FtsH Protease Research Articles

The FtsH protease is involved in development, stress response and heat shock control in Caulobacter crescentus.

The ftsH gene of Caulobacter crescentus has been isolated and identified as a component of the general stress response of this organism. In C. crescentus, ftsH expression is transiently induced after temperature upshift and in stationary phase. Consistent with this, mutants deprived of the FtsH protease are viable at normal growth conditions, but are highly sensitive to elevated temperature, increased salt concentration or the presence of antibiotics. Overexpression of ftsH resulted in an increased salt but not thermotolerance, emphasizing the importance of the FtsH protease in stress response. Mutants lacking FtsH were unable to undergo morphological and physiological adaptation in stationary phase and, upon starvation, experienced a more pronounced loss of viability than cells containing FtsH. In addition, cells lacking FtsH had an increased cellular concentration of the heat shock sigma factor sigma32, indicating that, as in Escherichia coli, the FtsH protease is involved in the control of the C. crescentus heat shock response. In agreement with this, transcription of the heat-induced sigma32-dependent gene dnaK was derepressed at normal temperature when FtsH was absent. In contrast, the groEL gene, which is controlled in response to heat stress by both sigma32 and a HcrA/CIRCE mechanism, was not derepressed in an ftsH mutant. Finally, FtsH is involved in C. crescentus development and cell cycle control. ftsH mutants were unable to synthesize stalks efficiently and had a severe cell division phenotype. In the absence of FtsH, swarmer cells differentiated into stalked cells faster than when FtsH was present, even though the entire cell cycle was longer under these conditions. Thus, directly or indirectly, the FtsH protease is involved in the inherent biological clock mechanism, which controls the timing of cell differentiation in C. crescentus.

Integration of Global Regulation of Two Aromatic-Responsive σ54-Dependent Systems: a Common Phenotype by Different Mechanisms

Pseudomonas-derived regulators DmpR and XylR are structurally and mechanistically related sigma(54)-dependent activators that control transcription of genes involved in catabolism of aromatic compounds. The binding of distinct sets of aromatic effectors to these regulatory proteins results in release of a repressive interdomain interaction and consequently allows the activators to promote transcription from their cognate target promoters. The DmpR-controlled Po promoter region and the XylR-controlled Pu promoter region are also similar, although homology is limited to three discrete DNA signatures for binding sigma(54) RNA polymerase, the integration host factor, and the regulator. These common properties allow cross-regulation of Pu and Po by DmpR and XylR in response to appropriate aromatic effectors. In vivo, transcription of both the DmpR/Po and XylR/Pu regulatory circuits is subject to dominant global regulation, which results in repression of transcription during growth in rich media. Here, we comparatively assess the contribution of (p)ppGpp, the FtsH protease, and a component of an alternative phosphoenolpyruvate-sugar phosphotransferase system, which have been independently implicated in mediating this level of regulation. Further, by exploiting the cross-regulatory abilities of these two circuits, we identify the target component(s) that are intercepted in each case. The results show that (i) contrary to previous speculation, FtsH is not universally required for transcription of sigma(54)-dependent systems; (ii) the two factors found to impact the XylR/Pu regulatory circuit do not intercept the DmpR/Po circuit; and (iii) (p)ppGpp impacts the DmpR/Po system to a greater extent than the XylR/Pu system in both the native Pseudomonas putida and a heterologous Escherichia coli host. The data demonstrate that, despite the similarities of the specific regulatory circuits, the host global regulatory network latches onto and dominates over these specific circuits by exploiting their different properties. The mechanistic implications of how each of the host factors exerts its action are discussed.

In Vivo Proteolytic Degradation of the Escherichia coli Acyltransferase HlyC

Escherichia coli hemolysin (HlyA) is the prototype toxin of a major family of exoproteins produced by Gram-negative bacteria known as "repeats in toxins." Only fatty acid-acylated HlyA molecules at residues Lys564 and Lys690 are able to damage the target cell membrane. Fatty acylation of pro-HlyA is dependent on the co-synthesized acyltransferase HlyC and the acylated form of acyl-carrier protein. By using a collection of hlyA and hlyC mutant strains, the processing of HlyC was investigated. HlyC was not detected by Western blot in an E. coli strain encoding hlyC and hlyA, but it was present in a strain encoding only hlyC. The hlyC mRNA pattern, however, was similar in both strains indicating that the turnover of HlyC does not occur at the transcriptional level. HlyC was detected in Western blots of cell lysates from an E. coli strain encoding HlyC and a HlyA derivative where both acylation sites were substituted. Similar results were obtained when HlyC was expressed in a hlyA mutant strain lacking part of a putative HlyC binding domain, indicating that this particular HlyA region affects HlyC stability. We did not detect HlyC in cell lysates from hlyC mutants with different abilities to acylate pro-HlyA, suggesting that the degradation of HlyC is not related to the HlyA acylation process. The protease systems ClpAP, ClpXP, and FtsH were found to be responsible for the HlyA-dependent processing of HlyC.

Defective plasmid partition in ftsH mutants of Escherichia coli.

FtsH is an ATP-dependent protease that is essential for cell viability in Escherichia coli. The essential function of FtsH is to maintain the proper balance of biosynthesis of major membrane components, lipopolysaccharide and phospholipids. F plasmid uses a partitioning system and is localized at specific cell positions, which may be related to the cell envelope, to ensure accurate partitioning. We have examined the effects of ftsH mutations on the maintenance of a mini-F plasmid, and have found that temperature-sensitive ftsH mutants are defective in mini-F plasmid partition, but not replication, at permissive temperature for cell growth. A significant fraction of replicated plasmid molecules tend to localize close together on one side of the cell, which may result in failure to pass the plasmid to one of the two daughter cells upon cell division. By contrast, an ftsH null mutant carrying the suppressor mutation sfhC did not affect partitioning of the plasmid. The sfhC mutation also suppressed defective maintenance in temperature-sensitive ftsH mutants. Using this new phenotype caused by ftsH mutations, we also isolated a new temperature-sensitive ftsH mutant. Mutations in ftsH cause an increase in the lipopolysaccharide/ phospholipid ratio due to stabilization of the lpxC gene product, which is involved in lipopolysaccharide synthesis and is a substrate for proteolysis by the FtsH protease. It is likely that altered membrane structure affects the localization or activity of a putative plasmid partitioning apparatus located at positions equivalent to 1/4 and 3/4 of the cell length.

Chloroplast and mitochondrial proteases in Arabidopsis. A proposed nomenclature.

The identity and scope of chloroplast and mitochondrial proteases in higher plants has only started to become apparent in recent years. Biochemical and molecular studies suggested the existence of Clp, FtsH, and DegP proteases in chloroplasts, and a Lon protease in mitochondria, although currently the full extent of their role in organellar biogenesis and function remains poorly understood. Rapidly accumulating DNA sequence data, especially from Arabidopsis, has revealed that these proteolytic enzymes are found in plant cells in multiple isomeric forms. As a consequence, a systematic approach was taken to catalog all these isomers, to predict their intracellular location and putative processing sites, and to propose a standard nomenclature to avoid confusion and facilitate scientific communication. For the Clp protease most of the ClpP isomers are found in chloroplasts, whereas one is mitochondrial. Of the ATPase subunits, the one ClpD and two ClpC isomers are located in chloroplasts, whereas both ClpX isomers are present in mitochondria. Isomers of the Lon protease are predicted in both compartments, as are the different forms of FtsH protease. DegP, the least characterized protease in plant cells, has the most number of isomers and they are predicted to localize in several cell compartments. These predictions, along with the proposed nomenclature, will serve as a framework for future studies of all four families of proteases and their individual isomers.

Revisiting the Lysogenization Control of Bacteriophage λ: IDENTIFICATION AND CHARACTERIZATION OF A NEW HOST COMPONENT, HflD

Upon infection to the Escherichia coli cell, the genome of bacteriophage lambda either replicates to form new progenies (lytic growth) or integrates into the host chromosome (lysogenization). The lambda CII protein is a key determinant in the lysis-lysogeny decision. It is a short-lived transcription activator for the lambda genes essential for lysogeny establishment. In this study, we isolated a new class of hfl (high frequency lysogenization) mutants of E. coli, using a new selection for enhancement of CII-stimulated transcription. The gene affected was termed hflD, which encodes a protein of 213 amino acids. An hflD-disrupted mutant indeed showed an Hfl phenotype, indicating that HflD acts to down-regulate lysogenization. HflD is associated peripherally with the cytoplasmic membrane. Its interaction with CII was demonstrated in vitro using purified proteins as well as in vivo using the bacterial two-hybrid system. Pulse-chase examinations demonstrated that the HflD function is required for the rapid in vivo degradation of CII, although it interfered with FtsH-mediated CII proteolysis in an in vitro reaction system using detergent-solubilized components. We suggest that HflD is a factor that sequesters CII from the target promoters and recruits it to the membrane where the FtsH protease is localized.

An internal region of the RpoH heat shock transcription factor is critical for rapid degradation by the FtsH protease

The proteolysis of regulatory proteins plays an important role in the control of gene expression. The Escherichia coli heat shock sigma factor RpoH (σ 32) is highly unstable. Its instability is determined by interactions with the DnaK chaperone machine, RNA polymerase and the ATP-dependent protease FtsH. Bradyrhizobium japonicum expresses three RpoH proteins of which RpoH 1 is highly stable. To determine which regions of E. coli RpoH determine protein lability, we generated a number of truncated versions and hybrid proteins. Truncation of N-terminal amino acids had no, and deletion of C-terminal amino acids only a minor effect on stability of RpoH. A major determinant of RpoH lability was mapped to a region of about 85 amino acids (residues 36–122) roughly comprising the sigma factor region 2. This is the first demonstration of an internal RpoH region being responsible for FtsH-mediated degradation.

Probing the mechanism of ATP hydrolysis and substrate translocation in the AAA protease FtsH by modelling and mutagenesis.

We have built a homology model of the AAA domain of the ATP-dependent protease FtsH of Escherichia coli based on the crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein. The resulting model of the hexameric ring of the ATP-bound form of the AAA ATPase suggests a plausible mechanism of ATP binding and hydrolysis, in which invariant residues of Walker motifs A and B and the second region of homology, characteristic of the AAA ATPases, play key roles. The importance of these invariant residues was confirmed by site-directed mutagenesis. Further modelling suggested a mechanism by which ATP hydrolysis alters the conformation of the loop forming the central hole of the hexameric ring. It is proposed that unfolded polypeptides are translocated through the central hole into the protease chamber upon cycles of ATP hydrolysis. Degradation of polypeptides by FtsH is tightly coupled to ATP hydrolysis, whereas ATP binding alone is sufficient to support the degradation of short peptides. Furthermore, comparative structural analysis of FtsH and a related ATPase, HslU, reveals interesting similarities and differences in mechanism.

Bacteriophage lambda cIII gene product has an additional function apart from inhibition of cII degradation.

For lysogenization of Escherichia coli cells by bacteriophage lambda, functions of three lambda genes called c are necessary. The cI gene codes for a repressor that blocks activities of lytic promoters. However, early after infection, expression of cI is dependent on the function of the cII gene, coding for a specific transcriptional activator. The cII protein is unstable in E. coli cells due to FtsH-mediated proteolysis. The cIII gene product is an inhibitor of the FtsH protease. Here we demonstrate that cIII may have another function apart from inhibition of cII degradation. We found that overexpression of the cII gene results in impaired lysogenization by phage lambda, however simultaneous overexpression of the cIII gene abolished this negative effect on lysogenization. Analysis of cII-mediated transcriptional activation of certain promoters at different levels of cII and cIII proteins in cells confirmed that observed effects cannot be explained assuming that the only role of cIII is inhibition of FtsH-mediated degradation of cII. We propose that cIII has an additional role apart from its well-known function in indirect stabilization of cII. Apparently, cIII influences not only cII level but also activity of this transcriptional stimulator, especially at its high concentrations.

The role of FtsH proteins in photosystem biosynthesis and turnover in Arabidopsis and Synechocystis

Both Arabidopsis thaliana and Synechocystis PCC 6803 genomes encode several homologues of Escherichia coli FtsH. These proteins are AAA-family proteases (ATPases associated with a variety of cellular activities), which are widely distributed in prokaryotes and eukaryotes. Among their diverse roles, FtsH proteins are important in photosynthesis. Of four found in Synechocystis, one (encoded by open-reading frame slr0228) appears to be involved in photosystem I (PSI) biosynthesis, and it has been suggested that it may also participate in photosystem II (PSII) D1-protein turnover (see S8 poster by P. Silva et al.). In Arabidopsis, mutation of the `Var2?-ftsH leads to severe leaf-variegation [1]. Whereas fluorescence-emission spectra of Synechocystis show a higher PSI:PSII ratio in wild-type (WT) than in slr0228? cells, in Arabidopsis, WT and Var2 have similar PSI:PSII peaks under normal growth conditions. High-light treatment, however, results in greater photoinhibition of Var2-FtsH? plants, measured by Fv/Fm. Fluorescence emission spectra also show reduced recovery from photoinhibitory high light in Var2 compared with WT. These experiments and Western blots indicate that the D1 repair cycle is impeded in the Arabidopsis FtsH mutant as in the cyanobacterium. Sequence similarities in the slr0228 and Var2 ftsH genes indeed suggest they may be closely-related proteins. To study the role of slr0228 in reaction centre biosynthesis, site-directed mutants have been made in PSII? and chlorophyll-synthesis-deficient Synechocystis strains. Further work characterising the role of plant and cyanobacterial FtsH proteins is presented. 1. Chen M, Choi Y, Voytas DF, Rodermel S: Mutations in the Arabidopsis VAR2 locus cause leaf variegation due to the loss of a chloroplast FtsH protease. Plant J 2000, 22:303.

Involvement of an FtsH homologue in the assembly of functional photosystem I in the cyanobacterium Synechocystis sp. PCC 6803

The Synechocystis sp. PCC 6803 genome encodes four putative homologues of the AAA protease FtsH, two of which (slr0228 and sll1463) have been subjected to insertional mutagenesis in this study. Disruption of sll1463 had no discernible effect but disruption of slr0228 caused a 60% reduction in the abundance of functional photosystem I, without affecting the cellular content of photosystem II or phycobilisomes. Fluorescence and immunoblotting analyses show reductions in PS I polypeptides and possible structural alterations in the residual PS I, indicating an important role for slr0228 in PS I biogenesis.

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.

Differential degradation of Escherichia coli sigma32 and Bradyrhizobium japonicum RpoH factors by the FtsH protease.

The Escherichia coli heat shock sigma factor sigma32 (RpoH) is rapidly degraded under non-stress conditions. The integrity of the DnaK chaperone machinery and the ATP-dependent FtsH protease are required for sigma32 proteolysis in vivo. Bradyrhizobium japonicum expresses three sigma32-type transcription factors, RpoH1, RpoH2, and RpoH3, which are functional in E. coli. We compared the stability of these sigma factors with E. coli sigma32 stability. In E. coli C600 (wild-type), the half-lives of sigma32, RpoH1, RpoH2 and RpoH3 were 30 s, 7 min, 4 min and 4 min, respectively. The first three proteins were stabilized in ftsH mutant backgrounds, indicating that they are degraded by FtsH in the wild-type. Proteolysis of RpoH3 was FtsH-independent because this sigma factor was not stabilized in ftsH mutants. Interestingly, in a purified in vitro system, all four RpoH proteins were degraded by FtsH, indicating that in vivo protein degradation depends on additional cellular factors. Rationally designed point mutations of sigma32 and RpoH1 suggested that the highly conserved RpoH box does not play a major role in conferring stability to RpoH factors. Presumably, several regions distributed along the primary sequence of the sigma factor are important for FtsH-mediated proteolysis. Finally, we provide evidence that proteolysis of RpoH factors in vivo depends on the DnaK machinery, irrespective of the protease involved.

Evidence for an active role of the DnaK chaperone system in the degradation of σ 32

Under non-stressed conditions in Escherichia coli, the heat shock transcription factor σ 32 is rapidly degraded by the AAA protease FtsH. The DnaK chaperone system is also required for the rapid turnover of σ 32 in the cell. It has been hypothesized that the DnaK chaperone system facilitates the degradation of σ 32 by sequestering it from RNA polymerase core. This hypothesis predicts that mutant σ 32 proteins, which are deficient in binding to RNA polymerase core, will be degraded independently of the DnaK chaperone system. We examined the in vivo stability of such mutant σ 32 proteins. Results indicated that the mutant σ 32 proteins as similar as authentic σ 32 were stabilized in Δ dnaK and Δ dnaJ/Δ cbpA cells. The interaction between σ 32 and DnaK/DnaJ/GrpE was not affected by these mutations. These results strongly suggest that the degradation of σ 32 requires an unidentified active role of the DnaK chaperone system.

Structural and functional studies on an FtsH inhibitor from Bacillus subtilis

The small 3 kDa SpoVM protein is essential for development of the spore in Bacillus subtilis. Genetic and biochemical experiments have shown that the function of SpoVM is to inhibit the proteolytic activity of FtsH during sporulation. We have used a combination of genetic and biophysical techniques to characterise the role of this small polypeptide. SpoVM was found to be widespread in Bacillus as well as in two Clostridia species, suggesting that SpoVM provides a common mechanism for inactivating the FtsH protease during spore differentiation. Using site-specific mutagenesis, we have identified C-terminal residues of SpoVM essential for biological activity. Analysis of SpoVM’s structure showed that it is able to assume an α-helical conformation in the presence of a lipid interface which may be important in interacting with FtsH.

Characterization of a conserved α-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 α 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 σ32 and the bacteriophage λ 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 α helical region is an important structural element of FtsH (HflB).

Mutations in the Arabidopsis VAR2 locus cause leaf variegation due to the loss of a chloroplast FtsH protease

Variegated plants have green- and white-sectored leaves. Cells in the green sectors contain morphologically normal chloroplasts, whereas cells in the white sectors contain non-pigmented plastids that lack organized lamellar structures. Many variegations are caused by mutations in nuclear genes that affect plastid function, yet in only a few cases have the responsible genes been cloned. We show that mutations in the nuclear VAR2 locus of Arabidopsis cause variegation due to loss of a chloroplast thylakoid membrane protein that bears similarity to the FtsH family of AAA proteins (ATPases associated with diverse cellular activities). Escherichia coli FtsH is a chaperone metalloprotease that functions in a number of diverse membrane-associated events. Although FtsH homologs have been identified in multicellular organisms, their functions and activities are largely unknown; we provide genetic in vivo evidence that VAR2 functions in thylakoid membrane biogenesis. We have isolated four var2 alleles and they have allowed us to define domains of the protein that are required for activity. These include two putative ATP-binding sites. VAR2 protein amounts generally correlate with the severity of the var2 mutant phenotype. One allele lacks detectable VAR2 protein, suggesting that the mechanism of var2 variegation involves the action of a redundant activity in the green sectors. We conclude that redundant activities may be a general mechanism to explain nuclear gene-induced plant variegations.

The thylakoid FtsH protease plays a role in the light-induced turnover of the photosystem II D1 protein.

The photosystem II reaction center D1 protein is known to turn over frequently. This protein is prone to irreversible damage caused by reactive oxygen species that are formed in the light; the damaged, nonfunctional D1 protein is degraded and replaced by a new copy. However, the proteases responsible for D1 protein degradation remain unknown. In this study, we investigate the possible role of the FtsH protease, an ATP-dependent zinc metalloprotease, during this process. The primary light-induced cleavage product of the D1 protein, a 23-kD fragment, was found to be degraded in isolated thylakoids in the dark during a process dependent on ATP hydrolysis and divalent metal ions, suggesting the involvement of FtsH. Purified FtsH degraded the 23-kD D1 fragment present in isolated photosystem II core complexes, as well as that in thylakoid membranes depleted of endogenous FtsH. In this study, we definitively identify the chloroplast protease acting on the D1 protein during its light-induced turnover. Unlike previously identified membrane-bound substrates for FtsH in bacteria and mitochondria, the 23-kD D1 fragment represents a novel class of FtsH substrate-functionally assembled proteins that have undergone irreversible photooxidative damage and cleavage.

The Thylakoid FtsH Protease Plays a Role in the Light-Induced Turnover of the Photosystem II D1 Protein

The Thylakoid FtsH Protease Plays a Role in the Light-Induced Turnover of the Photosystem II D1 Protein

Dissecting the Role of a Conserved Motif (the Second Region of Homology) in the AAA Family of ATPases: SITE-DIRECTED MUTAGENESIS OF THE ATP-DEPENDENT PROTEASE FtsH

Escherichia coli FtsH is an ATP-dependent protease that belongs to the AAA protein family. The second region of homology (SRH) is a highly conserved motif among AAA family members and distinguishes these proteins in part from the wider family of Walker-type ATPases. Despite its conservation across the AAA family of proteins, very little is known concerning the function of the SRH. To address this question, we introduced point mutations systematically into the SRH of FtsH and studied the activities of the mutant proteins. Highly conserved amino acid residues within the SRH were found to be critical for the function of FtsH, with mutations at these positions leading to decreased or abolished ATPase activity. The effects of the mutations on the protease activity of FtsH correlated strikingly with their effects on the ATPase activity. The ATPase-deficient SRH mutants underwent an ATP-induced conformational change similar to wild type FtsH, suggesting an important role for the SRH in ATP hydrolysis but not ATP binding. Analysis of the data in the light of the crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein suggests a plausible mechanism of ATP hydrolysis by the AAA ATPases, which invokes an intermolecular catalytic role for the SRH.