scholarly journals Roles of H+-ATPase and proton motive force in ATP-dependent protein translocation in vitro.

1986 ◽  
Vol 167 (1) ◽  
pp. 389-392 ◽  
Author(s):  
L L Chen ◽  
P C Tai
Keyword(s):  
Protein Translocation ◽  
Proton Motive Force ◽  
Motive Force ◽  
Dependent Protein

scholarly journals Biochemical Characterization of a Mutationally Altered Protein Translocase: Proton Motive Force Stimulation of the Initiation Phase of Translocation

2003 ◽  
Vol 185 (2) ◽  
pp. 405-412 ◽  
Author(s):  
Hiroyuki Mori ◽  
Koreaki Ito
Keyword(s):  
Biochemical Characterization ◽  
Protein Translocation ◽  
Proton Motive Force ◽  
Motive Force ◽  
Peptide Cleavage ◽  
Initiation Phase ◽  
Membrane Complex ◽  
Absolute Dependence

ABSTRACT Protein translocation across the Escherichia coli plasma membrane is facilitated by concerted actions of the SecYEG integral membrane complex and the SecA ATPase. A secY mutation (secY39) affects Arg357, an evolutionarily conserved and functionally important residue, and impairs the translocation function in vivo and in vitro. In this study, we used the “superactive” mutant forms of SecA, which suppress the SecY39 deficiency, to characterize the mutationally altered SecY39EG translocase. It was found that SecY39-mediated preprotein translocation exhibited absolute dependence on the proton motive force. The proton motive force-dependent step proved to lie before signal peptide cleavage. We suggest that the proton motive force assists in the initiation phase of protein translocation.

scholarly journals In Vivo Synthesis of the Periplasmic Domain of TonB Inhibits Transport through the FecA and FhuA Iron Siderophore Transporters of Escherichia coli

2001 ◽  
Vol 183 (20) ◽  
pp. 5885-5895 ◽  
Author(s):  
S. Peter Howard ◽  
Christina Herrmann ◽  
Chad W. Stratilo ◽  
V. Braun
Keyword(s):  
Escherichia Coli ◽  
Cytoplasmic Membrane ◽  
Signal Sequence ◽  
Outer Membrane Proteins ◽  
Proton Motive Force ◽  
Motive Force ◽  
In Vivo Studies ◽  
Siderophore Transport

ABSTRACT The siderophore transport activities of the two outer membrane proteins FhuA and FecA of Escherichia coli require the proton motive force of the cytoplasmic membrane. The energy of the proton motive force is postulated to be transduced to the transport proteins by a protein complex that consists of the TonB, ExbB, and ExbD proteins. In the present study, TonB fragments lacking the cytoplasmic membrane anchor were exported to the periplasm by fusing them to the cleavable signal sequence of FecA. Overexpressed TonB(33-239), TonB(103-239), and TonB(122-239) fragments inhibited transport of ferrichrome by FhuA and of ferric citrate by FecA, transcriptional induction of the fecABCDE transport genes by FecA, infection by phage φ80, and killing of cells by colicin M via FhuA. Transport of ferrichrome by FhuAΔ5-160 was also inhibited by TonB(33-239), although FhuAΔ5-160 lacks the TonB box which is involved in TonB binding. The results show that TonB fragments as small as the last 118 amino acids of the protein interfere with the function of wild-type TonB, presumably by competing for binding sites at the transporters or by forming mixed dimers with TonB that are nonfunctional. In addition, the interactions that are inhibited by the TonB fragments must include more than the TonB box, since transport through corkless FhuA was also inhibited. Since the periplasmic TonB fragments cannot assume an energized conformation, these in vivo studies also agree with previous cross-linking and in vitro results, suggesting that neither recognition nor binding to loaded siderophore receptors is the energy-requiring step in the TonB-receptor interactions.

scholarly journals Additional In Vitro and In Vivo Evidence for SecA Functioning as Dimers in the Membrane: Dissociation into Monomers Is Not Essential for Protein Translocation in Escherichia coli

2007 ◽  
Vol 190 (4) ◽  
pp. 1413-1418 ◽  
Author(s):  
Hongyun Wang ◽  
Bing Na ◽  
Hsiuchin Yang ◽  
Phang C. Tai
Keyword(s):  
Escherichia Coli ◽  
Lipid Bilayers ◽  
Cytoplasmic Membrane ◽  
Protein Translocation ◽  
Secretory Proteins ◽  
Oligomeric State ◽  
Dependent Protein ◽  
Ts Mutant

ABSTRACT SecA is an essential component in the Sec-dependent protein translocation pathway and, together with ATP, provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. Previous studies established that SecA undergoes monomer-dimer equilibrium in solution. However, the oligomeric state of functional SecA during the protein translocation process is controversial. In this study, we provide additional evidence that SecA functions as a dimer in the membrane by (i) demonstration of the capability of the presumably monomeric SecA derivative to be cross-linked as dimers in vitro and in vivo, (ii) complementation of the growth of a secA(Ts) mutant with another nonfunctional SecA or (iii) in vivo complementation and in vitro function of a genetically tandem SecA dimer that does not dissociate into monomers, and (iv) formation of similar ring-like structures by the tandem SecA dimer and SecA in the presence of lipid bilayers. We conclude that SecA functions as a dimer in the membrane and dissociation into monomers is not necessary during protein translocation.

scholarly journals Dynamics of the localization of the plastid terminal oxidase inside the chloroplast

2020 ◽  
Vol 71 (9) ◽  
pp. 2661-2669 ◽  
Author(s):  
Susanne Bolte ◽  
Elodie Marcon ◽  
Mélanie Jaunario ◽  
Lucas Moyet ◽  
Maité Paternostre ◽  
...  
Keyword(s):  
Thylakoid Membrane ◽  
Fluorescent Protein ◽  
Proton Motive Force ◽  
Motive Force ◽  
Terminal Oxidase ◽  
Valve Function ◽  
Partial Dissociation ◽  
Green Fluorescent ◽  
Plastid Terminal Oxidase

Abstract The plastid terminal oxidase (PTOX) is a plastohydroquinone:oxygen oxidoreductase that shares structural similarities with alternative oxidases (AOXs). Multiple roles have been attributed to PTOX, such as involvement in carotene desaturation, a safety valve function, participation in the processes of chlororespiration, and setting the redox poise for cyclic electron transport. PTOX activity has been previously shown to depend on its localization at the thylakoid membrane. Here we investigate the dynamics of PTOX localization dependent on the proton motive force. Infiltrating illuminated leaves with uncouplers led to a partial dissociation of PTOX from the thylakoid membrane. In vitro reconstitution experiments showed that the attachment of purified recombinant maltose-binding protein (MBP)–OsPTOX to liposomes and isolated thylakoid membranes was strongest at slightly alkaline pH values in the presence of lower millimolar concentrations of KCl or MgCl2. In Arabidopsis thaliana overexpressing green fluorescent protein (GFP)–PTOX, confocal microscopy images showed that PTOX formed distinct spots in chloroplasts of dark-adapted or uncoupler-treated leaves, while the protein was more equally distributed in a network-like structure in the light. We propose a dynamic PTOX association with the thylakoid membrane depending on the presence of a proton motive force.

scholarly journals Chaperone mediated coupling of subunit availability to activation of flagellar Type III Secretion

2020 ◽  
Author(s):  
Owain J. Bryant ◽  
Betty Y-W. Chung ◽  
Gillian M. Fraser
Keyword(s):  
Cell Membrane ◽  
Electric Potential ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Proton Motive Force ◽  
Secretion System ◽  
Motive Force ◽  
Type Iii

AbstractBacterial flagellar subunits are exported across the cell membrane by the flagellar Type III Secretion System (fT3SS), powered by the proton motive force (pmf) and a specialized ATPase that enables the flagellar export gate to utilise the pmf electric potential (ΔΨ). Export gate activation is mediated by the ATPase stalk, FliJ, but how this process is regulated to prevent wasteful dissipation of pmf in the absence of subunit cargo is not known. Here, we show that FliJ activation of the export gate is regulated by flagellar export chaperones. FliJ binds unladen chaperones and, using novel chaperone variants specifically defective for FliJ binding, we show that disruption of this interaction attenuates motility and cognate subunit export. We demonstrate in vitro that chaperones and the FlhA export gate component compete for binding to FliJ, and show in vivo that unladen chaperones, which would be present in the cell when subunit levels are low, sequester FliJ to prevent activation of the export gate and attenuate subunit export. Our data indicate a mechanism whereby chaperones couple availability of subunit cargo to pmf-driven export by the fT3SS.

scholarly journals Small-Molecule Acetylation Controls the Degradation of Benzoate and Photosynthesis inRhodopseudomonas palustris

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Chelsey M. VanDrisse ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Rhodopseudomonas Palustris ◽  
Photosynthetic Apparatus ◽  
Reducing Power ◽  
Proton Motive Force ◽  
Motive Force ◽  
Light Harvesting Complexes ◽  
Pigment Synthesis ◽  
Content Type ◽  
Genes Encoding

ABSTRACTThe degradation of lignin-derived aromatic compounds such as benzoate has been extensively studied inRhodopseudomonas palustris, and the chemistry underpinning the conversion of benzoate to acetyl coenzyme A (acetyl-CoA) is well understood. Here we characterize the last unknown gene, badL,of thebad(benzoic acid degradation) cluster. BadL function is required for growth under photoheterotrophic conditions with benzoate as the organic carbon source (i.e., light plus anoxia). On the basis of bioinformatics andin vivoandin vitrodata, we show that BadL, aGcn5-relatedN-acetyltransferase (GNAT) (PF00583), acetylates aminobenzoates to yield acetamidobenzoates. The latter relieved repression of thebadDEFGABoperon by binding to BadM, triggering the synthesis of enzymes that activate and dearomatize the benzene ring. We also show that acetamidobenzoates are required for the expression of genes encoding the photosynthetic reaction center light-harvesting complexes through a BadM-independent mechanism. The effect of acetamidobenzoates on pigment synthesis is new and different than their effect on the catabolism of benzoate.IMPORTANCEThis work shows that the BadL protein ofRhodopseudomonas palustrishasN-acetyltransferase activity and that this activity is required for the catabolism of benzoate under photosynthetic conditions in this bacterium.R. palustrisoccupies lignin-rich habitats, making its benzoate-degrading capability critical for the recycling of this important, energy-rich biopolymer. This work identifies the product of the BadL enzyme as acetamidobenzoates, which were needed to derepress genes encoding benzoate-degrading enzymes and proteins of the photosynthetic apparatus responsible for the generation of the proton motive force under anoxia in the presence of light. In short, acetamidobenzoates potentially coordinate the use of benzoate as a source of reducing power and carbon with the generation of a light-driven proton motive force that fuels ATP synthesis, motility, transport, and many other processes in the metabolically versatile bacteriumR. palustris.

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