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2.
PLoS One ; 14(2): e0211584, 2019.
Article in English | MEDLINE | ID: mdl-30716090

ABSTRACT

The Cpx-envelope stress system regulates the expression of virulence factors in many Gram-negative pathogens. In Salmonella enterica serovar Typhimurium deletion of the sensor kinase CpxA but not of the response regulator CpxR results in the down regulation of the key regulator for invasion, HilA encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we provide evidence that cpxA deletion interferes with dephosphorylation of CpxR resulting in increased levels of active CpxR and consequently in misregulation of target genes. 14 potential operons were identified to be under direct control of CpxR. These include the virulence determinants ecotin, the omptin PgtE, and the SPI-2 regulator SsrB. The Tat-system and the PocR regulator that together promote anaerobic respiration of tetrathionate on 1,2-propanediol are also under direct CpxR control. Notably, 1,2-propanediol represses hilA expression. Thus, our work demonstrates for the first time the involvement of the Cpx system in a complex network mediating metabolism and virulence function.


Subject(s)
Bacterial Proteins/metabolism , Protein Kinases/metabolism , Salmonella typhi/metabolism , Salmonella typhi/pathogenicity , Anaerobiosis , Gene Expression Regulation, Bacterial , Genomics , Mutation , Phosphorylation , Salmonella typhi/genetics , Virulence
3.
BMC Microbiol ; 17(1): 197, 2017 Sep 18.
Article in English | MEDLINE | ID: mdl-28923010

ABSTRACT

BACKGROUND: The aminoglycoside antibiotic gentamicin was supposed to induce a crosstalk between the Cpx- and the Arc-two-component systems (TCS). Here, we investigated the physical interaction of the respective TCS components and compared the results with their respective gene expression and protein abundance. The findings were interpreted in relation to the global proteome profile upon gentamicin treatment. RESULTS: We observed specific interaction between CpxA and ArcA upon treatment with the aminoglycoside gentamicin using Membrane-Strep-tagged protein interaction experiments (mSPINE). This interaction was neither accompanied by detectable phosphorylation of ArcA nor by activation of the Arc system via CpxA. Furthermore, no changes in absolute amounts of the Cpx- and Arc-TCS could be determined with the sensitive single reaction monitoring (SRM) in presence of gentamicin. Nevertheless, upon applying shotgun mass spectrometry analysis after treatment with gentamicin, we observed a reduction of ArcA ~ P-dependent protein synthesis and a significant Cpx-dependent alteration in the global proteome profile of E. coli. CONCLUSIONS: This study points to the importance of the Cpx-TCS within the complex regulatory network in the E. coli response to aminoglycoside-caused stress.


Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Escherichia coli Proteins/drug effects , Escherichia coli Proteins/metabolism , Escherichia coli/drug effects , Escherichia coli/metabolism , Gentamicins/metabolism , Protein Kinases/metabolism , Aminoglycosides/metabolism , Bacterial Outer Membrane Proteins/drug effects , Bacterial Proteins , Escherichia coli/genetics , Gene Expression Regulation, Bacterial/drug effects , Membrane Proteins/drug effects , Membrane Proteins/metabolism , Protein Interaction Maps , Protein Kinases/drug effects , Proteome/analysis , Repressor Proteins/drug effects , Repressor Proteins/metabolism , Stress, Physiological , Transcription Factors
4.
Microbiologyopen ; 5(4): 582-96, 2016 08.
Article in English | MEDLINE | ID: mdl-27039284

ABSTRACT

Two-component systems (TCS) play a pivotal role for bacteria in stress regulation and adaptation. However, it is not well understood how these systems are modulated to meet bacterial demands. Especially, for those TCS using an accessory protein to integrate additional signals, no data concerning the role of the accessory proteins within the coordination of the response is available. The Cpx envelope stress two-component system, composed of the sensor kinase CpxA and the response regulator CpxR, is orchestrated by the periplasmic protein CpxP which detects misfolded envelope proteins and inhibits the Cpx system in unstressed cells. Using selected reaction monitoring, we observed that the amount of CpxA and CpxR, as well as their stoichiometry, are only marginally affected, but that a 10-fold excess of CpxP over CpxA is needed to switch off the Cpx system. Moreover, the relative quantification of the proteome identified not only acid stress response as a new indirect target of the Cpx system, but also suggests a general function of the Cpx system for cell wall stability.


Subject(s)
Adaptation, Physiological/physiology , Bacterial Proteins/metabolism , Cell Wall/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Membrane Proteins/metabolism , Protein Kinases/metabolism , Stress, Physiological/physiology , Escherichia coli/genetics , Mass Spectrometry , Proteome/analysis , Proteomics/methods , Transcription Factors/metabolism
5.
PLoS One ; 11(2): e0149187, 2016.
Article in English | MEDLINE | ID: mdl-26882435

ABSTRACT

Two-component systems are the major means by which bacteria couple adaptation to environmental changes. All utilize a phosphorylation cascade from a histidine kinase to a response regulator, and some also employ an accessory protein. The system-wide signaling fidelity of two-component systems is based on preferential binding between the signaling proteins. However, information on the interaction kinetics between membrane embedded histidine kinase and its partner proteins is lacking. Here, we report the first analysis of the interactions between the full-length membrane-bound histidine kinase CpxA, which was reconstituted in nanodiscs, and its cognate response regulator CpxR and accessory protein CpxP. Using surface plasmon resonance spectroscopy in combination with interaction map analysis, the affinity of membrane-embedded CpxA for CpxR was quantified, and found to increase by tenfold in the presence of ATP, suggesting that a considerable portion of phosphorylated CpxR might be stably associated with CpxA in vivo. Using microscale thermophoresis, the affinity between CpxA in nanodiscs and CpxP was determined to be substantially lower than that between CpxA and CpxR. Taken together, the quantitative interaction data extend our understanding of the signal transduction mechanism used by two-component systems.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Nanoparticles/chemistry , Protein Kinases/metabolism , Signal Transduction , Adenosine Triphosphate/metabolism , Escherichia coli Proteins/chemistry , Histidine Kinase , Lipid Bilayers/metabolism , Nanoparticles/ultrastructure , Protein Binding , Protein Kinases/chemistry
6.
Biochemistry ; 54(23): 3670-6, 2015 Jun 16.
Article in English | MEDLINE | ID: mdl-25993101

ABSTRACT

The Cpx stress response system is induced by various environmental and cellular stimuli. It is also activated in Escherichia coli strains lacking the major phospholipid, phosphatidylethanolamine (PE). However, it is not known whether CpxA directly senses changes in the lipid bilayer or the presence of misfolded proteins due to the lack of PE in their membranes. To address this question, we used an in vitro reconstitution system and vesicles with different lipid compositions to track modulations in the activity of CpxA in different lipid bilayers. Moreover, the Cpx response was validated in vivo by monitoring expression of a PcpxP-gfp reporter in lipid-engineered strains of E. coli. Our combined data indicate that CpxA responds specifically to different lipid compositions.


Subject(s)
Bacterial Proteins/chemistry , Escherichia coli Proteins/chemistry , Lipid Bilayers/chemistry , Models, Molecular , Phosphatidylethanolamines/chemistry , Protein Kinases/chemistry , Protein Processing, Post-Translational , Signal Transduction , Acholeplasma laidlawii/enzymology , Acholeplasma laidlawii/metabolism , Arabidopsis/enzymology , Arabidopsis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cardiolipins/chemistry , Cardiolipins/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Genes, Reporter , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Lipid Bilayers/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Phosphatidylethanolamines/metabolism , Phosphatidylglycerols/chemistry , Phosphatidylglycerols/metabolism , Phosphorylation , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Kinases/metabolism , Recombinant Fusion Proteins/metabolism , Surface Properties
7.
PLoS One ; 9(9): e107383, 2014.
Article in English | MEDLINE | ID: mdl-25207645

ABSTRACT

Two-component systems, consisting of an inner membrane sensor kinase and a cytosolic response regulator, allow bacteria to respond to changes in the environment. Some two-component systems are additionally orchestrated by an accessory protein that integrates additional signals. It is assumed that spatial and temporal interaction between an accessory protein and a sensor kinase modifies the activity of a two-component system. However, for most accessory proteins located in the bacterial envelope the mechanistic details remain unclear. Here, we analyzed the interaction between the periplasmic accessory protein CpxP and the sensor kinase CpxA in Escherichia coli in dependency of three specific stimuli. The Cpx two-component system responds to envelope stress and plays a pivotal role for the quality control of multisubunit envelope structures, including type three secretion systems and pili of different pathogens. In unstressed cells, CpxP shuts off the Cpx response by a yet unknown mechanism. We show for the first time the physical interaction between CpxP and CpxA in unstressed cells using bacterial two-hybrid system and membrane-Strep-tagged protein interaction experiments. In addition, we demonstrate that a high salt concentration and the misfolded pilus subunit PapE displace CpxP from the sensor kinase CpxA in vivo. Overall, this study provides clear evidence that CpxP modulates the activity of the Cpx system by dynamic interaction with CpxA in response to specific stresses.


Subject(s)
Escherichia coli Proteins/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Membrane Proteins/genetics , Periplasm/metabolism , Protein Kinases/genetics , Signal Transduction , Bacterial Secretion Systems/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Fimbriae, Bacterial/genetics , Fimbriae, Bacterial/metabolism , Membrane Proteins/metabolism , Osmotic Pressure , Protein Binding , Protein Kinases/metabolism , Proton-Translocating ATPases/genetics , Proton-Translocating ATPases/metabolism
8.
J Bacteriol ; 196(5): 1084-93, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24375102

ABSTRACT

The DctSR two-component system of Bacillus subtilis controls the expression of the aerobic C4-dicarboxylate transporter DctA. Deletion of DctA leads to an increased dctA expression. The inactivation of DctB, an extracellular binding protein, is known to inhibit the expression of dctA. Here, interaction between the sensor kinase DctS and the transporter DctA as well as the binding protein DctB was demonstrated in vivo using streptavidin (Strep) or His protein interaction experiments (mSPINE or mHPINE), and the data suggest that DctA and DctB act as cosensors for DctS. The interaction between DctS and DctB was also confirmed by the bacterial two-hybrid system (BACTH). In contrast, no indication was obtained for a direct interaction between the transporter DctA and the binding protein DctB. Activity levels of uptake of [(14)C]succinate by bacteria that expressed DctA from a plasmid were similar in the absence and the presence of DctB, demonstrating that the binding protein DctB is not required for transport. Thus, DctB is involved not in transport but in cosensing with DctS, highlighting DctB as the first example of a TRAP-type binding protein that acts as a cosensor. The simultaneous presence of DctS/DctB and DctS/DctA sensor pairs and the lack of direct interaction between the cosensors DctA and DctB indicate the formation of a tripartite complex via DctS. It is suggested that the DctS/DctA/DctB complex forms the functional unit for C4-dicarboxylate sensing in B. subtilis.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Dicarboxylic Acids/metabolism , Membrane Proteins/metabolism , Bacillus subtilis/classification , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Dicarboxylic Acids/chemistry , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Enzymologic , Membrane Proteins/genetics , Plasmids , Protein Binding
9.
J Vis Exp ; (81): e50810, 2013 Nov 07.
Article in English | MEDLINE | ID: mdl-24300168

ABSTRACT

Membrane proteins are essential for cell viability and are therefore important therapeutic targets(1-3). Since they function in complexes(4), methods to identify and characterize their interactions are necessary(5). To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE(6). This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane- SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.


Subject(s)
Chromatography, Affinity/methods , Cross-Linking Reagents/chemistry , Immunoblotting/methods , Mass Spectrometry/methods , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Cell Membrane/chemistry , Cell Membrane/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Formaldehyde/chemistry , Membrane Proteins/isolation & purification , Protein Interaction Mapping , Protein Kinases/chemistry , Protein Kinases/metabolism , Silver Staining/methods
10.
FEMS Microbiol Lett ; 326(1): 12-22, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22092888

ABSTRACT

The Cpx-envelope stress system coordinates the expression and assembly of surface structures important for the virulence of Gram-negative pathogenic bacteria. It is comprised of the membrane-anchored sensor kinase CpxA, the cytosolic response regulator CpxR and the accessory protein CpxP. Characteristic of the group of two-component systems, the Cpx system responds to a broad range of stimuli including pH, salt, metals, lipids and misfolded proteins that cause perturbation in the envelope. Moreover, the Cpx system has been linked to inter-kingdom signalling and bacterial cell death. However, although signal specificity has been assumed, for most signals the mechanism of signal integration is not understood. Recent structural and functional studies provide the first insights into how CpxP inhibits CpxA and serves as sensor for misfolded pilus subunits, pH and salt. Here, we summarize and reflect on the current knowledge on signal integration by the Cpx-envelope stress system.


Subject(s)
Bacterial Proteins/physiology , Cell Membrane/physiology , Gram-Negative Bacteria/physiology , Membrane Proteins/physiology , Protein Kinases/physiology , Stress, Physiological , Bacterial Proteins/chemistry , Cell Membrane/chemistry , Cell Membrane/metabolism , Fimbriae, Bacterial/genetics , Fimbriae, Bacterial/metabolism , Gram-Negative Bacteria/pathogenicity , Membrane Proteins/chemistry , Protein Folding , Protein Kinases/chemistry , Signal Transduction
11.
Proteomics ; 11(10): 2124-8, 2011 May.
Article in English | MEDLINE | ID: mdl-21472855

ABSTRACT

Membrane proteins are crucial for many essential cellular processes. As membrane proteins function in complexes, methods to detect and to characterize membrane protein-protein interactions are undoubtedly required. Therefore, we developed the "Membrane-Strep-tagged protein interaction experiment" (Membrane-SPINE) that combines the specific purification of a Strep-tagged membrane protein with the reversible fixation of protein complexes by formaldehyde cross-linking. In combination with MS analysis, we suggest Membrane-SPINE as a powerful tool to identify unknown interaction partners of membrane proteins in vivo.


Subject(s)
Membrane Proteins/metabolism , Protein Interaction Mapping/methods , Proteins/analysis , Proteomics/methods , Streptavidin/metabolism , Affinity Labels , Bacterial Proteins/metabolism , Cloning, Molecular , Formaldehyde , Immunoblotting , Mass Spectrometry , Protein Binding , Proteins/chemistry , Proteins/metabolism , Salmonella typhimurium , Streptavidin/chemistry
12.
Res Microbiol ; 162(4): 405-9, 2011 May.
Article in English | MEDLINE | ID: mdl-21349329

ABSTRACT

In Gram-negative bacteria, the Cpx signal transduction pathway (CpxAR) has an overall regulatory function, but the mechanism of its activation is still poorly understood. Here we investigated the impact of the periplasmic sensor domain of CpxA on signalling by mutational analysis in vitro. Substitutions (R33C and L38P) in the N-terminal region significantly increased CpxA autophosphorylation, whereas a substitution in a predicted beta sheet (E92K), which impaired the inhibitory effect of the auxiliary CpxP protein, had no significant effect on catalytic activity. Thus, our data suggest different regions on the periplasmic domain of CpxA, which impact to modulate signal transduction.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Protein Kinases/chemistry , Protein Kinases/metabolism , Signal Transduction , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Phosphorylation , Protein Kinases/genetics , Protein Structure, Tertiary
13.
J Biol Chem ; 286(11): 9805-14, 2011 Mar 18.
Article in English | MEDLINE | ID: mdl-21239493

ABSTRACT

Bacteria are equipped with two-component systems to cope with environmental changes, and auxiliary proteins provide response to additional stimuli. The Cpx two-component system is the global modulator of cell envelope stress in gram-negative bacteria that integrates very different signals and consists of the kinase CpxA, the regulator CpxR, and the dual function auxiliary protein CpxP. CpxP both inhibits activation of CpxA and is indispensable for the quality control system of P pili that are crucial for uropathogenic Escherichia coli during kidney colonization. How these two essential biological functions of CpxP are linked is not known. Here, we report the crystal structure of CpxP at 1.45 Å resolution with two monomers being interdigitated like "left hands" forming a cap-shaped dimer. Our combined structural and functional studies suggest that CpxP inhibits the kinase CpxA through direct interaction between its concave polar surface and the negatively charged sensor domain on CpxA. Moreover, an extended hydrophobic cleft on the convex surface suggests a potent substrate recognition site for misfolded pilus subunits. Altogether, the structural details of CpxP provide a first insight how a periplasmic two-component system inhibitor blocks its cognate kinase and is released from it.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Fimbriae, Bacterial/chemistry , Membrane Proteins/chemistry , Periplasmic Proteins/chemistry , Protein Kinase Inhibitors/chemistry , Animals , DNA-Binding Proteins , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Fimbriae, Bacterial/genetics , Fimbriae, Bacterial/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Periplasmic Proteins/genetics , Periplasmic Proteins/metabolism , Protein Kinase Inhibitors/metabolism , Protein Kinases/chemistry , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Structure, Quaternary , Protein Structure, Tertiary
14.
Res Microbiol ; 160(6): 396-400, 2009.
Article in English | MEDLINE | ID: mdl-19596441

ABSTRACT

In Escherichia coli, the CpxRA sensor regulator system is induced by a variety of signals, including pH, osmolarity, metals and misfolded envelope proteins. Here, we analyzed the effect of the folding defective maltose binding protein MalE219 on the reconstituted Cpx signalling pathway in detail. Surprisingly, autokinase and phosphatase activities of the reconstituted CpxA-6His protein remained unaffected, whereas phosphotransfer to CpxR became activated by MalE219. Since stimulation occurred only in CpxA-containing proteoliposomes, our data provide the first biochemical indication of allosteric stimulation due to direct contact between MalE219 and the sensor kinase CpxA. Consequently, we suggest that this direct interaction is a new mechanism enabling the Cpx pathway to sense misfolded proteins.


Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/metabolism , Carrier Proteins/chemistry , Escherichia coli Proteins/metabolism , Protein Folding , Protein Kinases/metabolism , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Maltose-Binding Proteins , Mutation , Phosphorylation , Protein Kinases/genetics , Signal Transduction
15.
J Biol Chem ; 282(12): 8583-93, 2007 Mar 23.
Article in English | MEDLINE | ID: mdl-17259177

ABSTRACT

In Escherichia coli the Cpx sensor regulator system senses different kinds of envelope stress and responds by triggering the expression of periplasmic folding factors and proteases. It consists of the membrane-anchored sensor kinase CpxA, the response regulator CpxR, and the periplasmic protein CpxP. The Cpx pathway is induced in vivo by a variety of signals including pH variation, osmotic stress, and misfolded envelope proteins and is inhibited by overproduced CpxP. Because it is not clear how the Cpx pathway is able to recognize and correspond to so many different signals we overproduced, solubilized, purified, and incorporated the complete membrane-integral CpxA protein into proteoliposomes to analyze its biochemical properties in more detail. Autokinase and phosphotransfer activities of the reconstituted CpxA-His6 protein were stimulated by KCl. NaCl also stimulated the activities but to a lesser extent. Other osmotic active solutes as glycine betaine, sucrose, and proline had no effect. The system was further characterized by testing for susceptibility to sensor kinase inhibitors. Among these, Closantel inhibited the activities of solubilized but not of the reconstituted CpxA-His6 protein. We further analyzed the effect of CpxP on CpxA activities. Purified tagless CpxP protein reduced the phosphorylation status of CpxA to 50% but had no effect on CpxA phosphotransfer or phosphatase activities. As the in vitro system excludes the involvement of other factors our finding is the first biochemical evidence for direct protein-protein interaction between the sensor kinase CpxA and the periplasmic protein CpxP resulting in a down-regulation of the autokinase activity of CpxA.


Subject(s)
Bacterial Proteins/physiology , Escherichia coli Proteins/physiology , Escherichia coli/metabolism , Membrane Proteins/physiology , Protein Kinases/physiology , Betaine/pharmacology , Histidine Kinase , Hydrogen-Ion Concentration , Liposomes/chemistry , Osmosis , Potassium Chloride/chemistry , Proline/pharmacology , Protein Denaturation , Protein Folding , Protein Kinases/chemistry , Signal Transduction , Sodium Chloride/chemistry
16.
Mol Microbiol ; 50(5): 1579-89, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14651640

ABSTRACT

We previously characterized a defective-folding mutant of maltose-binding protein of Escherichia coli, MalE31, which formed periplasmic inclusion bodies. Here, we show that MalE31 aggregation does not affect bacterial growth at 30 degrees C but is lethal at 37 degrees C. Surprisingly, under mild heat shock conditions at 42 degrees C, inclusion bodies are degraded and bacterial growth is restored. One physiological consequence for the cells overproducing MalE31 was to induce an extracytoplasmic stress response by increasing the expression of the heat shock protease DegP via the CpxA/CpxR two-component signalling pathway. Furthermore, we show that the Cpx response is required to rescue the cells from the toxicity mediated by MalE31. Finally, expression of highly destabilized MalE variants that do not aggregate in the periplasm also induces the Cpx pathway, indicating that inclusion body formation is not necessary to activate this specific extracytoplasmic stress regulatory system.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/physiology , Heat-Shock Response , Inclusion Bodies/physiology , Periplasm/metabolism , Periplasmic Binding Proteins/metabolism , Temperature , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Periplasmic Binding Proteins/genetics , Periplasmic Proteins/genetics , Periplasmic Proteins/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Signal Transduction
17.
Res Microbiol ; 153(7): 399-404, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12405345

ABSTRACT

We previously characterized a defective-folding variant of the periplasmic maltose-binding protein, MalE31. To examine the alternative folding pathways open to the MalE31 precursor, we have analyzed the cellular fates of this aggregation-prone protein carrying altered signal sequences. Our results are most easily interpreted by a kinetic competition between exportation, folding, and degradation.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Mutation , Periplasmic Binding Proteins/chemistry , Periplasmic Binding Proteins/metabolism , Protein Folding , Biological Transport , Escherichia coli/genetics , Escherichia coli/physiology , Escherichia coli Proteins/genetics , Genetic Variation , Heat-Shock Proteins/metabolism , Heat-Shock Response , Mutagenesis, Site-Directed , Periplasmic Binding Proteins/genetics , Protein Conformation , Protein Sorting Signals/genetics , Subcellular Fractions/metabolism
18.
Biochim Biophys Acta ; 1565(1): 64-72, 2002 Sep 20.
Article in English | MEDLINE | ID: mdl-12225853

ABSTRACT

The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK(2)) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK(2) complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a V(max) of 1.25 micromol P(i)/min/mg and a K(m) of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIA(Glc), a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes.


Subject(s)
ATP-Binding Cassette Transporters/biosynthesis , Escherichia coli Proteins , Maltose/metabolism , Monosaccharide Transport Proteins/biosynthesis , Salmonella typhimurium/metabolism , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/isolation & purification , ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Escherichia coli/metabolism , Genetic Vectors , Monosaccharide Transport Proteins/chemistry , Monosaccharide Transport Proteins/isolation & purification , Proteolipids , Salmonella typhimurium/chemistry , Salmonella typhimurium/genetics , Urea , Vanadates/pharmacology
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