When, how and why? Regulated proteolysis by the essential FtsH protease in Escherichia coli

2017 ◽  
Vol 398 (5-6) ◽  
pp. 625-635 ◽  
Author(s):  
Lisa-Marie Bittner ◽  
Jan Arends ◽  
Franz Narberhaus
Keyword(s):  
Escherichia Coli ◽  
Atpase Domain ◽  
Protease Domain ◽  
Ftsh Protease ◽  
Lipopolysaccharide Biosynthesis ◽  
General Quality ◽  
Key Enzyme ◽  
Periplasmic Chaperone ◽  
Folded Proteins ◽  
Proteolytic Chamber

AbstractCellular proteomes are dynamic and adjusted to permanently changing conditions by ATP-fueled proteolytic machineries. Among the five AAA+proteases inEscherichia coliFtsH is the only essential and membrane-anchored metalloprotease. FtsH is a homohexamer that uses its ATPase domain to unfold and translocate substrates that are subsequently degraded without the need of ATP in the proteolytic chamber of the protease domain. FtsH eliminates misfolded proteins in the context of general quality control and properly folded proteins for regulatory reasons. Recent trapping approaches have revealed a number of novel FtsH substrates. This review summarizes the substrate diversity of FtsH and presents details on the surprisingly diverse recognition principles of three well-characterized substrates: LpxC, the key enzyme of lipopolysaccharide biosynthesis; RpoH, the alternative heat-shock sigma factor and YfgM, a bifunctional membrane protein implicated in periplasmic chaperone functions and cytoplasmic stress adaptation.

scholarly journals Role of the PDZ Domains in Escherichia coli DegP Protein

2007 ◽  
Vol 189 (8) ◽  
pp. 3176-3186 ◽  
Author(s):  
Jack Iwanczyk ◽  
Daniela Damjanovic ◽  
Joel Kooistra ◽  
Vivian Leong ◽  
Ahmad Jomaa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protease Activity ◽  
Pdz Domain ◽  
Structural Features ◽  
Periplasmic Protein ◽  
Pdz Domains ◽  
Protease Domain ◽  
Interaction Domains ◽  
And Function

ABSTRACT PDZ domains are modular protein interaction domains that are present in metazoans and bacteria. These domains possess unique structural features that allow them to interact with the C-terminal residues of their ligands. The Escherichia coli essential periplasmic protein DegP contains two PDZ domains attached to the C-terminal end of the protease domain. In this study we examined the role of each PDZ domain in the protease and chaperone activities of this protein. Specifically, DegP mutants with either one or both PDZ domains deleted were generated and tested to determine their protease and chaperone activities, as well as their abilities to sequester unfolded substrates. We found that the PDZ domains in DegP have different roles; the PDZ1 domain is essential for protease activity and is responsible for recognizing and sequestering unfolded substrates through C-terminal tags, whereas the PDZ2 domain is mostly involved in maintaining the hexameric cage of DegP. Interestingly, neither of the PDZ domains was required for the chaperone activity of DegP. In addition, we found that the loops connecting the protease domain to PDZ1 and connecting PDZ1 to PDZ2 are also essential for the protease activity of the hexameric DegP protein. New insights into the roles of the PDZ domains in the structure and function of DegP are provided. These results imply that DegP recognizes substrate molecules targeted for degradation and substrate molecules targeted for refolding in different manners and suggest that the substrate recognition mechanisms may play a role in the protease-chaperone switch, dictating whether the substrate is degraded or refolded.

scholarly journals YejM Modulates Activity of the YciM/FtsH Protease Complex To Prevent Lethal Accumulation of Lipopolysaccharide

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Randi L. Guest ◽  
Daniel Samé Guerra ◽  
Maria Wissler ◽  
Jacqueline Grimm ◽  
Thomas J. Silhavy
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type ◽  
Essential Protein ◽  
Ftsh Protease ◽  
Acyl Chains ◽  
Lipid Balance

ABSTRACT Lipopolysaccharide (LPS) is an essential glycolipid present in the outer membrane (OM) of many Gram-negative bacteria. Balanced biosynthesis of LPS is critical for cell viability; too little LPS weakens the OM, while too much LPS is lethal. In Escherichia coli, this balance is maintained by the YciM/FtsH protease complex, which adjusts LPS levels by degrading the LPS biosynthesis enzyme LpxC. Here, we provide evidence that activity of the YciM/FtsH protease complex is inhibited by the essential protein YejM. Using strains in which LpxC activity is reduced, we show that yciM is epistatic to yejM, demonstrating that YejM acts upstream of YciM to prevent toxic overproduction of LPS. Previous studies have shown that this toxicity can be suppressed by deleting lpp, which codes for a highly abundant OM lipoprotein. It was assumed that deletion of lpp restores lipid balance by increasing the number of acyl chains available for glycerophospholipid biosynthesis. We show that this is not the case. Rather, our data suggest that preventing attachment of lpp to the peptidoglycan sacculus allows excess LPS to be shed in vesicles. We propose that this loss of OM material allows continued transport of LPS to the OM, thus preventing lethal accumulation of LPS within the inner membrane. Overall, our data justify the commitment of three essential inner membrane proteins to avoid toxic over- or underproduction of LPS. IMPORTANCE Gram-negative bacteria are encapsulated by an outer membrane (OM) that is impermeable to large and hydrophobic molecules. As such, these bacteria are intrinsically resistant to several clinically relevant antibiotics. To better understand how the OM is established or maintained, we sought to clarify the function of the essential protein YejM in Escherichia coli. Here, we show that YejM inhibits activity of the YciM/FtsH protease complex, which regulates synthesis of the essential OM glycolipid lipopolysaccharide (LPS). Our data suggest that disrupting proper communication between LPS synthesis and transport to the OM leads to accumulation of LPS within the inner membrane (IM). The lethality associated with this event can be suppressed by increasing OM vesiculation. Our research has identified a completely novel signaling pathway that we propose coordinates LPS synthesis and transport.

Expression of correctly folded proteins in Escherichia coli

1996 ◽  
Vol 7 (2) ◽  
pp. 190-197 ◽  
Author(s):  
George Georgiou ◽  
Pascal Valax
Keyword(s):  
Escherichia Coli ◽  
Folded Proteins

DnaK from Vibrio proteolyticus: Complementation of a dnaK-null mutant of Escherichia coli and the role of its atpase domain

2005 ◽  
Vol 99 (2) ◽  
pp. 136-142
Author(s):  
Kazuaki Yoshimune ◽  
Andrey Galkin ◽  
Ljudmila Kulakova ◽  
Tohru Yoshimura ◽  
Nobuyoshi Esaki
Keyword(s):  
Escherichia Coli ◽  
Null Mutant ◽  
Atpase Domain ◽  
Vibrio Proteolyticus

scholarly journals The role of monovalent cations in the ATPase reaction of DNA gyrase

2015 ◽  
Vol 71 (4) ◽  
pp. 996-1005 ◽  
Author(s):  
Stephen James Hearnshaw ◽  
Terence Tsz-Hong Chung ◽  
Clare Elizabeth Mary Stevenson ◽  
Anthony Maxwell ◽  
David Mark Lawson
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Dna Gyrase ◽  
Monovalent Cation ◽  
Cation Binding ◽  
Monovalent Cations ◽  
Atpase Domain ◽  
Atpase Reaction ◽  
Resolution Structure

Four new crystal structures of the ATPase domain of the GyrB subunit ofEscherichia coliDNA gyrase have been determined. One of these, solved in the presence of K+, is the highest resolution structure reported so far for this domain and, in conjunction with the three other structures, reveals new insights into the function of this domain. Evidence is provided for the existence of two monovalent cation-binding sites: site 1, which preferentially binds a K+ion that interacts directly with the α-phosphate of ATP, and site 2, which preferentially binds an Na+ion and the functional significance of which is not clear. The crystallographic data are corroborated by ATPase data, and the structures are compared with those of homologues to investigate the broader conservation of these sites.

scholarly journals Spontaneous Deletion of a 209-Kilobase-Pair Fragment from the Escherichia coli Genome Occurs with Acquisition of Resistance to an Assortment of Infectious Phages

2008 ◽  
Vol 74 (14) ◽  
pp. 4256-4263 ◽  
Author(s):  
Yasunori Tanji ◽  
Kenji Hattori ◽  
Kohichi Suzuki ◽  
Kazuhiko Miyanaga
Keyword(s):  
Escherichia Coli ◽  
Parent Strain ◽  
Protein C ◽  
Protein Production ◽  
Growth Characteristics ◽  
Phage Resistance ◽  
Continuous Cultures ◽  
A Genome ◽  
Lipopolysaccharide Biosynthesis ◽  
Pair Fragment

ABSTRACT To breed resistance to an assortment of infectious phages, continuous cultures of Escherichia coli JM109 grown in a chemostat were exposed to phage mixtures prepared from sewage influent. Four sequential chemostat-grown cultures were each infected with a different phage mixture. At the end of a chemostat run, one phage-resistant colony was isolated and used to inoculate the subsequent culture. This process was repeated, and increased phage resistance of the input bacterial strain resulted from the successive challenges with different phage cocktails. Multiple mutations apparently accumulated progressively. A mutant isolated at the end of the four runs, designated D198, showed resistance to 38 of 40 phages that infect the parent strain, JM109. D198 produced less outer membrane protein C (OmpC) than JM109. However, restoration of the OmpC protein by plasmid-mediated complementation did not completely restore the susceptibility of D198 to the 38 phages. Therefore, alterations beyond the level of OmpC protein production contribute to the phage resistance of D198. PCR-based genetic analysis revealed that D198 has a genome that is 209 kbp (about 200 genes) smaller than JM109. The deletion includes the chromosomal section from ompC to wbbL that encodes the rhamnosyl transferase involved in lipopolysaccharide biosynthesis. Strains D198 and JM109 were comparable in their growth characteristics and their abilities to express a recombinant protein.

scholarly journals Purification and properties of cystathionine β-lyase from Arabidopsis thaliana overexpressed in Escherichia coli

1996 ◽  
Vol 320 (2) ◽  
pp. 383-392 ◽  
Author(s):  
Stéphane RAVANEL ◽  
Dominique JOB ◽  
Roland DOUCE
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Biochemical Properties ◽  
Coli Strain ◽  
High Yield ◽  
Purification And Properties ◽  
Key Enzyme ◽  
Schiff Base Formation ◽  
Insight Into ◽  
Base Formation

Cystathionine β-lyase is a key enzyme in sulphur metabolism that catalyses the second reaction specific for methionine biosynthesis, the pyridoxal 5´-phosphate-dependent β-cleavage of cystathionine to produce homocysteine. To obtain insight into the biochemical properties of the plant enzyme, the cDNA encoding cystathionine β-lyase from Arabidopsis thaliana was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated at high yield (29 mg of pure protein/litre of cell culture) using an efficient two-step purification procedure. Physicochemical properties of the Arabidopsis cystathionine β-lyase were similar to those previously reported for the bacterial enzymes. In particular, the native recombinant protein is a tetramer composed of four identical subunits of 46 kDa, each being associated with one molecule of pyridoxal 5´-phosphate. Interaction between the apoenzyme and pyridoxal 5´-phosphate is extremely tight, being characterized by a Kd value of 0.5 µM. Purification and sequencing of the phosphopyridoxyl peptide established that Schiff base formation between the cofactor and the holoenzyme occurs at lysine-278. The substrate specificity of the recombinant cystathionine β-lyase resembles that of the enzyme isolated from other sources, cystathionine and djenkolate being the most effective substrates. The cystathionine analogue aminoethoxyvinylglycine irreversibly inactivates the recombinant cystathionine β-lyase. The inactivation is accompanied by dramatic modification of the spectral properties of the enzyme that can be attributed to the attack of the azomethine linkage between pyridoxal 5´-phosphate and lysine-278 of the polypeptide by aminoethoxyvinylglycine.

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