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John R Collier - One of the best experts on this subject based on the ideXlab platform.
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polylysine mediated translocation of the diphtheria toxin catalytic domain through the anthrax protective antigen pore
2014Co-Authors: Onkar Sharma, John R CollierAbstract:Anthrax toxin is a tripartite system consisting of two catalytic moieties, lethal factor (LF) and edema factor (EF),1,2 and a receptor binding/pore forming moiety, protective antigen (PA; MW = 83 kDa).3−6 These three individually nontoxic proteins combine to elicit many of the disease manifestations caused by Bacillus anthracis. After release from the bacteria, PA binds to its cellular receptors7−12 and is cleaved by cell-surface furin to a 63 kDa form (PA63).13,14 PA63 self-assembles to form a heptameric4,5,10 or octameric prepore,15 which then binds the enzymatic LF and EF moieties, yielding a series of complexes at the cell surface.15−18 These complexes are endocytosed,19,20 and exposure to acidic conditions of the endosomal compartment causes the PA prepore to undergo a conformational change to the pore state.3,21,22 The pore, inserted into the endosomal membrane, translocates the LF and EF moieties to the cytoplasm,23−25 where they modify their respective intracellular targets to the benefit of the bacterium. The anthrax toxin system has been studied extensively to learn how a proteinaceous toxin pore is able to translocate a protein across a phospholipid bilayer. Certain heterologous proteins may be potentiated to undergo PA-dependent translocation by fusion with the PA binding26 N-terminal domain of LF (LFn, residues 1–263) or the corresponding domain of EF. Thus, for example, fusing LFn to the N-terminus allows heterologous proteins and peptides (e.g., the catalytic domain of Pseudomonas exotoxin A,27 diphtheria toxin,28 ricin29 or shiga toxin,30 and the cytotoxic T lymphocyte epitope from Listeria monocytogenes(31)) to be delivered to the cytosol via PA. The ability of LFn to potentiate proteins for PA-dependent translocation stems from its ability to bind to the mouth of the PA pore and orient its disordered, highly charged leader into the lumen of the pore.32 N- to C-terminal translocation occurs in vitro in planar bilayers in the presence of a transmembrane pH gradient corresponding to that between the acidic lumen of the endosome and the neutral cytosol,33 and a charge state-dependent Brownian Ratchet Mechanism has been proposed.34 In cellular assays of PA-dependent translocation, we have often used DTA, the catalytic domain of diphtheria toxin, as a heterologous effector protein, as its delivery to the cytosol may be readily detected by measuring the inhibition of protein synthesis. Many years ago, we observed that DTA with a hexa-His tag at the N-terminus undergoes significant PA-dependent entry into cells, and we then showed that short N-terminal tracts of Lys or Arg, as well as His, also fostered translocation of DTA via the PA pore.35 Consistent with this finding, it has been reported that an N-terminal His6 tag can promote PA-dependent entry of an active domain of the C2 toxin and epidermal cell differentiation inhibitor of Staphylococcus aureus.36 In the study presented here, we fused tracts of up to 15 Lys residues to the N- or C-terminus of DTA and examined their ability to promote translocation of DTA into cells. These studies, together with experiments performed in parallel in planar bilayers, suggest that the PA pore is able to translocate appropriately tagged proteins across membranes in both N to C and C to N direction, although translocation is less efficient in the C to N direction.
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membrane translocation by anthrax toxin
2009Co-Authors: John R CollierAbstract:Much attention has been focused on anthrax toxin recently, both because of its central role in the pathogenesis of Bacillus anthracis and because it has proven to be one of the most tractable toxins for studying how enzymic moieties of intracellularly acting toxins traverse membranes. The Protective Antigen (PA) moiety of the toxin, after being proteolytically activated at the cell surface, self-associates to form a heptameric pore precursor (prepore). The prepore binds up to three molecules of Edema Factor (EF), Lethal Factor (LF), or both, forming a series of complexes that are then endocytosed. Under the influence of acidic pH within the endosome, the prepore undergoes a conformational transition to a mushroom-shaped pore, with a globular cap and 100A-long stem that spans the membrane. Electrophysiological studies in planar bilayers indicate that EF and LF translocate through the pore in unfolded form and in the N- to C-terminal direction. The pore serves as an active transporter, which translocates its proteinaceous cargo across the endosomal membrane in response to DeltapH and perhaps, to a degree, Deltapsi. A ring of seven Phe residues (Phe427) in the lumen of the pore forms a seal around the translocating polypeptide and blocks the passage of ions, presumably preserving the pH gradient. A charge state-dependent Brownian Ratchet Mechanism has been proposed to explain how the pore translocates EF and LF. This transport Mechanism of the pore may function in concert with molecular chaperonins to effect delivery of effector proteins in catalytically active form to the cytosolic compartment of host cells.
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membrane translocation by anthrax toxin
2009Co-Authors: John R CollierAbstract:Much attention has been focused on anthrax toxin recently, both because of its central role in the pathogenesis of Bacillus anthracis and because it has proven to be one of the most tractable toxins for studying how enzymic moieties of intracellularly acting toxins traverse membranes. The Protective Antigen (PA) moiety of the toxin, after being proteolytically activated at the cell surface, self-associates to form a heptameric pore precursor (prepore). The prepore binds up to three molecules of Edema Factor (EF), Lethal Factor (LF), or both, forming a series of complexes that are then endocytosed. Under the influence of acidic pH within the endosome, the prepore undergoes a conformational transition to a mushroom-shaped pore, with a globular cap and 100 A-long stem that spans the membrane. Electrophysiological studies in planar bilayers indicate that EF and LF translocate through the pore in unfolded form and in the N- to C-terminal direction. The pore serves as an active transporter, which translocates its proteinaceous cargo across the endosomal membrane in response to ΔpH and perhaps, to a degree, Δψ. A ring of seven Phe residues (Phe427) in the lumen of the pore forms a seal around the translocating polypeptide and blocks the passage of ions, presumably preserving the pH gradient. A charge state-dependent Brownian Ratchet Mechanism has been proposed to explain how the pore translocates EF and LF. This transport Mechanism of the pore may function in concert with molecular chaperonins to effect delivery of effector proteins in catalytically active form to the cytosolic compartment of host cells.
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protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient
2006Co-Authors: Bryan A Krantz, Alan Finkelstein, John R CollierAbstract:Protective antigen (PA) from anthrax toxin assembles into a homoheptamer on cell surfaces and forms complexes with the enzymatic components: lethal factor (LF) and edema factor (EF). Endocytic vesicles containing these complexes are acidified, causing the heptamer to transform into a transmembrane pore that chaperones the passage of unfolded LF and EF into the cytosol. We show in planar lipid bilayers that a physiologically relevant proton gradient (DeltapH, where the endosome is acidified relative to the cytosol) is a potent driving force for translocation of LF, EF and the LF amino-terminal domain (LFN) through the PA63 pore. DeltapH-driven translocation occurs even under a negligible membrane potential. We found that acidic endosomal conditions known to destabilize LFN correlate with an increased translocation rate. The hydrophobic heptad of lumen-facing Phe427 residues in PA (or phi clamp) drives translocation synergistically under a DeltapH. We propose that a Brownian Ratchet Mechanism proposed earlier for the phi clamp is cooperatively linked to a protonation-state, DeltapH-driven Ratchet acting trans to the phi-clamp site. In a sense, the channel functions as a proton/protein symporter.
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protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient
2006Co-Authors: Bryan A Krantz, Alan Finkelstein, John R CollierAbstract:Protective antigen (PA) from anthrax toxin assembles into a homoheptamer on cell surfaces and forms complexes with the enzymatic components: lethal factor (LF) and edema factor (EF). Endocytic vesicles containing these complexes are acidified, causing the heptamer to transform into a transmembrane pore that chaperones the passage of unfolded LF and EF into the cytosol. We show in planar lipid bilayers that a physiologically relevant proton gradient (ΔpH, where the endosome is acidified relative to the cytosol) is a potent driving force for translocation of LF, EF and the LF amino-terminal domain (LFN) through the PA63 pore. ΔpH-driven translocation occurs even under a negligible membrane potential. We found that acidic endosomal conditions known to destabilize LFN correlate with an increased translocation rate. The hydrophobic heptad of lumen-facing Phe427 residues in PA (or ϕ clamp) drives translocation synergistically under a ΔpH. We propose that a Brownian Ratchet Mechanism proposed earlier for the ϕ clamp is cooperatively linked to a protonation-state, ΔpH-driven Ratchet acting trans to the ϕ-clamp site. In a sense, the channel functions as a proton/protein symporter.
Bryan A Krantz - One of the best experts on this subject based on the ideXlab platform.
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electrostatic Ratchet in the protective antigen channel promotes anthrax toxin translocation
2012Co-Authors: Sarah L Wyniasmith, Michael J. Brown, Gina Chirichella, Gigi Kemalyan, Bryan A KrantzAbstract:Abstract Central to the power-stroke and Brownian-Ratchet Mechanisms of protein translocation is the process through which nonequilibrium fluctuations are rectified or Ratcheted by the molecular motor to transport substrate proteins along a specific axis. We investigated the Ratchet Mechanism using anthrax toxin as a model. Anthrax toxin is a tripartite toxin comprised of the protective antigen (PA) component, a homooligomeric transmembrane translocase, which translocates two other enzyme components, lethal factor (LF) and edema factor (EF), into the host cell's cytosol under the proton motive force (PMF). The PA-binding domains of LF and EF (LFN and EFN) possess identical folds and similar solution stabilities; however, EFN translocates ~10- to 200-fold slower than LFN, depending on the electrical-potential (Δψ) and chemical-potential (ΔpH) compositions of the PMF. From an analysis of LFN/EFN chimera proteins, we identified two 10-residue cassettes comprised of charged sequence that were responsible for the impaired translocation kinetics of EFN. These cassettes have nonspecific electrostatic requirements: one surprisingly prefers acidic residues when driven by either a Δψ or a ΔpH; the second requires basic residues only when driven by a Δψ. Through modeling and experiment, we identified a charged surface in the PA channel responsible for charge selectivity. The charged surface latches the substrate and promotes PMF-driven transport. We propose an electrostatic Ratchet in the channel, comprised of opposing rings of charged residues, enforces directionality by interacting with charged cassettes in the substrate, thereby generating forces sufficient to drive unfolding.
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protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient
2006Co-Authors: Bryan A Krantz, Alan Finkelstein, John R CollierAbstract:Protective antigen (PA) from anthrax toxin assembles into a homoheptamer on cell surfaces and forms complexes with the enzymatic components: lethal factor (LF) and edema factor (EF). Endocytic vesicles containing these complexes are acidified, causing the heptamer to transform into a transmembrane pore that chaperones the passage of unfolded LF and EF into the cytosol. We show in planar lipid bilayers that a physiologically relevant proton gradient (DeltapH, where the endosome is acidified relative to the cytosol) is a potent driving force for translocation of LF, EF and the LF amino-terminal domain (LFN) through the PA63 pore. DeltapH-driven translocation occurs even under a negligible membrane potential. We found that acidic endosomal conditions known to destabilize LFN correlate with an increased translocation rate. The hydrophobic heptad of lumen-facing Phe427 residues in PA (or phi clamp) drives translocation synergistically under a DeltapH. We propose that a Brownian Ratchet Mechanism proposed earlier for the phi clamp is cooperatively linked to a protonation-state, DeltapH-driven Ratchet acting trans to the phi-clamp site. In a sense, the channel functions as a proton/protein symporter.
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protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient
2006Co-Authors: Bryan A Krantz, Alan Finkelstein, John R CollierAbstract:Protective antigen (PA) from anthrax toxin assembles into a homoheptamer on cell surfaces and forms complexes with the enzymatic components: lethal factor (LF) and edema factor (EF). Endocytic vesicles containing these complexes are acidified, causing the heptamer to transform into a transmembrane pore that chaperones the passage of unfolded LF and EF into the cytosol. We show in planar lipid bilayers that a physiologically relevant proton gradient (ΔpH, where the endosome is acidified relative to the cytosol) is a potent driving force for translocation of LF, EF and the LF amino-terminal domain (LFN) through the PA63 pore. ΔpH-driven translocation occurs even under a negligible membrane potential. We found that acidic endosomal conditions known to destabilize LFN correlate with an increased translocation rate. The hydrophobic heptad of lumen-facing Phe427 residues in PA (or ϕ clamp) drives translocation synergistically under a ΔpH. We propose that a Brownian Ratchet Mechanism proposed earlier for the ϕ clamp is cooperatively linked to a protonation-state, ΔpH-driven Ratchet acting trans to the ϕ-clamp site. In a sense, the channel functions as a proton/protein symporter.
Kiyoshi Mizuuchi - One of the best experts on this subject based on the ideXlab platform.
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brownian Ratchet Mechanism for faithful segregation of low copy number plasmids
2017Co-Authors: Anthony G Vecchiarelli, Kiyoshi Mizuuchi, Keir C Neuman, Jian LiuAbstract:Bacterial plasmids are extrachromosomal DNA that provides selective advantages for bacterial survival. Plasmid partitioning can be remarkably robust. For high-copy-number plasmids, diffusion ensures that both daughter cells inherit plasmids after cell division. In contrast, most low-copy-number plasmids need to be actively partitioned by a conserved tripartite ParA-type system. ParA is an ATPase that binds to chromosomal DNA; ParB is the stimulator of the ParA ATPase and specifically binds to the plasmid at a centromere-like site, parS. ParB stimulation of the ParA ATPase releases ParA from the bacterial chromosome, after which it takes a long time to reset its DNA-binding affinity. We previously demonstrated in vitro that the ParA system can exploit this biochemical asymmetry for directed cargo transport. Multiple ParA-ParB bonds can bridge a parS-coated cargo to a DNA carpet, and they can work collectively as a Brownian Ratchet that directs persistent cargo movement with a ParA-depletion zone trailing behind. By extending this model, we suggest that a similar Brownian Ratchet Mechanism recapitulates the full range of actively segregated plasmid motilities observed in vivo. We demonstrate that plasmid motility is tuned as the replenishment rate of the ParA-depletion zone progressively increases relative to the cargo speed, evolving from diffusion to pole-to-pole oscillation, local excursions, and, finally, immobility. When the plasmid replicates, the daughters largely display motilities similar to that of their mother, except that when the single-focus progenitor is locally excursive, the daughter foci undergo directed segregation. We show that directed segregation maximizes the fidelity of plasmid partition. Given that local excursion and directed segregation are the most commonly observed modes of plasmid motility in vivo, we suggest that the operation of the ParA-type partition system has been shaped by evolution for high fidelity of plasmid segregation.
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directed and persistent movement arises from mechanochemistry of the para parb system
2015Co-Authors: Longhua Hu, Kiyoshi Mizuuchi, Anthony G Vecchiarelli, Keir C NeumanAbstract:The segregation of DNA before cell division is essential for faithful genetic inheritance. In many bacteria, segregation of low-copy number plasmids involves an active partition system composed of a nonspecific DNA-binding ATPase, ParA, and its stimulator protein ParB. The ParA/ParB system drives directed and persistent movement of DNA cargo both in vivo and in vitro. Filament-based models akin to actin/microtubule-driven motility were proposed for plasmid segregation mediated by ParA. Recent experiments challenge this view and suggest that ParA/ParB system motility is driven by a diffusion Ratchet Mechanism in which ParB-coated plasmid both creates and follows a ParA gradient on the nucleoid surface. However, the detailed Mechanism of ParA/ParB-mediated directed and persistent movement remains unknown. Here, we develop a theoretical model describing ParA/ParB-mediated motility. We show that the ParA/ParB system can work as a Brownian Ratchet, which effectively couples the ATPase-dependent cycling of ParA–nucleoid affinity to the motion of the ParB-bound cargo. Paradoxically, this resulting processive motion relies on quenching diffusive plasmid motion through a large number of transient ParA/ParB-mediated tethers to the nucleoid surface. Our work thus sheds light on an emergent phenomenon in which nonmotor proteins work collectively via mechanochemical coupling to propel cargos—an ingenious solution shaped by evolution to cope with the lack of processive motor proteins in bacteria.
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cell free study of f plasmid partition provides evidence for cargo transport by a diffusion Ratchet Mechanism
2013Co-Authors: Anthony G Vecchiarelli, Ling Chin Hwang, Kiyoshi MizuuchiAbstract:Increasingly diverse types of cargo are being found to be segregated and positioned by ParA-type ATPases. Several minimalistic systems described in bacteria are self-organizing and are known to affect the transport of plasmids, protein machineries, and chromosomal loci. One well-studied model is the F plasmid partition system, SopABC. In vivo, SopA ATPase forms dynamic patterns on the nucleoid in the presence of the ATPase stimulator, SopB, which binds to the sopC site on the plasmid, demarcating it as the cargo. To understand the relationship between nucleoid patterning and plasmid transport, we established a cell-free system to study plasmid partition reactions in a DNA-carpeted flowcell. We observed depletion zones of the partition ATPase on the DNA carpet surrounding partition complexes. The findings favor a diffusion-Ratchet model for plasmid motion whereby partition complexes create an ATPase concentration gradient and then climb up this gradient toward higher concentrations of the ATPase. Here, we report on the dynamic properties of the Sop system on a DNA-carpet substrate, which further support the proposed diffusion-Ratchet Mechanism.
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phage mu transposition immunity protein pattern formation along dna by a diffusion Ratchet Mechanism
2010Co-Authors: Yongwoon Han, Kiyoshi MizuuchiAbstract:DNA transposons integrate into host chromosomes with limited target sequence specificity. Without Mechanisms to avoid insertion into themselves, transposons risk self-destruction. Phage Mu avoids this problem by transposition immunity, involving MuA-transposase and MuB ATP-dependent DNA-binding protein. MuB-bound DNA acts as an efficient transposition target, but MuA clusters bound to Mu DNA ends activate the MuB-ATPase and dissociate MuB from their neighborhood before target site commitment, making the regions near Mu ends a poor target. This MuA-cluster-MuB interaction requires formation of DNA loops between the MuA- and the MuB-bound DNA sites. At early times, MuB clusters are disassembled via loops with smaller average size, and at later times, MuA clusters find distantly located MuB clusters by forming loops with larger average sizes. We demonstrate that iterative loop formation/disruption cycles with intervening diffusional steps result in larger DNA loops, leading to preferential insertion of the transposon at sites distant from the transposon ends.
Jian Liu - One of the best experts on this subject based on the ideXlab platform.
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treadmilling ftsz polymers drive the directional movement of spg synthesis enzymes via a brownian Ratchet Mechanism
2019Co-Authors: Joshua W Mccausland, Xinxing Yang, Zhixin Lyu, Bill Soderstrom, Jie Xiao, Jian LiuAbstract:Abstract FtsZ, a highly conserved bacterial tubulin GTPase homolog, is a central component of the cell division machinery in nearly all walled bacteria. FtsZ polymerizes at the future division site and recruits greater than 30 proteins to assemble into a macromolecular complex termed the divisome. Many of these divisome proteins are involved in septal cell wall peptidoglycan (sPG) synthesis. Recent studies found that FtsZ polymers undergo GTP hydrolysis-coupled treadmilling dynamics along the circumference the division site, driving the processive movement of sPG synthesis enzymes. How FtsZ’s treadmilling drives the directional transport of sPG enzymes and what its precise role is in bacterial cell division are unknown. Combining theoretical modeling and experimental testing, we show that FtsZ’s treadmilling drives the directional movement of sPG-synthesis enzymes via a Brownian Ratchet Mechanism, where the shrinking end of FtsZ polymers introduces an asymmetry to rectify diffusions of single sPG enzymes into persistent end-tracking movement. Furthermore, we show that the processivity of this directional movement is dependent on the binding potential between FtsZ and the enzyme, and hinges on the balance between the enzyme’s diffusion and FtsZ’s treadmilling speed. This interplay could provide a Mechanism to control the level of available enzymes for active sPG synthesis both in time and space, explaining the distinct roles of FtsZ treadmilling in modulating cell wall constriction rate observed in different bacterial species.
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protein gradients on the nucleoid position the carbon fixing organelles of cyanobacteria
2018Co-Authors: Joshua S Maccready, Anthony G Vecchiarelli, Jian Liu, Pusparanee Hakim, Eric J Young, Katherine W Osteryoung, Daniel C DucatAbstract:Carboxysomes are protein-based bacterial organelles encapsulating key enzymes of the Calvin-Benson-Bassham cycle. Previous work has implicated a ParA-like protein (hereafter McdA) as important for spatially organizing carboxysomes along the longitudinal axis of the model cyanobacterium Synechococcus elongatus PCC 7942. Yet, how self-organization of McdA emerges and contributes to carboxysome positioning is unknown. Here, we identify a small protein, termed McdB that localizes to carboxysomes and drives emergent oscillatory patterning of McdA on the nucleoid. Our results demonstrate that McdB directly stimulates McdA ATPase activity and its release from DNA, driving carboxysome-dependent depletion of McdA locally on the nucleoid and promoting directed motion of carboxysomes towards increased concentrations of McdA. We propose that McdA and McdB are a previously unknown class of self-organizing proteins that utilize a Brownian-Ratchet Mechanism to position carboxysomes in cyanobacteria, rather than a cytoskeletal system. These results have broader implications for understanding spatial organization of protein mega-complexes and organelles in bacteria.
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protein gradients on the nucleoid position the carbon fixing organelles of cyanobacteria
2018Co-Authors: Joshua S Maccready, Anthony G Vecchiarelli, Jian Liu, Pusparanee Hakim, Eric J Young, Katherine W Osteryoung, Daniel C DucatAbstract:Carboxysomes are protein-based bacterial organelles that encapsulate a key enzyme of the Calvin-Benson-Bassham cycle. Previous work has implicated a ParA-like protein (hereafter McdA) as important for spatially organizing carboxysomes along the longitudinal axis of the model cyanobacterium Synechococcus elongatus PCC 7942. Yet, how self-organization of McdA emerges and contributes to carboxysome positioning is unknown. Here, we show that a small protein, termed McdB, localizes to carboxysomes through interactions with carboxysome shell proteins to drive emergent oscillatory patterning of McdA on the nucleoid. Our results demonstrate that McdB directly interacts to stimulate McdA ATPase activity, and indicate that carboxysome-dependent McdA depletion zone formation on the nucleoid is required for directed motion of carboxysomes towards increased concentrations of McdA. We propose that McdA and McdB are a new class of self-organizing proteins that follow a Brownian-Ratchet Mechanism, challenging the cytoskeletal model of organelle transport, for equidistant positioning of carboxysomes in cyanobacteria. These results have broader implications for understanding spatial organization of protein mega-complexes and organelles in bacteria more broadly.
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brownian Ratchet Mechanism for faithful segregation of low copy number plasmids
2017Co-Authors: Anthony G Vecchiarelli, Kiyoshi Mizuuchi, Keir C Neuman, Jian LiuAbstract:Bacterial plasmids are extrachromosomal DNA that provides selective advantages for bacterial survival. Plasmid partitioning can be remarkably robust. For high-copy-number plasmids, diffusion ensures that both daughter cells inherit plasmids after cell division. In contrast, most low-copy-number plasmids need to be actively partitioned by a conserved tripartite ParA-type system. ParA is an ATPase that binds to chromosomal DNA; ParB is the stimulator of the ParA ATPase and specifically binds to the plasmid at a centromere-like site, parS. ParB stimulation of the ParA ATPase releases ParA from the bacterial chromosome, after which it takes a long time to reset its DNA-binding affinity. We previously demonstrated in vitro that the ParA system can exploit this biochemical asymmetry for directed cargo transport. Multiple ParA-ParB bonds can bridge a parS-coated cargo to a DNA carpet, and they can work collectively as a Brownian Ratchet that directs persistent cargo movement with a ParA-depletion zone trailing behind. By extending this model, we suggest that a similar Brownian Ratchet Mechanism recapitulates the full range of actively segregated plasmid motilities observed in vivo. We demonstrate that plasmid motility is tuned as the replenishment rate of the ParA-depletion zone progressively increases relative to the cargo speed, evolving from diffusion to pole-to-pole oscillation, local excursions, and, finally, immobility. When the plasmid replicates, the daughters largely display motilities similar to that of their mother, except that when the single-focus progenitor is locally excursive, the daughter foci undergo directed segregation. We show that directed segregation maximizes the fidelity of plasmid partition. Given that local excursion and directed segregation are the most commonly observed modes of plasmid motility in vivo, we suggest that the operation of the ParA-type partition system has been shaped by evolution for high fidelity of plasmid segregation.
John L Rubinstein - One of the best experts on this subject based on the ideXlab platform.
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structure and conformational states of the bovine mitochondrial atp synthase by cryo em
2015Co-Authors: Anna Zhou, Alexis Rohou, Daniel G Schep, John V Bason, Martin G Montgomery, John E Walker, Nikolaus Grigorieff, John L RubinsteinAbstract:Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic Mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian Ratchet Mechanism for proton-translocation-driven rotation in ATP synthases. DOI: http://dx.doi.org/10.7554/eLife.10180.001
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structure and conformational states of the bovine mitochondrial atp synthase by cryo em
2015Co-Authors: Anna Zhou, Alexis Rohou, Daniel G Schep, John V Bason, Martin G Montgomery, John E Walker, Nikolaus Grigorieff, John L RubinsteinAbstract:Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic Mechanism where proton translocation through the membrane-bound FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor. Here we report single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO¬ region that involves both the a and b subunits. 3D classification of images revealed seven different states of the enzyme that show different modes of bending and twisting of the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian Ratchet Mechanism for proton-translocation driven rotation in ATP synthases.
