Contribution of Electric Field (Δψ) to Steady-State Transthylakoid Proton Motive Force (pmf) in Vitro and in Vivo. Control ofpmfParsing into Δψ and ΔpH by Ionic Strength†

Biochemistry ◽  
2001 ◽  
Vol 40 (5) ◽  
pp. 1226-1237 ◽  
Author(s):  
Jeffrey A. Cruz ◽  
Colette A. Sacksteder ◽  
Atsuko Kanazawa ◽  
David M. Kramer
Keyword(s):  
Steady State ◽  
Ionic Strength ◽  
Electric Field ◽  
Proton Motive Force ◽  
Motive Force

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 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 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.

Factors Controlling Electropermeabilisation of Cell Membranes

2002 ◽  
Vol 1 (5) ◽  
pp. 319-327 ◽  
Author(s):  
M. P. Rols ◽  
M. Golzio ◽  
B. Gabriel ◽  
J. Teissié
Keyword(s):  
Cell Surface ◽  
Electric Field ◽  
Pulse Duration ◽  
Cell Membranes ◽  
Physical Parameters ◽  
Local Exchange ◽  
Physical Mechanisms ◽  
A Cell

Electric field pulses are a new approach for drug and gene delivery for cancer therapy. They induce a localized structural alteration of cell membranes. The associated physical mechanisms are well explained and can be safely controlled. A position dependent modulation of the membrane potential difference is induced when an electric field is applied to a cell. Electric field pulses with an overcritical intensity evoke a local membrane alteration. A free exchange of hydrophilic low molecular weight molecules takes place across the membrane. A leakage of cytosolic metabolites and a loading of polar drugs into the cytoplasm are obtained. The fraction of the cell surface which is competent for exchange is a function of the field intensity. The level of local exchange is strongly controlled by the pulse duration and the number of successive pulses. The permeabilised state is long lived. Its lifetime is under the control of the cumulated pulse duration. Cell viability can be preserved. Gene transfer is obtained but its mechanism is not a free diffusion. Plasmids are electrophoretically accumulated against the permeabilised cell surface and form aggregates due to the field effect. After the pulses, several steps follow: translocation to the cytoplasm, traffic to the nucleus and expression. Molecular structural and metabolic changes in cells remain mostly poorly understood. Nevertheless, while most studies were established on cells in culture ( in vitro), recent experiments show that similar effects are obtained on tissue ( in vivo). Transfer remains controlled by the physical parameters of the electrical treatment.

scholarly journals Activating Signal Cointegrator 2 Belongs to a Novel Steady-State Complex That Contains a Subset of Trithorax Group Proteins

2003 ◽  
Vol 23 (1) ◽  
pp. 140-149 ◽  
Author(s):  
Young-Hwa Goo ◽  
Young Chang Sohn ◽  
Dae-Hwan Kim ◽  
Seung-Whan Kim ◽  
Min-Jung Kang ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Steady State ◽  
Nuclear Receptors ◽  
Transcriptional Activation ◽  
Retinoic Acid Receptor ◽  
Dependent Manner ◽  
Trithorax Group ◽  
Trithorax Group Proteins

ABSTRACT Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. These include activating signal cointegrator 2 (ASC-2), a recently isolated transcriptional coactivator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors and numerous other transcription factors. In this report, we show that ASC-2 belongs to a steady-state complex of approximately 2 MDa (ASC-2 complex [ASCOM]) in HeLa nuclei. ASCOM contains retinoblastoma-binding protein RBQ-3, α/β-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2. In particular, ALR-1/2 and HALR contain a highly conserved 130- to 140-amino-acid motif termed the SET domain, which was recently implicated in histone H3 lysine-specific methylation activities. Indeed, recombinant ALR-1, HALR, and immunopurified ASCOM exhibit very weak but specific H3-lysine 4 methylation activities in vitro, and transactivation by retinoic acid receptor appears to involve ligand-dependent recruitment of ASCOM and subsequent transient H3-lysine 4 methylation of the promoter region in vivo. Thus, ASCOM may represent a distinct coactivator complex of nuclear receptors. Further characterization of ASCOM will lead to a better understanding of how nuclear receptors and other transcription factors mediate transcriptional activation.

The anterior and posterior ‘stomach’ regions of larval Aedes aegypti midgut: regional specialization of ion transport and stimulation by 5-hydroxytryptamine

1999 ◽  
Vol 202 (3) ◽  
pp. 247-252 ◽  
Author(s):  
T.M. Clark ◽  
A. Koch ◽  
D.F. Moffett
Keyword(s):  
Steady State ◽  
Aedes Aegypti ◽  
Ion Transport ◽  
Malpighian Tubules ◽  
Transport Mechanisms ◽  
Regional Specialization ◽  
Negative Deflection ◽  
Electrical Polarity

The ‘stomach’ region of the larval mosquito midgut is divided into histologically distinct anterior and posterior regions. Anterior stomach perfused symmetrically with saline in vitro had an initial transepithelial potential (TEP) of −66 mV (lumen negative) that decayed within 10–15 min to a steady-state TEP near −10 mV that was maintained for at least 1 h. Lumen-positive TEPs were never observed in the anterior stomach. The initial TEP of the perfused posterior stomach was opposite in polarity, but similar in magnitude, to that of the anterior stomach, measuring +75 mV (lumen positive). This initial TEP of the posterior stomach decayed rapidly at first, then more slowly, eventually reversing the electrical polarity of the epithelium as lumen-negative TEPs were recorded in all preparations within 70 min. Nanomolar concentrations of the biogenic amine 5-hydroxytryptamine (5-HT, serotonin) stimulated both regions, causing a negative deflection of the TEP of the anterior stomach and a positive deflection of the TEP of the posterior stomach. Phorbol 12,13-diacetate also caused a negative deflection of the TEP of the anterior stomach, but had no effect on the TEP of the posterior stomach. These data demonstrate that 5-HT stimulates region-specific ion-transport mechanisms in the stomach of Aedes aegypti and suggest that 5-HT coordinates the actions of the Malpighian tubules and midgut in the maintenance of an appropriate hemolymph composition in vivo.

GABA receptors precede glutamate receptors in hypothalamic development; differential regulation by astrocytes

1995 ◽  
Vol 74 (4) ◽  
pp. 1473-1484 ◽  
Author(s):  
G. Chen ◽  
P. Q. Trombley ◽  
A. N. van den Pol
Keyword(s):  
Steady State ◽  
Amino Acid ◽  
Glutamate Receptors ◽  
Gaba Receptors ◽  
Receptor Expression ◽  
Glycine Receptors ◽  
Hypothalamic Neurons ◽  
Amino Acid Receptors

1. The developmental changes in gamma-aminobutyrate (GABA)-, glutamate-, and glycine-mediated currents in cultured embryonic neurons (n = 134) from rat hypothalamus were studied with the use of whole cell voltage-clamp recording. 2. GABA-evoked currents were detected in neurons cultured from 15-day embryos (E15) a few hours after plating. Every neuron studied from the time of plating at E15 to 2 wk later responded to GABA (30 microM). The peak and steady-state currents evoked by GABA increased by four- to fivefold within 2 wk in culture. The time constants of the desensitization of GABA currents did not change during this period. The properties of the responses to GABA were not altered by different culture densities or substrates. 3. Glycine activated receptors that were pharmacologically distinct from GABA receptors on hypothalamic neurons. The glycine responses increased by > 50-fold within 2 wk in culture. The percentage of cells responding to glycine (500 microM) was 20% at 0 days in vitro (DIV), and increased to 100% at 6 DIV. Astrocytes increased both the amplitude of glycine-mediated currents and the percentage of cells responding to glycine. 4. Glutamate-mediated currents developed later than GABA-mediated currents. The percentage of cells responding to glutamate (500 microM) increased within the 1st wk, from 20% on the day of plating to 100% after 6 DIV. Both the peak currents and the steady-state currents mediated by glutamate increased by 20-fold during the 2 wk in culture. Both the amplitude of the responses to glutamate and the percentage of cells responding to glutamate were increased by growing neurons either on an astrocyte substrate or in high-density cultures. 5. The currents and conductance changes elicited by GABA were greater than those generated by glutamate or glycine throughout the period examined. This difference was particularly evident in younger cells. After 3 days in vitro, GABA (30 microM) elicited a mean current of 1,648 pA, whereas glutamate (500 microM) only elicited a 266-pA current, and glycine (500 microM) elicited a 278-pA current from neurons growing on an astrocyte layer. 6. The expression of amino acid receptors was heterogeneous among hypothalamic neurons in younger cultures. Whereas all neurons expressed GABA receptors, some developing neurons did not express detectable glutamate receptors or glycine receptors. 7. Each of the three amino acid-evoked currents increased from E15 (1 DIV) to E20 (1 DIV), indicating an intrinsic development in the expression of the amino acid receptors in vivo. The GABA, glutamate, and glycine currents at E15, 10 DIV were similar to the currents at E20, 5 DIV (both 25 days after conception), suggesting parallel developmental patterns for amino acid receptor expression in vitro and in vivo. 8. Together, these data suggest that GABA may play a major role in early development because hypothalamic neurons are more sensitive to GABA than to either glutamate or glycine. However, glutamate and glycine receptors appear more sensitive to regulation by the local environment than GABA receptors because culture density and the astrocyte substrate have greater inductive effects on glutamate and glycine receptors than on GABA receptors.

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