced twisted cells which eventually lysed. Even when GFP–FtsAΔ15 is expressed at a high enough level to form fluorescent rods in the cytoplasm, the cells remained about wild type in length. As the presence of these rods did not significantly affect cell division, FtsZ and FtsA must not be present in these rods. Immunofluorescence experiments confirmed that FtsZ was not present in these fluorescent rods but was still present in rings at the division site (data not shown). If we assume that the truncated FtsA protein retains the FtsZ interaction domain (and we assume that the MTS does not have a direct role in the interaction with FtsZ because it can be replaced with a MinD MTS) then the lack of targeting to the membrane results in a low affinity for FtsZ. In other words, these results strongly argue that FtsA must first to be targeted to the membrane before it interacts with FtsZ.
Interestingly, FtsA truncated for the MTS forms an organized structure in the cytoplasm which appear as fluorescent rods when the protein is fused to GFP. Other bacterial actin homologues, MreB and ParM are known to polymerize (Jensen and Gerdes, 1999; van den Ent et al., 2001; Jones et al., 2001); however, FtsA is unusual because one subdomain has an altered position and topology. Although FtsA has been shown to self-interact by yeast two hybrid (Yim et al., 2000) and purify as a dimer (Feucht et al., 2001), polymerization in vitro has not been demonstrated. One possibility is that interaction of FtsA with the membrane regulates the self-interaction and that removal of the membrane targeting motif allows the protein to undergo unregulated polymerization in the cytoplasm.
Yim et al. (2000) observed that removing 5–27 residues from the carboxy end of FtsA led to loss of complementation and a failure to localize to the Z ring. They also observed, as Gayda et al. (1992) had, that overexpression of C-terminally truncated FtsA results in a curved cell phenotype. Using the yeast two hybrid system they showed that the truncated mutants self-interacted but did not interact with the full-length protein. From this they proposed that the conserved C-terminal motif regulated the FtsA self-interaction. By integrating all of these results we suggest a model in which the interaction of FtsA with the membrane promotes self-interaction and interaction with FtsZ resulting in cooperative linkage of FtsZ polymers to form the Z ring.
FtsA in evolution and tethering the Z ring to the membrane
The ftsA gene is highly conserved among cell division genes and is almost always found in tandem with ftsZ. This relationship could reflect the need to maintain a set ratio between these two proteins for cell division to take place (Dai and Lutkenhaus, 1992) and possibly reflects coevolution of these two proteins because they work together.
The direct interaction of FtsA with FtsZ has been well characterized from the FtsZ perspective but it is not clear yet what domain of FtsA is involved in this interaction (Haney et al., 2001). Also, the interaction is well conserved in evolution because it is observed between proteins from as distantly related bacterial species as E. coli and B. subtilis (Wang et al., 1997). Interestingly, subdomain IC of FtsA, the subdomain that occupies a unique position among actin-like proteins, is essential for cell division but not required for FtsA to localize to the Z ring (Rico et al., 2004).
Our findings indicate that FtsA interacts directly with the membrane through a well conserved C-terminal amphiphatic helix that acts as an MTS. This result is significant because it suggests, along with the widespread presence of FtsA in bacteria, that FtsA might be the predominant mechanism for tethering the Z ring to the membrane in bacteria. However, some bacteria have a homologue of FtsZ, but no obvious FtsA, such as mycobacteria, mycoplasma and cyanobacteria. In these bacteria another protein must perform this function. In such bacteria or eukaryotic organelles (chloroplasts) with no FtsA, other proteins able to interact with both FtsZ and the membrane have been identified. One such protein, ZipN in cyanobacteria and its homologue Arc6 in chloroplasts, appears to be essential for the cell division process of these organisms (Vitha et al., 2003; Mazouni et al., 2004). In addition, FtsZ from Mycobacterium tuberculosis interacts with FtsW, an integral membrane protein, through sequences in these proteins that appear to be unique to this genus (Datta et al., 2002). These examples might represent the evolution of the anchoring of the Z ring to the membrane to other proteins.
Possible role of FtsA in Z-ring formation
Since FtsA is targeted to the membrane by an amphipathic helix, it raises questions as to whether the binding is reversible and regulated by ATP hydrolysis similar to MinD from E. coli? FtsA binds and hydrolyses ATP (Feucht et al., 2001) and can undergo phosphorylation (Sanchez et al., 1994). Fractionation studies indicate that FtsA bound to the membrane is unphosphorylated and cannot bind ATP while the cytoplasmic form is phosphorylated and binds ATP (Sanchez et al., 1994). This would suggest that the interaction of FtsA with the membrane is somehow regulated; however, mutants deficient in phosphorylation and ATP binding appeared functional.
Our studies demonstrate that FtsA's MTS can tether GFP to the membrane and is more similar in sequence to the MinD MTS from B. subtilis than E. coli, suggesting that oligomerization is not required for membrane binding. On the other hand, the MTS from the E. coli MinD, which requires oligomerization for membrane binding (Szeto et al., 2003), can functionally substitute for FtsA MTS indicating that oligomerization at the membrane is occurring.
Our results indicate that binding of FtsA to the membrane increases the affinity of FtsA for FtsZ and possibly itself suggesting a new model for Z ring formation. A major difference between this and an earlier model (Lutkenhaus, 1993) is that Z ring formation is not controlled at the level of a nucleation site but instead depends upon a reduction in the influence of negative factors that limit FtsZ polymers from associating to form the Z ring. In the new model FtsZ is mostly associated with FtsA and ZipA at the membrane anywhere in the cell. Importantly, this membrane tethered FtsZ constantly undergoes assembly throughout the cell to form dynamic membrane-tethered polymers. Evidence for such highly dynamic FtsZ polymers occurring throughout the cell has been provided recently (Thanedar and Margolin, 2004). The preferential assembly of the Z ring at midcell is driven by the Min and Noc systems (Wu and Errington, 2004), which act to limit polymer length at the membrane away from midcell (Fig. 7). At midcell, the combined influence of these negative effectors is reduced as cells elongate. Polymers emerging from midcell but growing parallel to the long axis will encounter the negative effect of Min/Noc thus limiting their growth. However, polymers growing perpendicular to the long axis will continue to grow. The increased polymer length allows FtsZ polymers to associate laterally resulting in formation of a Z ring. The lateral association could be driven by ZipA, which can bundle FtsZ filaments in vitro (RayChaudhuri, 1999; Hale et al., 2000) and possibly by FtsA through interaction between FtsA molecules bound to different FtsZ polymers. As we have shown previously (Pichoff and Lutkenhaus, 2002), either is sufficient to support Z ring formation.
Figure 7. Model for assembly of the Z ring. In this model FtsZ polymers are linked to the membrane through FtsA or ZipA (only FtsA is depicted because either can support Z-ring assembly). These membrane-linked polymers are formed throughout the cell; however, their length is limited by the combined negative action of the Min and Noc systems (indicated by the shading). As the cell division cycle progresses the influence of Min and Noc at midcell is reduced allowing longer FtsZ polymers to form. Polymer growth parallel to the long axis of the cell is limited by Min/Noc whereas growth perpendicular to the long axis is not. Formation of the Z ring may be favoured by interaction between the longer FtsZ protofilaments as well as ZipA-induced bundling and interaction between FtsA molecules bound to different FtsZ polymers.
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