Molecular characterization of Escherichia coli FtsE and FtsX

The genes ftsE and ftsX are organized in one operon together with ftsY. FtsY codes for the receptor of the signal recognition particle (SRP) that functions in targeting a subset of inner membrane proteins. We have found no indications for a structural relationship between FtsE/X and FtsY. Evidence is presented that FtsE and FtsX form a complex in the inner membrane that bears the characteristics of an ATP-binding cassette (ABC)-type transporter. FtsE is a hydrophilic nucleotide-binding protein that has a tendency to dimerize and associates with the inner membrane through an interaction with the integral membrane protein FtsX. An FtsE null mutant showed filamentous growth and appeared viable on high salt medium only, indicating a role for FtsE in cell division and/or salt transport.     

Interaction between cell division proteins FtsE and FtsZ

FtsE and FtsX, which are widely conserved homologs of ABC transporters and interact with each other, have important but unknown functions in bacterial cell division. Coimmunoprecipitation of Escherichia coli cell extracts revealed that a functional FLAG-tagged version of FtsE, the putative ATP-binding component, interacts with FtsZ, the bacterial tubulin homolog required to assemble the cytokinetic Z ring and recruit the components of the divisome. This interaction is independent of FtsX, the predicted membrane component of the ABC transporter, which has been shown previously to interact with FtsE. The interaction also occurred independently of FtsA or ZipA, two other E. coli cell division proteins that interact with FtsZ. In addition, FtsZ copurified with FLAG-FtsE. Surprisingly, the conserved C-terminal tail of FtsZ, which interacts with other cell division proteins, such as FtsA and ZipA, was dispensable for interaction with FtsE. In support of a direct interaction with FtsZ, targeting of a green fluorescent protein (GFP)-FtsE fusion to Z rings required FtsZ, but not FtsA. Although GFP-FtsE failed to target Z rings in the absence of ZipA, its localization was restored in the presence of the ftsA* bypass suppressor, indicating that the requirement for ZipA is indirect. Coexpression of FLAG-FtsE and FtsX under certain conditions resulted in efficient formation of minicells, also consistent with an FtsE-FtsZ interaction and with the idea that FtsE and FtsX regulate the activity of the divisome.     

The Escherichia coli cell division proteins FtsY, FtsE and FtsX are inner membrane-associated

Summary The cell division genes ftsY, ftsE and ftsX form an operon mapping at 76 min on the Escherichia coli chromosome. The protein products of these genes have been indentified previously. We have studied the cellular location of the radiolabelled Fts proteins using maxicells and standard fractionation procedures. Previous protein sequence homologies suggested an inner membrane location for FtsE. We have confirmed this predicted location and have shown that FtsY and FtsX are also inner membrane-associated. These results are igreement with the hypothesis that FtsE may act at the inner membrane, in a septalsome complex, by coupling ATP hydrolysis to the process of bacterial cell division.     

Biochemical characterization of an E. coli cell division factor FtsE shows ATPase cycles similar to the NBDs of ABC-transporters

The peptidoglycan (PG) layer is an intricate and dynamic component of the bacterial cell wall, which requires a constant balance between its synthesis and hydrolysis. FtsEX complex present on the inner membrane is shown to transduce signals to induce PG hydrolysis. FtsE has sequence similarity with the nucleotide-binding domains (NBDs) of ABC transporters. The NBDs in most of the ABC transporters couple ATP hydrolysis to transport molecules inside or outside the cell. Also, this reaction cycle is driven by the dimerization of NBDs. Though extensive studies have been carried out on the Escherchia coli FtsEX complex, it remains elusive regarding how FtsEX complex helps in signal transduction or transportation of molecules. Also, very little is known about the biochemical properties and ATPase activities of FtsE. Because of its strong interaction with the membrane-bound protein FtsX, FtsE stays insoluble upon overexpression in E. coli, and thus, most studies on E. coli FtsE (FtsE_Ec) in the past have used refolded FtsE. Here in the present paper, for the first time, we report the soluble expression, purification, and biochemical characterization of FtsE from E. coli. The purified soluble FtsE exhibits high thermal stability, exhibits ATPase activity and has more than one ATP-binding site. We have also demonstrated a direct interaction between FtsE and the cytoplasmic loop of FtsX. Together, our findings suggest that during bacterial division, the ATPase cycle of FtsE and its interaction with the FtsX cytoplasmic loop may help to regulate the PG hydrolysis at the mid cell.     

Genomic, transcriptional and phenotypic analysis of ftsE and ftsX of Neisseria gonorrhoeae.

Although ftsE and ftsX are not universally present in bacteria, they are present in various Neisseria species as determined by Southern hybridization. The ftsE and ftsX genes of Neisseria gonorrhoeae (Ng) CH811 were cloned, sequenced and were shown to be co-transcribed from two promoters (P(E)1 and P(E)2) which were identified upstream of ftsE(Ng) by primer extension. Sequence analysis of FtsE(Ng) and alignment with other FtsE indicated that it contained the conserved motifs of ABC domains while sequence alignment of FtsX(Ng) with other published FtsX sequences predicted that they all contain four transmembrane segments and a conserved motif (Leu-hydrophobic aa-Gly-Ala/Gly) which may prove to be important for FtsX function. The viability of ftsE(Ng) and ftsX(Ng) mutants that were constructed by insertional inactivation indicated that these genes are not essential. The role of FtsE and FtsX is controversial. Analysis of ftsE(Ng) and ftsX(Ng) mutants by transmission electron microscopy showed that both exhibited morphological abnormalities indicative of defective division sites and in some cases aberrant condensation of DNA.     

Effect of depletion of FtsY on spore morphology and the protein composition of the spore coat layer in Bacillus subtilis

Bacillus subtilis FtsY is a homolog of the alpha-subunit of mammalian signal recognition particle (SRP) receptor, and is essential for protein translocation and vegetative cell growth. An FtsY conditional null mutant (strain ISR39) can express ftsY during the vegetative stage but not during spore formation. Spores of ISR39 have the same resistance to heat and chloroform as the wild-type, while their resistance to lysozyme is reduced. Electron microscopy showed that the outer coat of spores was incompletely assembled. The coat protein profile of the ftsY mutant spores was different from that of wild-type spores. The amounts of CotA, and CotE were reduced in spore coat proteins of ftsY mutant spores and the molecular mass of CotB was reduced. In addition, CotA, CotB, and CotE are present in normal form at T(8) of sporulation in ftsY mutant cells. These results suggest that FtsY has a pivotal role in assembling coat proteins onto the coat layer during spore morphogenesis.     

Filamentous morphology in GroE-depleted Escherichia coli induced by impaired folding of FtsE

The chaperonin GroE (GroEL and the cochaperonin GroES) is the only chaperone system that is essential for the viability of Escherichia coli. It is known that GroE-depleted cells exhibit a filamentous morphology, suggesting that GroE is required for the folding of proteins involved in cell division. Although previous studies, including proteome-wide analyses of GroE substrates, have suggested several targets of GroE in cell division, there is no direct in vivo evidence to identify which substrates exhibit obligate dependence on GroE for folding. Among the candidate substrates, we found that prior excess production of FtsE, a protein engaged in cell division, completely suppressed the filamentation of GroE-depleted E. coli. The GroE depletion led to a drastic decrease in FtsE, and the cells exhibited a known phenotype associated with impaired FtsE function. In the GroE-depleted filamentous cells, the localizations of FtsA and ZipA, both of which assemble with the FtsZ septal ring before FtsE, were normal, whereas FtsX, the interaction partner of FtsE, and FtsQ, which is recruited after FtsE, did not localize to the ring, suggesting that the decrease in FtsE is a cause of the filamentous morphology. Finally, a reconstituted cell-free translation system revealed that the folding of newly translated FtsE was stringently dependent on GroEL/GroES. Based on these findings, we concluded that FtsE is a target substrate of the GroE system in E. coli cell division.     

The Haloferax volcanii FtsY homolog is critical for haloarchaeal growth but does not require the A domain

The targeting of many Sec substrates to the membrane-associated translocation pore requires the cytoplasmic signal recognition particle (SRP). In Eukarya and Bacteria it has been shown that membrane docking of the SRP-substrate complex occurs via the universally conserved SRP receptor (Sralpha/beta and FtsY, respectively). While much has been learned about the archaeal SRP in recent years, few studies have examined archaeal Sralpha/FtsY homologs. In the present study the FtsY homolog of Haloferax volcanii was characterized in its native host. Disruption of the sole chromosomal copy of ftsY in H. volcanii was possible only under conditions where either the full-length haloarchaeal FtsY or an amino-terminally truncated version of this protein lacking the A domain, was expressed in trans. Subcellular fractionation analysis of H. volcanii ftsY deletion strains expressing either one of the complementing proteins revealed that in addition to a cytoplasmic pool, both proteins cofractionate with the haloarchaeal cytoplasmic membrane. Moreover, membrane localization of the universally conserved SRP subunit SRP54, the key binding partner of FtsY, was detected in both H. volcanii strains. These analyses suggest that the H. volcanii FtsY homolog plays a crucial role but does not require its A domain for haloarchaeal growth.     

How FtsEX localizes to the Z ring and interacts with FtsA to regulate cell division

In Escherichia coli, FtsEX, a member of the ABC transporter superfamily, is involved in regulating the assembly and activation of the divisome to couple cell wall synthesis to cell wall hydrolysis at the septum. Genetic studies indicate FtsEX acts on FtsA to begin the recruitment of the downstream division proteins but blocks septal PG synthesis until a signal is received that divisome assembly is complete. However, the details of how FtsEX localizes to the Z ring and how it interacts with FtsA are not clear. Our results show that recruitment of FtsE and FtsX is codependent and suggest that the FtsEX complex is recruited through FtsE interacting with the conserved tail of FtsZ (CCTP), thus adding FtsEX to a growing list of proteins that interacts with the CCTP of FtsZ. Furthermore, we find that the N‐terminus of FtsX is not required for FtsEX localization to the Z ring but is required for its functions in cell division indicating that it interacts with FtsA. Taken together, these results suggest that FtsEX first interacts with FtsZ to localize to the Z ring and then interacts with FtsA to promote divisome assembly and regulate septal PG synthesis.     

A predicted ABC transporter, FtsEX, is needed for cell division in Escherichia coli

FtsE and FtsX have homology to the ABC transporter superfamily of proteins and appear to be widely conserved among bacteria. Early work implicated FtsEX in cell division in Escherichia coli, but this was subsequently challenged, in part because the division defects in ftsEX mutants are often salt remedial. Strain RG60 has an ftsE::kan null mutation that is polar onto ftsX. RG60 is mildly filamentous when grown in standard Luria-Bertani medium (LB), which contains 1% NaCl, but upon shift to LB with no NaCl growth and division stop. We found that FtsN localizes to potential division sites, albeit poorly, in RG60 grown in LB with 1% NaCl. We also found that in wild-type E. coli both FtsE and FtsX localize to the division site. Localization of FtsX was studied in detail and appeared to require FtsZ, FtsA, and ZipA, but not the downstream division proteins FtsK, FtsQ, FtsL, and FtsI. Consistent with this, in media lacking salt, FtsA and ZipA localized independently of FtsEX, but the downstream proteins did not. Finally, in the absence of salt, cells depleted of FtsEX stopped dividing before any change in growth rate (mass increase) was apparent. We conclude that FtsEX participates directly in the process of cell division and is important for assembly or stability of the septal ring, especially in salt-free media.     

Role of FtsEX in cell division of Escherichia coli: viability of ftsEX mutants is dependent on functional SufI or high osmotic strength

In Escherichia coli, at least 12 proteins, FtsZ, ZipA, FtsA, FtsE/X, FtsK, FtsQ, FtsL, FtsB, FtsW, FtsI, FtsN, and AmiC, are known to localize to the septal ring in an interdependent and sequential pathway to coordinate the septum formation at the midcell. The FtsEX complex is the latest recruit of this pathway, and unlike other division proteins, it is shown to be essential only on low-salt media. In this study, it is shown that ftsEX null mutations are not only salt remedial but also osmoremedial, which suggests that FtsEX may not be involved in salt transport as previously thought. Increased coexpression of cell division proteins FtsQ-FtsA-FtsZ or FtsN alone restored the growth defects of ftsEX mutants. ftsEX deletion exacerbated the defects of most of the mutants affected in Z ring localization and septal assembly; however, the ftsZ84 allele was a weak suppressor of ftsEX. The viability of ftsEX mutants in high-osmolarity conditions was shown to be dependent on the presence of a periplasmic protein, SufI, a substrate of twin-arginine translocase. In addition, SufI in multiple copies could substitute for the functions of FtsEX. Taken together, these results suggest that FtsE and FtsX are absolutely required for the process of cell division in conditions of low osmotic strength for the stability of the septal ring assembly and that, during high-osmolarity conditions, the FtsEX and SufI functions are redundant for this essential process.     

The FtsEX ABC transporter directs cellular differentiation in Bacillus subtilis

A fundamental challenge in developmental biology is to elucidate the regulatory events that trigger cellular differentiation. Sporulation in the Gram-positive bacterium Bacillus subtilis serves as a simple experimental model system to address this challenge. The hallmark of sporulation is the formation of an asymmetrically positioned septum that divides the cell into unequally sized progeny. Here we describe the role of an ABC transporter, comprising the FtsE and FtsX proteins, in the initiation of spore formation. We discovered that in the absence of this transporter, entry into sporulation is delayed and an atypical symmetric septum is formed instead of a polar one. We show that this phenotype can be suppressed by artificially activating the master regulator of sporulation, Spo0A, or by activating the histidine kinases that function upstream of Spo0A. Our data indicate that the FtsEX transporter is one of the top components in the hierarchy of factors required to initiate sporulation, and thus it is essential for establishing proper temporal activation of the process.     

An alternative protein targeting pathway in Escherichia coli: studies on the role of FtsY.

In Escherichia coli, a signal recognition particle (SRP) has been identified which binds specifically to the signal sequence of presecretory proteins and which appears to be essential for efficient translocation of a subset of proteins. In this study we have investigated the function of E. coli FtsY which shares sequence similarity with the alpha-subunit of the eukaryotic SRP receptor ('docking protein') in the membrane of the endoplasmic reticulum. A strain was constructed which allows the conditional expression of FtsY. Depletion of FtsY is shown to cause the accumulation of the precursor form of beta-lactamase, OmpF and ribose binding protein in vivo, whereas the processing of various other presecretory proteins is unaffected. Furthermore, FtsY-depleted inverted cytoplasmic membrane vesicles are shown to be defective in the translocation of pre-beta-lactamase using an in vitro import assay. Subcellular localization studies revealed that FtsY is located in part at the cytoplasmic membrane with which it seems peripherally associated. These observations suggest that FtsY is the functional E. coli homolog of the mammalian SRP receptor.     

The structure of the chloroplast signal recognition particle (SRP) receptor reveals mechanistic details of SRP GTPase activation and a conserved membrane targeting site

Two GTPases in the signal recognition particle and its receptor (FtsY) regulate protein targeting to the membrane by formation of a heterodimeric complex. The activation of both GTPases in the complex is essential for protein translocation. We present the crystal structure of chloroplast FtsY (cpFtsY) at 1.75 A resolution. The comparison with FtsY structures in different nucleotide bound states shows structural changes relevant for GTPase activation and provides insights in how cpFtsY is pre-organized for complex formation with cpSRP54. The structure contains an amino-terminal amphipathic helix similar to the membrane targeting sequence of Escherichia coli FtsY. In cpFtsY this motif is extended, which might be responsible for the enhanced attachment of the protein to the thylakoid membrane.     

Identification of ZipA,a signal recognition particle-dependent protein from Neisseria gonorrhoeae

A genetic screen designed to identify proteins that utilize the signal recognition particle (SRP) for targeting in Escherichia coli was used to screen a Neisseria gonorrhoeae plasmid library. Six plasmids were identified in this screen, and each is predicted to encode one or more putative cytoplasmic membrane (CM) proteins. One of these, pSLO7, has three open reading frames (ORFs), two of which have no similarity to known proteins in GenBank other than sequences from the closely related N. meningitidis. Further analyses showed that one of these, SLO7ORF3, encodes a protein that is dependent on the SRP for localization. This gene also appears to be essential in N. gonorrhoeae since it was not possible to generate null mutations in the gene. Although appearing unique to Neisseria at the DNA sequence level, SLO7ORF3 was found to share some features with the cell division gene zipA of E. coli. These features included similar chromosomal locations (with respect to linked genes) as well as similarities in the predicted protein domain structures. Here, we show that SLO7ORF3 can complement an E. coli conditional zipA mutant and therefore encodes a functional ZipA homolog in N. gonorrhoeae. This observation is significant in that it is the first ZipA homolog identified in a non-rod-shaped organism. Also interesting is that this is the fourth cell division protein (the others are FtsE, FtsX, and FtsQ) shown to utilize the SRP for localization, which may in part explain why the genes encoding the three SRP components are essential in bacteria.     

Lipids trigger a conformational switch that regulates signal recognition particle (SRP)-mediated protein targeting

Co-translational protein targeting to the membrane is mediated by the signal recognition particle and its receptor (FtsY). Their homologous GTPase domains interact at the membrane and form a heterodimer in which both GTPases are activated. The prerequisite for protein targeting is the interaction of FtsY with phospholipids. However, the mechanism of FtsY regulation by phospholipids remained unclear. Here we show that the N terminus of FtsY (A domain) is natively unfolded in solution and define the complete membrane-targeting sequence. We show that the membrane-targeting sequence is highly dynamic in solution, independent of nucleotides and directly responds to the density of anionic phospholipids by a random coil-helix transition. This conformational switch is essential for tethering FtsY to membranes and activates the GTPase for its subsequent interaction with the signal recognition particle. Our results underline the dynamics of lipid-protein interactions and their importance in the regulation of protein targeting and translocation across biological membranes.     

The very early evolution of protein translocation across membranes

In this study, we used a computational approach to investigate the early evolutionary history of a system of proteins that, together, embed and translocate other proteins across cell membranes. Cell membranes comprise the basis for cellularity, which is an ancient, fundamental organizing principle shared by all organisms and a key innovation in the evolution of life on Earth. Two related requirements for cellularity are that organisms are able to both embed proteins into membranes and translocate proteins across membranes. One system that accomplishes these tasks is the signal recognition particle (SRP) system, in which the core protein components are the paralogs, FtsY and Ffh. Complementary to the SRP system is the Sec translocation channel, in which the primary channel-forming protein is SecY. We performed phylogenetic analyses that strongly supported prior inferences that FtsY, Ffh, and SecY were all present by the time of the last universal common ancestor of life, the LUCA, and that the ancestor of FtsY and Ffh existed before the LUCA. Further, we combined ancestral sequence reconstruction and protein structure and function prediction to show that the LUCA had an SRP system and Sec translocation channel that were similar to those of extant organisms. We also show that the ancestor of Ffh and FtsY that predated the LUCA was more similar to FtsY than Ffh but could still have comprised a rudimentary protein translocation system on its own. Duplication of the ancestor of FtsY and Ffh facilitated the specialization of FtsY as a membrane bound receptor and Ffh as a cytoplasmic protein that could bind nascent proteins with specific membrane-targeting signal sequences. Finally, we analyzed amino acid frequencies in our ancestral sequence reconstructions to infer that the ancestral Ffh/FtsY protein likely arose prior to or just after the completion of the canonical genetic code. Taken together, our results offer a window into the very early evolutionary history of cellularity.