Polypeptide binding of Escherichia coli FtsH (HflB)

The Escherichia coli FtsH protein is a membrane-bound and ATP-dependent protease. In this study, we describe ATP-dependent conformational changes in FtsH as well as a polypeptide binding ability of this protein. A 33 kDa segment of FtsH became trypsin resistant in the presence of ATP. ATP and ATPγS prevented self-aggregation of detergent-solubilized FtsH-His₆-Myc at 37°C, again suggesting that the binding of ATP induces a conformational change in FtsH. Affinity chromatography showed that FtsH-His₆-Myc can associate with denatured alkaline phosphatase (PhoA) but not with the native enzyme. Denatured PhoA also prevented the aggregation of FtsH, and these two proteins co-sedimented through a sucrose gradient. Binding between FtsH-His₆-Myc and detergent-solubilized SecY was also demonstrated. Although FtsH-bound SecY was processed further for ATP-dependent proteolysis, FtsH-bound PhoA was not. Thus, FtsH association with denatured PhoA is uncoupled from proteolysis. Overproduction of FtsH significantly increased the cytoplasmic localization of the PhoA moiety of a MalF–PhoA hybrid protein, in which a charged residue had been introduced into a transmembrane segment. Thus, denatured PhoA binding of FtsH may also occur in vivo .     

Direct CIII-HflB interaction is responsible for the inhibition of the HflB (FtsH)-mediated proteolysis of Escherichia coli sigma(32) by lambdaCIII

The CIII protein of bacteriophage lambda exhibits antiproteolytic activity against the ubiquitous metalloprotease HflB (FtsH) of Escherichia coli, thereby stabilizing the lambdaCII protein and promoting lysogenic development of the phage. CIII also protects E.coli sigma(32), another substrate of HflB. We have recently shown that the protection of CII from HflB by CIII involves direct CIII-HflB binding, without any interaction between CII and CIII [HalderS, DattaAB & Parrack P (2007) J Bacteriol189, 8130-8138]. Such a mode of action for lambdaCIII would be independent of the HflB substrate. In this study, we tested the ability of CIII to protect sigma(32) from HflB digestion. The inhibition of HflB-mediated proteolysis of sigma(32) by CIII is very similar to that of lambdaCII, characterized by an enhanced protection by the core CIII peptide CIIIC (amino acids 14-41 of lambdaCIII) and a lack of interaction between sigma(32) and CIII.     

Escherichia coli FtsH (HflB) degrades a membrane-associated TolAI-II-beta-lactamase fusion protein under highly denaturing conditions

TolAI--II--beta-lactamase, a fusion protein consisting of the inner membrane and transperiplasmic domains of TolA followed by TEM--beta-lactamase associated with the inner membrane but remained confined to the cytoplasm when expressed at high level in Escherichia coli. Although the fusion protein was resistant to proteolysis in vivo, it was hydrolyzed during preparative SDS-polyacrylamide electrophoresis and when insoluble cellular fractions unfolded with 5 M urea were subjected to microdialysis. Inhibitor profiling studies revealed that both a metallo- and serine protease were involved in TolAI--II--beta-lactamase degradation under denaturing conditions. The in vitro degradation rates of the fusion protein were not affected when insoluble fractions were harvested from a strain lacking protease IV, but were significantly reduced when microdialysis experiments were conducted with material isolated from an isogenic ftsH1 mutant. Adenine nucleotides were not required for degradation, and ATP supplementation did not accelerate the apparent rate of TolAI--II--beta-lactamase hydrolysis under denaturing conditions. Our results indicate that the metalloprotease active site of FtsH remains functional in the presence of 3--5 M urea and suggest that the ATPase and proteolytic activities of FtsH can be uncoupled if the substrate is sufficiently unstructured. Thus, a key role of the FtsH AAA module appears to be the net unfolding of bound substrates so that they can be efficiently engaged by the protease active site.     

Characterization of a conserved alpha-helical, coiled-coil motif at the C-terminal domain of the ATP-dependent FtsH (HflB) protease of Escherichia coli

FtsH (HflB) is an ATP-dependent protease found in prokaryotic cells, mitochondria and chloroplasts. Here, we have identified, in the carboxy-terminal region of FtsH (HfIB), a short alpha helix predicted of forming a coiled-coil, leucine zipper, structure. This region appears to be structurally conserved. The presence of the coiled-coil motif in the Escherichia coli FtsH (HflB) was demonstrated by circular dichroism and cross-linking experiments. Mutational analysis showed that three highly conserved leucine residues are essential for FtsH (HfIB) activity in vivo and in vitro. Purified proteins mutated in the conserved leucine residues, were found to be defective in the degradation of E. coli sigma(32) and the bacteriophage lambda CII proteins. In addition, the mutant proteins were defective in the binding of CII The mutations did not interfere with the ATPase activity of FtsH (HflB). Finally, the mutant proteins were found to be more sensitive to trypsin degradation than the wild-type enzyme suggesting that the alpha helical region is an important structural element of FtsH (HflB).     

A protease complex in the Escherichia coli plasma membrane: HflKC (HflA) forms a complex with FtsH (HflB), regulating its proteolytic activity against SecY.

Escherichia coli FtsH (HflB), a membrane-bound ATPase is required for proteolytic degradation of uncomplexed forms of the protein translocase SecY subunit. We have now isolated SecY-stabilizing mutations that cause an amino acid substitution in the HflK-HflC membrane protein complex. Although HflKC protein was believed to have a proteolytic activity against lambda cII protein, deletion of hflK-hflC did not stabilize SecY. Instead, the mutant alleles were partially dominant and overexpression of ftsH suppressed the mutational effects, suggesting that the mutant proteins antagonized the degradation of SecY. These results raise the possibility that even the wild-type HflKC protein acts to antagonize FtsH. Consistent with this notion, the hflkC null mutation accelerated degradation of the SecY24 protein. Furthermore cross-linking, co-immunoprecipitation, histidine-tagging and gel filtration experiments all indicated that FtsH and HflKC form a complex in vivo and in vitro. Finally, purified HflKC protein inhibited the SecY-degrading activity of purified FtsH protein in vitro. These results indicate that the proteolytic activity of FtsH is modulated negatively by its association with HflKC.     

Escherichia coli requires the protease activity of FtsH for growth

FtsH protease, the product of the essential ftsH gene, is a membrane-bound ATP-dependent metalloprotease of Escherichia coli that has been shown to be involved in the rapid turnover of key proteins, secretion of proteins into and through the membrane, and mRNA decay. The pleiotropic effects of ftsH mutants have led to the suggestion that FtsH possesses an ATP-dependent chaperone function that is independent of its protease function. When considering FtsH as a target for novel antibacterials, it is necessary to determine which of these functions is critical for the growth and survival of bacteria. To address this, we constructed the FtsH mutants E418Q, which retains significant ATPaseactivity but lacks protease activity, and K201N, which lacks both protease and ATPase activities. These mutants were introduced into an E. coli ftsH knockout strain which has wild-type FtsH supplied from a plasmid under control of the inducible araBAD promoter. Since neither mutant would complement the ftsH defect produced in the absence of arabinose, we conclude that the protease function of FtsH is required for bacterial growth.     

A colicin-tolerant Escherichia coli mutant that confers hfl phenotype carries two mutations in the region coding for the C-terminal domain of FtsH (HflB)

An Escherichia coli mutant, ER437, which was originally isolated for colicin tolerance, was found to carry two amino acid changes in the C-terminal portion of FtsH (HflB). These mutations were demonstrated to reduce the ability of FtsH to degrade the phage lambda CII protein in vivo and in vitro, providing a rationalization for the mutant Hfl phenotype.     

The HflB protease of Escherichia coli degrades its inhibitor lambda cIII.

The cIII protein of bacteriophage lambda is known to protect two regulatory proteins from degradation by the essential Escherichia coli protease HflB (also known as FtsH), viz., the lambda cII protein and the host heat shock sigma factor sigma32. lambda cIII, itself an unstable protein, is partially stabilized when the HflB concentration is decreased, and its half-life is decreased when HflB is overproduced, strongly suggesting that it is degraded by HflB in vivo. The in vivo degradation of lambda cIII (unlike that of sigma32) does not require the molecular chaperone DnaK. Furthermore, the half-life of lambda cIII is not affected by depletion of the endogenous ATP pool, suggesting that lambda cIII degradation is ATP independent (unlike that of lambda cII and sigma32). The lambda cIII protein, which is predicted to contain a 22-amino-acid amphipathic helix, is associated with the membrane, and nonlethal overproduction of lambda cIII makes cells hypersensitive to the detergent sodium dodecyl sulfate. This could reflect a direct lambda cIII-membrane interaction or an indirect association via the membrane-bound HflB protein, which is known to be involved in the assembly of certain periplasmic and outer membrane proteins.     

Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli

The suppressor mutation, named sfhC21, that allows Escherichia coli ftsH null mutant cells to survive was found to be an allele of fabZ encoding R-3-hydroxyacyl-ACP dehydrase, involved in a key step of fatty acid biosynthesis, and appears to upregulate the dehydrase. The ftsH1(Ts) mutation increased the amount of lipopolysaccharide at 42 degrees C. This was accompanied by a dramatic increase in the amount of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase [the IpxC (envA) gene product] involved in the committed step of lipid A biosynthesis. Pulse-chase experiments and in vitro assays with purified components showed that FtsH, the AAA-type membrane-bound metalloprotease, degrades the deacetylase. Genetic evidence also indicated that the FtsH protease activity for the deacetylase might be affected when acyl-ACP pools were altered. The biosynthesis of phospholipids and the lipid A moiety of lipopolysaccharide, both of which derive their fatty acyl chains from the same R-3-hydroxyacyl-ACP pool, is regulated by FtsH.     

Roles of multimerization and membrane association in the proteolytic functions of FtsH (HflB)

FtsH (HflB) is an Escherichia coli ATP-dependent protease that degrades some integral membrane and cytoplasmic proteins. While anchored to the cytoplasmic membrane by the two transmembrane (TM) segments near the N-terminus, it has a large cytoplasmic domain. The N-terminal region also has a role in homo-oligomerization of this protein. To study the significance of the membrane integration and oligomer formation, we constructed FtsH derivatives in which the N-terminal region had been deleted or replaced with either the leucine zipper sequence from Saccharomyces cerevisiae GCN4 protein or TM regions from other membrane proteins. The cytoplasmic domain, which was monomeric and virtually inactive, was converted, by the attachment of the leucine zipper, to an oligomer with proteolytic function against a soluble, but not a membrane-bound substrate. In contrast, chimeric TM-FtsH proteins were active against both substrate classes. We suggest that the cytoplasmic domain has intrinsic but weak self-interaction ability, which becomes effective with the aid of the leucine zipper or membrane tethering, and that membrane association is essential for FtsH to degrade integral membrane proteins.     

Identification of glutamic acid 479 as the gluzincin coordinator of zinc in FtsH (HflB)

Escherichia coli FtsH (HflB) is a membrane-bound and ATP-dependent metalloprotease. Its cytoplasmic domain contains a zinc-binding motif, H(417)EXXH, whose histidine residues have been shown to be functionally important. Although they are believed to be involved directly in zinc coordination, nothing is known about the third zinc ligand of this protease. Sequence alignment indicates that glutamic acid residues are conserved among the FtsH homologues at positions corresponding to Glu(479) and Glu(585) of E. coli FtsH. We replaced each of them by Gln, Asp, Lys, or Val. Mutations at position 479 compromised the proteolytic functions of FtsH in vivo. In vitro proteolytic activities of the E479Q, E479V, and E479D mutant enzymes were much lower than that of the wild-type protein and were significantly stimulated by a high concentration of zinc ion. These mutant proteins retained the wild-type levels of ATPase activities, and their trypsin susceptibilities as well as CD spectra were essentially indistinguishable from those of the wild-type protein, indicating that the mutations did not cause gross conformational changes in FtsH. They exhibited reduced zinc contents upon purification. From these results, we conclude that Glu(479) is a zinc-coordinating residue.     

Probing the adaptive response of Escherichia coli to extracellular Zn(II)

The adaptive response of Escherichia coli cells to differing intracellular and extracellular Zn(II) concentrations was evaluated by two-dimensional gel electrophoresis and peptide identifications. Twenty-one Zn(II)-responsive proteins, which were previously not known to be associated with Zn(II), were identified. Most of the proteins were related to cellular metabolism and include membrane transporters and glycolytic and TCA-associated enzymes. The expression levels of no known Zn(II) transporters were identified with these studies. The results of these studies suggest a role of Zn(II) in the expression levels of several E. coli proteins, and the results are discussed in light of recent genomic profiling studies on the adaptive response of E. coli cells to stress by Zn(II) excess.     

FtsH exists as an exceptionally large complex containing HflKC in the plasma membrane of Escherichia coli

FtsH is an ATP-dependent and membrane-associated protease, which exerts processive proteolysis against membrane-embedded and soluble substrate proteins. Although previous studies suggested that it functions as a homo-oligomer and it also interacts with HflK-HflC membrane protein complex (HflKC), it is still important to address the question of what kind of supramolecular assembly FtsH forms in wild-type cells. Now we show that FtsH in wild-type Escherichia coli cells exists exclusively as a large complex, termed FtsH holo-enzyme, which can be separated from bulk of membrane proteins after detergent solubilization and velocity sedimentation. This complex appears to have molecular mass of around 1000 kDa. A tentative model is presented that it is composed of hexamers of FtsH and of HflKC, with an ability to bind one or a few substrate molecules.     

Probing the conformational change of Escherichia coli undecaprenyl pyrophosphate synthase during catalysis using an inhibitor and tryptophan mutants.

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation reactions of eight isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C(55) undecaprenyl pyrophosphate (UPP). In the present study, site-directed mutagenesis, fluorescence quenching, and stopped-flow methods were utilized to examine the substrate binding and the protein conformational change. (S)-Farnesyl thiopyrophosphate (FsPP), a FPP analogue, was synthesized to probe the enzyme inhibition and events associated with the protein fluorescence change. This compound with a much less labile thiopyrophosphate shows K(i) value of 0.2 microm in the inhibition of Escherichia coli UPPS and serves as a poor substrate, with the k(cat) value (3.1 x 10(-7) s(-1)) 10(7) times smaller than using FPP as the substrate. Reduction of protein intrinsic fluorescence was observed upon addition of FPP (or FsPP) to the UPPS solution. Moreover, fluorescence studies carried out using W91F and other mutant UPPS with Trp replaced by Phe indicate that FPP binding mainly quenches the fluorescence of Trp-91, a residue in the alpha3 helix that moves toward the active site during substrate binding. Using stopped-flow apparatus, a three-phase protein fluorescence change with time was observed by mixing the E.FPP complex with IPP in the presence of Mg(2+). However, during the binding of E.FsPP with IPP, only the fastest phase was observed. These results suggest that the first phase is due to the IPP binding to E.FPP complex, and the other two slow phases are originated from the protein conformational change. The two slow phases coincide with the time course of FPP chain elongation from C(15) to C(55) and product release.     

Topology and subcellular localization of FtsH protein in Escherichia coli.

FtsH protein in Escherichia coli is an essential protein of 70.7 kDa (644 amino acid residues) with a putative ATP-binding sequence. Western blots (immunoblots) of proteins from fractionated cell extracts and immunoelectron microscopy of the FtsH-overproducing strain showed exclusive localization of the FtsH protein in the cytoplasmic membrane. Most of the FtsH-specific labeling with gold particles was observed in the cytoplasmic membrane and the adjacent cytoplasm; much less was observed in the outer membrane and in the bulk cytoplasm. Genetic analysis by TnphoA insertions into ftsH revealed that the 25- to 95-amino-acid region, which is flanked by two hydrophobic stretchs, protrudes into the periplasmic space. From these results, we concluded that FtsH protein is an integral cytoplasmic membrane protein spanning the membrane twice and that it has a large cytoplasmic carboxy-terminal part with a putative ATP-binding domain. The average number of FtsH molecules per cell was estimated to be approximately 400.