Phage‐display and correlated mutations identify an essential region of subdomain 1C involved in homodimerization of <i>Escherichia coli</i> FtsA

Carettoni, Daniele; Gómez‐Puertas, Paulino; Yim, Lucía; Mingorance, Jesús; Massidda, Orietta; Vicente, Miguel; Valencia, Alfonso; Domenici, Enrico; Anderluzzi, Daniela

doi:10.1002/prot.10244

Cited by 36 publications

(66 citation statements)

References 37 publications

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“…FtsZ is able to polymerize to form a ring at the cell center (3,38), and the C-terminal cytosolic domain of FtsZ has been shown to associate with ZipA and FtsA (24,26,32,34,44). Moreover, FtsA is able to dimerize (9,46). Characterization of the interactions involving the other Fts proteins has been limited, probably because these proteins are membrane bound and some of them are expressed at very low levels.…”

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confidence: 99%

Interaction Network amongEscherichia coliMembrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis

2005

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Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Several of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. Although these proteins appear to be recruited to the division site in a hierarchical order, the molecular interactions underlying the assembly of the cell division machinery remain mostly unspecified. In the present study, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to unravel the molecular basis of septum assembly by analyzing the protein interaction network among E. coli cell division proteins. Our results indicate that the Fts proteins are connected to one another through multiple interactions. A deletion mapping analysis carried out with two of these proteins, FtsQ and FtsI, revealed that different regions of the polypeptides are involved in their associations with their partners. Furthermore, we showed that the association between two Fts hybrid proteins could be modulated by the coexpression of a third Fts partner. Altogether, these data suggest that the cell division machinery assembly is driven by the cooperative association among the different Fts proteins to form a dynamic multiprotein structure at the septum site. In addition, our study shows that the cAMP-based two-hybrid system is particularly appropriate for analyzing molecular interactions between membrane proteins.In Escherichia coli, the cell division process, also referred to as cytokinesis, constriction, or septation, is one of the most central yet poorly understood aspects of the bacterial physiology (for reviews, see references 5, 33, and 45). The event takes place at the midcell and starts after the bacterial chromosomal DNA has been duplicated and segregated into two daughter nucleoids. Cell division genes, named fts, have been identified mainly through conditional mutants that form long filamentous cells at nonpermissive temperatures (4, 5). At present, at least fourteen proteins are known to be specifically required for the E. coli cell septation (for reviews, see references 1, 5, 33, 42, and 45). The majority of the Fts proteins are anchored to the cell membrane, and most of them appear to localize to the bacterial septum in a sequential order (for reviews, see references 5, 33, 35, and 40). Fluorescence microscopy studies using immunofluorescence or the green fluorescent protein (GFP) fused to the Fts proteins have revealed that assembly of the septum starts with the positioning of an FtsZ ring in the cell center. The FtsZ ring is stabilized by FtsA and ZipA, which localize to the septum independently of each other but only in the presence of the FtsZ protein. FtsQ follows FtsK, whose localization requires both FtsA and ZipA proteins, in this hierarchical assembly. Then FtsL, FtsB, FtsW, FtsI, FtsN, and AmiC are successively recruited to the FtsZ ring (for reviews, see refere...

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confidence: 99%

Interaction Network amongEscherichia coliMembrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis

2005

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“…It was recently proposed that subdomain 1c, in addition to the C terminus (33), is involved in dimerization of FtsA (7). If this were true, then one possible mechanism to explain the independent recruitment activity of DivIVA-1c would be that WT FtsA might actually bind to the 1c moiety on the DivIVA-1c protein, resulting in the recruitment of FtsI and FtsN by FtsA itself and not by subdomain 1c.…”

Section: Resultsmentioning

confidence: 99%

“…One such candidate domain is subdomain 1c, which, as deduced from the recent crystal structure of Thermotoga maritima FtsA (30), is conserved among other FtsAs and occupies a different spatial position within the molecule compared to other members of the ATPase superfamily, including MreB (29). Recently, subdomain 1c has been shown to be essential for FtsA function (7). In a novel adaptation of a bacterial two-hybrid assay based on polar recruitment of proteins to the cell poles by DivIVA (11), we show here that FtsA, as well as subdomain 1c, can recruit downstream cell division proteins FtsI and FtsN to the cell poles independently of the Z ring.…”

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Z-Ring-Independent Interaction between a Subdomain of FtsA and Late Septation Proteins as Revealed by a Polar Recruitment Assay

Corbin¹,

Geissler²,

Sadasivam³

et al. 2004

J Bacteriol

124

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FtsA, a member of the ATPase superfamily that includes actin and bacterial actin homologs, is essential for cell division of Escherichia coli and is recruited to the Z ring. In turn, recruitment of later essential division proteins to the Z ring is dependent on FtsA. In a polar recruitment assay, we found that FtsA can recruit at least two late proteins, FtsI and FtsN, to the cell poles independently of Z rings. Moreover, a unique structural domain of FtsA, subdomain 1c, which is divergent in the other ATPase superfamily members, is sufficient for this recruitment but not required for the ability of FtsA to localize to Z rings. Surprisingly, targeting the 1c subdomain to the Z ring by fusing it to FtsZ could partially suppress a thermosensitive ftsA mutation. These results suggest that subdomain 1c of FtsA is a completely independent functional domain with an important role in interacting with a septation protein subassembly.Cytokinesis in Escherichia coli is a complex process that relies on the intricate timing and placement of the Z ring. FtsZ assembly into the Z ring at mid-cell is proposed to provide a scaffold upon which at least 12 essential cell division proteins, including FtsA, ZipA, FtsEX, FtsK, FtsQ, FtsL, YgbQ, FtsW, FtsI, and FtsN, are recruited in a mostly linear order of dependency (6, 27). The resulting putative protein complex or divisome is required for the synthesis of the division septum and subsequent formation of new cell poles. FtsA, the second protein recruited to the Z ring, is a membrane-associated cytosolic protein (25) that is a member of a large family of ATPases that include actin, Hsp70, sugar kinases, and MreB (4). FtsA localization to mid-cell depends on prior assembly of FtsZ into the Z ring (3, 29) and is mediated via the C terminus of FtsZ (20,32).

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“…The FtsA membrane-targeting sequence (MTS) can be replaced by the MalF transmembrane domain or MinD MTS and remain functional for cell division, suggesting that the role of this sequence is limited to membrane binding (47,48). FtsA domain 1c has a role in self-interaction and the recruitment of downstream division proteins in E. coli (41,(48)(49)(50)(51)(52)(53)(54).…”

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confidence: 99%

FtsZ Ring Stability: of Bundles, Tubules, Crosslinks, and Curves

2013

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The first step in bacterial cytokinesis is the assembly of a stable but dynamic cytokinetic ring made up of the essential tubulin homolog FtsZ at the future site of division. Although FtsZ and its role in cytokinesis have been studied extensively, the precise architecture of the in vivo medial FtsZ ring (Z ring) is not well understood. Recent advances in superresolution imaging suggest that the Z ring comprises short, discontinuous, and loosely bundled FtsZ polymers, some of which are tethered to the membrane. A diverse array of regulatory proteins modulate the assembly, stability, and disassembly of the Z ring via direct interactions with FtsZ. Negative regulators of FtsZ play a critical role in ensuring the accurate positioning of FtsZ at the future site of division and in maintaining Z ring dynamics by controlling FtsZ polymer assembly/disassembly processes. Positive regulators of FtsZ are essential for tethering FtsZ polymers to the membrane and promoting the formation of stabilizing lateral interactions, permitting assembly of a mature Z ring. The past decade has seen the identification of several factors that promote FtsZ assembly, presumably through a variety of distinct molecular mechanisms. While a few of these proteins are broadly conserved, many positive regulators of FtsZ assembly are limited to small groups of closely related organisms, suggesting that FtsZ assembly is differentially modulated across bacterial species. In this review, we focus on the roles of positive regulators in Z ring assembly and in maintaining the integrity of the cytokinetic ring during the early stages of division. Bacterial cytokinesis is mediated by a macromolecular protein machine that is accurately positioned in space and time during the cell cycle. The earliest defined event during cytokinesis is the assembly of a polymeric FtsZ structure at the site of future division known as the FtsZ ring, or Z ring (1). The Z ring serves as a scaffold for the recruitment of other division proteins (2-5). FtsZ, a tubulin homolog, forms homopolymeric linear protofilaments upon binding GTP, and these filaments subsequently disassemble upon hydrolysis of the bound nucleotide (6-9). Approximately 30% of cellular FtsZ is present in the ring, and fluorescence recovery after photobleaching (FRAP) studies reveal that there is dynamic exchange between FtsZ molecules in the ring polymers and FtsZ monomers or short oligomers in the cytoplasm (10, 11).In Escherichia coli and Bacillus subtilis, cytokinesis can be separated chronologically into the following three steps: (i) assembly and stabilization of the Z ring at the future division site and, following a lag, (ii) recruitment of downstream division proteins, many of which are essential to form a constriction-competent division complex, and lastly, (iii) constriction of the Z ring coordinated with synthesis and splitting of septal peptidoglycan, leading to invagination of the cell membrane and division into two daughter cells (Fig. 1A) (2, 12, 13). The force required for constriction is likely...

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Phage‐display and correlated mutations identify an essential region of subdomain 1C involved in homodimerization of Escherichia coli FtsA

Cited by 36 publications

References 37 publications

Interaction Network amongEscherichia coliMembrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis

Interaction Network amongEscherichia coliMembrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis

Z-Ring-Independent Interaction between a Subdomain of FtsA and Late Septation Proteins as Revealed by a Polar Recruitment Assay

FtsZ Ring Stability: of Bundles, Tubules, Crosslinks, and Curves

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