scholarly journals An enhancer sequence in the intrinsically disordered region of the essential cell division protein FtsZ promotes conformation-guided substrate processing by ClpXP in Escherichia coli

2021 ◽  
Author(s):  
Marissa G. Viola ◽  
Theodora Myrto Perdikari ◽  
Catherine Trebino ◽  
Negar Rahmani ◽  
Kaylee L. Mathews ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Secondary Interaction ◽  
Interaction Site ◽  
Intrinsically Disordered ◽  
Degradation Reactions ◽  
Division Site ◽  
Intrinsically Disordered Region ◽  
Substrate Processing

AbstractThe essential bacterial division protein in Escherichia coli, FtsZ, assembles into the FtsZ-ring at midcell and recruits other proteins to the division site to promote septation. A region of the FtsZ amino acid sequence that links the conserved polymerization domain to a C-terminal protein interaction site was predicted to be intrinsically disordered and has been implicated in modulating spacing and architectural arrangements of FtsZ filaments. While the majority of cell division proteins that directly bind to FtsZ engage either the polymerization domain or the C-terminal interaction site, ClpX, the recognition and unfolding component of the bacterial ClpXP proteasome, has a secondary interaction with the predicted intrinsically disordered region (IDR) of FtsZ when FtsZ is polymerized. Here, we use NMR spectroscopy and reconstituted degradation reactions in vitro to demonstrate that this linker region is indeed disordered in solution and, further, that amino acids in the IDR of FtsZ enhance the degradation by conformationally-guided interactions.

scholarly journals Disassembly and degradation of MinD oscillator complexes by Escherichia coli ClpXP

2020 ◽  
Author(s):  
Christopher J. LaBreck ◽  
Catherine E. Trebino ◽  
Colby N. Ferreira ◽  
Josiah J. Morrison ◽  
Eric C. DiBiasio ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Linear Polymers ◽  
Atp Binding Site ◽  
Ftsz Ring ◽  
Degradation Reactions ◽  
A Cell ◽  
The Mind

AbstractMinD is a cell division ATPase in Escherichia coli that oscillates from pole to pole and regulates the spatial position of the cell division machinery. Together with MinC and MinE, the Min system restricts assembly of the FtsZ-ring to midcell, oscillating between the opposite ends of the cell and preventing FtsZ-ring misassembly at the poles. Here, we show that the ATP-dependent bacterial proteasome complex ClpXP degrades MinD in reconstituted degradation reactions in vitro, through direct recognition of the MinD N-terminal region, and in vivo. MinD degradation is enhanced during stationary phase, suggesting that ClpXP regulates levels of MinD in cells that are not actively dividing. MinC and MinD are known to co-assemble into linear polymers, therefore we monitored copolymers assembled in vitro after incubation with ClpXP and observed that ClpXP promotes rapid MinCD copolymer disassembly as a result of direct MinD degradation by ClpXP. The N-terminus of MinD, including residue Arg 3, which is near the ATP-binding site, is critical for degradation by ClpXP. Together, these results demonstrate that ClpXP degradation modifies conformational assemblies of MinD in vitro and depresses Min function in vivo during periods of reduced proliferation.

scholarly journals Phosphorylation in the intrinsically disordered region of F-BAR protein Imp2 regulates its contractile ring recruitment

2021 ◽  
Author(s):  
Alaina H. Willet ◽  
Maya G. Igarashi ◽  
Jun-Song Chen ◽  
Rahul Bhattacharjee ◽  
Liping Ren ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Contractile Ring ◽  
Casein Kinase 1 ◽  
Protein Levels ◽  
Intrinsically Disordered ◽  
Division Site ◽  
Protein Partners ◽  
Intrinsically Disordered Region ◽  
And Function

The F-BAR protein Imp2 is an important contributor to cytokinesis in the fission yeast, Schizosaccharomyces pombe. Because cell cycle regulated phosphorylation of the central intrinsically disordered region (IDR) of the Imp2 paralog, Cdc15, controls Cdc15 oligomerization state, localization, and ability to bind protein partners, we investigated whether Imp2 is similarly phosphoregulated. We found that Imp2 is endogenously phosphorylated on 28 sites within its IDR with the bulk of phosphorylation being constitutive. In vitro, casein kinase 1 (CK1) Hhp1 and Hhp2 can phosphorylate 17 sites and Cdk1 the remaining 11 sites. Mutations that prevent Cdk1 phosphorylation result in precocious Imp2 recruitment to the cell division site, and mutations designed to mimic these phosphorylation events delay Imp2 CR accumulation. Mutations that eliminated CK1 phosphorylation sites allowed CR sliding, and phosphomimetic substitutions at these sites reduced Imp2 protein levels and slowed CR constriction. Thus, like Cdc15, the Imp2 IDR is phosphorylated at many sites by multiple kinases. In contrast to Cdc15, for which phosphorylation plays a major cell cycle regulatory role, Imp2 phosphorylation is primarily constitutive with milder effects on localization and function.

scholarly journals Degradation of MinD oscillator complexes by Escherichia coli ClpXP

2020 ◽  
pp. jbc.RA120.013866
Author(s):  
Christopher J. LaBreck ◽  
Catherine E Trebino ◽  
Colby N Ferreira ◽  
Josiah J Morrison ◽  
Eric C DiBiasio ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Stationary Phase ◽  
Linear Polymers ◽  
Atp Binding Site ◽  
Ftsz Ring ◽  
Degradation Reactions ◽  
The Mind

MinD is a cell division ATPase in Escherichia coli that oscillates from pole to pole and regulates the spatial position of the cell division machinery. Together with MinC and MinE, the Min system restricts assembly of the FtsZ-ring to midcell, oscillating between the opposite ends of the cell and preventing FtsZ-ring misassembly at the poles. Here, we show that the ATP-dependent bacterial proteasome complex ClpXP degrades MinD in reconstituted degradation reactions in vitro and in vivo through direct recognition of the MinD N-terminal region. MinD degradation is enhanced during stationary phase, suggesting that ClpXP regulates levels of MinD in cells that are not actively dividing. ClpXP is a major regulator of growth-phase dependent proteins, and these results suggest that MinD levels are also controlled during stationary phase. In vitro, MinC and MinD are known to co-assemble into linear polymers, therefore we monitored copolymers assembled in vitro after incubation with ClpXP and observed that ClpXP promotes rapid MinCD copolymer destabilization and direct MinD degradation by ClpXP. The N-terminus of MinD, including residue Arg 3, which is near the ATP-binding site in sequence, is critical for degradation by ClpXP. Together, these results demonstrate that ClpXP degradation modifies conformational assemblies of MinD in vitro and depresses Min function in vivo during periods of reduced proliferation.

scholarly journals Analysis of MinC Reveals Two Independent Domains Involved in Interaction with MinD and FtsZ

2000 ◽  
Vol 182 (14) ◽  
pp. 3965-3971 ◽  
Author(s):  
Zonglin Hu ◽  
Joe Lutkenhaus
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Functional Domains ◽  
Wild Type ◽  
Terminal Domain ◽  
Ring Assembly ◽  
Z Ring ◽  
Ftsz Assembly

ABSTRACT In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by themin system. MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE. In this study we found that MinC consists of two functional domains connected by a short linker. When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro. The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts minfunction, resulting in a minicell phenotype. We also find that MinC is an oligomer, probably a dimer. Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization. These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD. The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly.

scholarly journals Structural Determinants Required To Target Penicillin-Binding Protein 3 to the Septum of Escherichia coli

2004 ◽  
Vol 186 (18) ◽  
pp. 6110-6117 ◽  
Author(s):  
André Piette ◽  
Claudine Fraipont ◽  
Tanneke den Blaauwen ◽  
Mirjam E. G. Aarsman ◽  
Soumya Pastoret ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Structural Determinants ◽  
Membrane Anchor ◽  
Interaction Sites ◽  
Division Site ◽  
Green Fluorescent

ABSTRACT In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septal peptidoglycan synthesis, consists of a short intracellular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-I236 noncatalytic module itself linked to a D237-V577 catalytic penicillin-binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome.

scholarly journals Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins

2002 ◽  
Vol 184 (3) ◽  
pp. 695-705 ◽  
Author(s):  
Joseph C. Chen ◽  
Michael Minev ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Random Mutagenesis ◽  
Dominant Negative ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Negative Effects ◽  
Mutant Proteins ◽  
Division Site

ABSTRACT FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

scholarly journals Contribution of the FtsQ Transmembrane Segment to Localization to the Cell Division Site

2007 ◽  
Vol 189 (20) ◽  
pp. 7273-7280 ◽  
Author(s):  
Dirk-Jan Scheffers ◽  
Carine Robichon ◽  
Gert Jan Haan ◽  
Tanneke den Blaauwen ◽  
Gregory Koningstein ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Membrane Protein ◽  
Cytoplasmic Membrane ◽  
Central Component ◽  
Transmembrane Segment ◽  
Division Site ◽  
Periplasmic Domain ◽  
Cell Division Protein

ABSTRACT The Escherichia coli cell division protein FtsQ is a central component of the divisome. FtsQ is a bitopic membrane protein with a large C-terminal periplasmic domain. In this work we investigated the role of the transmembrane segment (TMS) that anchors FtsQ in the cytoplasmic membrane. A set of TMS mutants was made and analyzed for the ability to complement an ftsQ mutant. Study of the various steps involved in FtsQ biogenesis revealed that one mutant (L29/32R;V38P) failed to functionally insert into the membrane, whereas another mutant (L29/32R) was correctly assembled and interacted with FtsB and FtsL but failed to localize efficiently to the cell division site. Our results indicate that the FtsQ TMS plays a role in FtsQ localization to the division site.

scholarly journals Characterization of YmgF, a 72-Residue Inner Membrane Protein That Associates with the Escherichia coli Cell Division Machinery

2008 ◽  
Vol 191 (1) ◽  
pp. 333-346 ◽  
Author(s):  
Gouzel Karimova ◽  
Carine Robichon ◽  
Daniel Ladant
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Dependent Manner ◽  
E Coli ◽  
Division Site ◽  
Inner Membrane Protein ◽  
Two Hybrid

ABSTRACT 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. Many of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. In the present study, we attempted to identify a novel putative component(s) of the E. coli cell division machinery by searching for proteins that could interact with known Fts proteins. To do that, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to perform a library screening in order to find putative partners of E. coli cell division protein FtsL. Here we report the characterization of YmgF, a 72-residue integral membrane protein of unknown function that was found to associate with many E. coli cell division proteins and to localize to the E. coli division septum in an FtsZ-, FtsA-, FtsQ-, and FtsN-dependent manner. Although YmgF was previously shown to be not essential for cell viability, we found that when overexpressed, YmgF was able to overcome the thermosensitive phenotype of the ftsQ1(Ts) mutation and restore its viability under low-osmolarity conditions. Our results suggest that YmgF might be a novel component of the E. coli cell division machinery.

scholarly journals Interaction Network among Escherichia coli Membrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis

2005 ◽  
Vol 187 (7) ◽  
pp. 2233-2243 ◽  
Author(s):  
Gouzel Karimova ◽  
Nathalie Dautin ◽  
Daniel Ladant
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Membrane Proteins ◽  
Hybrid System ◽  
Molecular Interactions ◽  
Interaction Network ◽  
Deletion Mapping ◽  
Division Site ◽  
Cooperative Association ◽  
Two Hybrid

ABSTRACT 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.

Simple topology: FtsK-directed recombination at the dif site

2013 ◽  
Vol 41 (2) ◽  
pp. 595-600 ◽  
Author(s):  
Ian Grainge
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Motor Domain ◽  
Site Specific ◽  
Multifunctional Protein ◽  
Supercoiled Plasmid ◽  
C Terminus ◽  
Dna Translocase

FtsK is a multifunctional protein, which, in Escherichia coli, co-ordinates the essential functions of cell division, DNA unlinking and chromosome segregation. Its C-terminus is a DNA translocase, the fastest yet characterized, which acts as a septum-localized DNA pump. FtsK's C-terminus also interacts with the XerCD site-specific recombinases which act at the dif site, located in the terminus region. The motor domain of FtsK is an active translocase in vitro, and, when incubated with XerCD and a supercoiled plasmid containing two dif sites, recombination occurs to give unlinked circular products. Despite years of research the mechanism for this novel form of topological filter remains unknown.

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