FtsH Protease Research Articles

Peptide-assisted degradation of the Salmonella MgtC virulence factor

MgtC is a virulence factor common to several intracellular pathogens that is required for intramacrophage survival and growth in magnesium-depleted medium. In Salmonella enterica, MgtC is coexpressed with the MgtB magnesium transporter and transcription of the mgtCB operon is induced by magnesium deprivation. Despite the high level of mgtCB transcriptional induction in magnesium-depleted medium, the MgtC protein is hardly detected in a wild-type Salmonella strain. Here, we show that downregulation of MgtC expression is dependent on a hydrophobic peptide, MgtR, which is encoded by the mgtCB operon. Our results suggest that MgtR promotes MgtC degradation by the FtsH protease, providing a negative regulatory feedback. Bacterial two-hybrid assays demonstrate that MgtR interacts with the inner-membrane MgtC protein. We identified mutant derivatives of MgtR and MgtC that prevent both regulation and interaction between the two partners. In macrophages, overexpression of the MgtR peptide led to a decrease of the replication rate of Salmonella. This study highlights the role of peptides in bacterial regulatory mechanisms and provides a natural antagonist of the MgtC virulence factor.

Expression of Protein Complexes and Individual Proteins Upon Transition of Etioplasts to Chloroplasts in Pea (Pisum sativum)

The protein complexes of pea (Pisum sativum L.) etioplasts, etio-chloroplasts and chloroplasts were examined using 2D Blue Native/SDS-PAGE. The most prominent protein complexes in etioplasts were the ATPase and the Clp and FtsH protease complexes which probably have a crucial role in the biogenesis of etioplasts and chloroplasts. Also the cytochrome b(6)f (Cyt b(6)f) complex was assembled in the etioplast membrane, as well as Rubisco, at least partially, in the stroma. These complexes are composed of proteins encoded by both the plastid and nuclear genomes, indicating that a functional cross-talk exists between pea etioplasts and the nucleus. In contrast, the proteins and protein complexes that bind chlorophyll, with the PetD subunit and the entire Cyt b(6)f complex as an exception, did not accumulate in etioplasts. Nevertheless, some PSII core components such as PsbE and the luminal oxygen-evolvong complex (OEC) proteins PsbO and PsbP accumulated efficiently in etioplasts. After 6 h de-etiolation, a complete PSII core complex appeared with 40% of the maximal photochemical efficiency, but a fully functional PSII was recorded only after 24 h illumination. Similarly, the core complex of PSI was assembled after 6 h illumination, whereas the PSI-light-harvesting complex I was stably assembled only in chloroplasts illuminated for 24 h. Moreover, a battery of proteins responsible for defense against oxidative stress accumulated particularly in etioplasts, including the stromal and thylakoidal forms of ascorbate peroxidase, glutathione reductase and PsbS.

光化学系IIのquality control：熱ストレス下でのホウレンソウチラコイド膜のD1タンパク質分解に関与するFtsHプロテアーゼの精製

光化学系IIのquality control：熱ストレス下でのホウレンソウチラコイド膜のD1タンパク質分解に関与するFtsHプロテアーゼの精製

Quality control of Photosystem II: the fate of heat-damaged D1 protein in spinach thylakoids depends on the distribution and action of FtsH proteases and phosphatases in the thylakoids

Quality control of Photosystem II: the fate of heat-damaged D1 protein in spinach thylakoids depends on the distribution and action of FtsH proteases and phosphatases in the thylakoids

Expression of Stress-Response Proteins Upon Whitefly-Mediated Inoculation of Tomato yellow leaf curl virus in Susceptible and Resistant Tomato Plants

To better understand the nature of resistance of tomato to the whitefly (Bemisia tabaci, B biotype)-transmitted Tomato yellow leaf curl virus (TYLCV), whiteflies and TYLCV were considered as particular cases of biotic stresses and virus resistance as a particular case of successful response to these stresses. Two inbred tomato lines issued from the same breeding program that used Solanum habrochaites as a TYLCV resistance source, one susceptible and the other resistant, were used to compare the expression of key proteins involved at different stages of the plant response with stresses: mitogen-activated protein kinases (MAPKs), cellular heat shock proteins (HSPs, proteases), and pathogenesis-related (PR) proteins. The two biotic stresses-non-viruliferous whitefly feeding and virus infection with viruliferous insects--led to a slow decline in abundance of MAPKs, HSPs, and chloroplast protease FtsH (but not chloroplast protease ClpC), and induced the activities of the PR proteins, beta-1,3-glucanase, and peroxidase. This decline was less pronounced in virus-resistant than in virus-susceptible lines. Contrary to whitefly infestation and virus infection, inoculation with the fungus Sclerotinia sclerotiorum induced a rapid accumulation of the stress proteins studied, followed by a decline; the virus-susceptible and -resistant tomato lines behaved similarly in response to the fungus.

The exposed N-terminal tail of the D1 subunit is required for rapid D1 degradation during photosystem II repair in Synechocystis sp PCC 6803.

The selective replacement of photodamaged D1 protein within the multisubunit photosystem II (PSII) complex is an important photoprotective mechanism in chloroplasts and cyanobacteria. FtsH proteases are involved at an early stage of D1 degradation, but it remains unclear how the damaged D1 subunit is recognized, degraded, and replaced. To test the role of the N-terminal region of D1 in PSII biogenesis and repair, we have constructed mutants of the cyanobacterium Synechocystis sp PCC 6803 that are truncated at the exposed N terminus. Removal of 5 or 10 residues blocked D1 synthesis, as assessed in radiolabeling experiments, whereas removal of 20 residues restored the ability to assemble oxygen-evolving dimeric PSII complexes but inhibited PSII repair at the level of D1 degradation. Overall, our results identify an important physiological role for the exposed N-terminal tail of D1 at an early step in selective D1 degradation. This finding has important implications for the recognition of damaged D1 and its synchronized replacement by a newly synthesized subunit.

The E3 ligase AtCHIP ubiquitylates FtsH1, a component of the chloroplast FtsH protease, and affects protein degradation in chloroplasts

The Arabidopsis E3 ligase AtCHIP was found to interact with FtsH1, a subunit of the chloroplast FtsH protease complex. FtsH1 can be ubiquitylated by AtCHIP in vitro, and the steady-state level of FtsH1 is reduced in AtCHIP-over-expressing plants under high-intensity light conditions, suggesting that the ubiquitylation of FtsH1 by AtCHIP might lead to the degradation of FtsH1 in vivo. Furthermore, the steady-state level of another subunit of the chloroplast FtsH protease complex, FtsH2, is also reduced in AtCHIP-over-expressing plants under high-intensity light conditions, and FtsH2 interacts physically with AtCHIP in vivo, suggesting the possibility that FtsH2 is also a substrate protein for AtCHIP in plant cells. A substrate of FtsH protease in vivo, the photosystem II reaction center protein D1, is not efficiently removed by FtsH in AtCHIP-over-expressing plants under high-intensity light conditions, supporting the assumption that FtsH subunits are substrates of AtCHIP in vivo, and that AtCHIP over-expression may lead to a reduced level of FtsH in chloroplasts. AtCHIP interacts with cytosolic Hsp70 and the precursors of FtsH1 and FtsH2 in the cytoplasm, and Hsp70 also interacts with FtsH1, and these protein-protein interactions appear to be increased under high-intensity light conditions, suggesting that Hsp70 might be partly responsible for the increased degradation of the substrates of Hsp70, such as FtsH1 and FtsH2, in AtCHIP-over-expressing plants under high-intensity light conditions. Therefore, AtCHIP, together with Hsp70, may play an important role in protein quality control in chloroplasts.

Region 2.1 of the Escherichia coli heat-shock sigma factor RpoH (σ 32) is necessary but not sufficient for degradation by the FtsH protease

The cellular level of the Escherichia coli heat-shock sigma factor RpoH (sigma32) is negatively controlled by chaperone-mediated proteolysis through the essential metalloprotease FtsH. Point mutations in the highly conserved region 2.1 stabilize RpoH in vivo. To assess the importance of this turnover element, hybrid proteins were constructed between E. coli RpoH and Bradyrhizobium japonicum RpoH1, a stable RpoH protein that differs from region 2.1 of E. coli RpoH at several positions. Nine amino acids forming a putative alpha-helix were exchanged between the two proteins. Both hybrids were active sigma factors and showed intermediate protein stability. Introduction of RpoH region 2.1 into the general stress sigma factor RpoS, which is a substrate of the ClpXP protease, did not render RpoS susceptible to FtsH. Hence, region 2.1 alone is not sufficient to confer FtsH sensitivity to other proteins. Region 2.1 is not a major chaperone-binding site since DnaK and DnaJ bound efficiently to all RpoH variants. The in vivo stability of the mutated RpoH proteins correlated with their stability in a purified in vitro degradation system, suggesting that region 2.1 might be directly involved in the interaction with the FtsH protease.

Sequence and Length Recognition of the C-terminal Turnover Element of LpxC, a Soluble Substrate of the Membrane-bound FtsH Protease

The membrane-anchored FtsH protease is essential in Escherichia coli as it adjusts the cellular amount of LpxC, the key enzyme in lipopolysaccharide (LPS) biosynthesis. Both accumulation and depletion of LpxC are toxic to E. coli. By continuous proteolysis of LpxC, FtsH maintains a low concentration of LpxC and, hence, the proper equilibrium between LPS and phospholipids. The C terminus of LpxC is required for turnover. By adding this tail to glutathione- S-transferase (GST) we show that it is necessary but not sufficient for FtsH-mediated degradation. A detailed mutational analysis revealed six non-polar residues in the C terminus of LpxC that are critical for degradation. Alteration of the C-terminal AVLA motif towards the SsrA-like sequence ALAA directed LpxC to other cellular proteases reinforcing the importance of the C-terminal tail for targeting to FtsH. Short C-terminal truncations stabilized LpxC. Most mutations in the C terminus of LpxC left its enzymatic activity intact as was shown by growth assays, microscopy and 2-keto-3-deoxyoctonate (KDO) determination. The critical length of the turnover element was defined by internal deletions. A C-terminal tail of about 20 amino acids length is required for proteolysis of LpxC by FtsH.

FtsH protease is required for induction of inorganic carbon acquisition complexes in Synechocystis sp. PCC 6803

Cyanobacteria possess a complex CO(2)-concentrating mechanism (CCM), which is induced by low inorganic carbon conditions. To investigate the involvement of proteases in the processes of induction and degradation of the CCM complexes, we studied the FtsH2 (DeltaSlr0228) and Deg-G (DeltaSlr1204/DeltaSll1679/DeltaSll1427) protease mutants of Synechocystis sp. PCC 6803. WT and protease mutant cells were grown under high CO(2) and then shifted to low CO(2), followed by a proteome analysis of the membrane protein complexes. Interestingly, in the FtsH2 protease mutant, inducible CCM complexes were not detected upon shift to low CO(2), whereas the Deg-G mutant behaved like WT. Also the transcripts of the inducible CCM genes and their regulator ndhR failed to accumulate upon shift of FtsH2 mutant cells from high to low CO(2), indicating that the regulation by the FtsH2 protease is upstream of NdhR. Moreover, functional photosynthesis was shown a prerequisite for induction of CCM in WT at low CO(2), possibly via generation of oxidative stress, which was shown here to enhance the expression of inducible CCM genes even at high CO(2) conditions. Once synthesized, the CCM complexes were not subject to proteolytic degradation, even when dispensable upon a shift of cells to high CO(2).

Bacterial cell division protein FtsZ is stable against degradation by AAA family protease FtsH in Escherichia coli cells

We have found that FtsH protease of Escherichia coli could degrade E. coli cell division protein FtsZ in an ATP- and Zn(2+)-dependent manner in vitro and that the degradation did not show specificity for the N-terminus or C-terminus of FtsZ, like in the case of degradation of its conventional substrate sigma(32) protein. In continuation of these observations, in the present study, we examined whether FtsH would affect the stability and turnover of FtsZ in vivo. We found that FtsZ levels were not elevated in E. coli AR754 (ftsH1 ts) cells at nonpermissive temperature as compared to the levels in an FtsH-active isogenic AR753 strain. Neither did FtsH degrade ectopically expressed FtsZ in AR754 strain nor did ectopic expression of FtsH reduced FtsZ levels in E. coli AR5090 ftsH null strain (ftsH::kan, sfhC21). Pulse chase experiments in AR754 and AR5090 strains showed that there were no compensatory changes in FtsZ turnover, in case FtsZ degradation had occurred. Even under cell division arrested conditions, wherein FtsZ was not required, FtsH protease did not degrade unutilized FtsZ. These experiments demonstrate that either FtsH protease may not have a role in regulating the levels of FtsZ in vivo under the conditions tested or that some cellular component(s) might be stabilising FtsZ against FtsH protease.

Quality control of Photosystem II: Cleavage and aggregation of heat-damaged D1 protein in spinach thylakoids

Moderate heat stress (40 °C, 30 min) on spinach thylakoids induced cleavage of the D1 protein, producing an N-terminal 23-kDa fragment, a C-terminal 9-kDa fragment, and aggregation of the D1 protein. A homologue of Arabidopsis FtsH2 protease, which is responsible for degradation of the damaged D1 protein, was abundant in the stroma thylakoids. Two processes occurred in the thylakoids in response to heat stress: dephosphorylation of the D1 protein in the stroma thylakoids, and aggregation of the phosphorylated D1 protein in the grana. Heat stress also induced the release of the extrinsic PsbO, P and Q proteins from Photosystem II, which affected D1 degradation and aggregation significantly. The cleavage and aggregation of the D1 protein appear to be two alternative processes influenced by protein phosphorylation/dephosphorylation, distribution of FtsH, and intactness of the thylakoids.

Phage Lambda CIII: A Protease Inhibitor Regulating the Lysis-Lysogeny Decision

The ATP-dependent protease FtsH (HflB) complexed with HflKC participates in post-translational control of the lysis-lysogeny decision of bacteriophage lambda by rapid degradation of lambda CII. Both phage-encoded proteins, the CII transcription activator and the CIII polypeptide, are required for efficient lysogenic response. The conserved CIII is both an inhibitor and substrate of FtsH. Here we show that the protease inhibitor CIII is present as oligomeric amphipathic α helical structures and functions as a competitive inhibitor of FtsH by preventing binding of the CII substrate. We identified single alanine substitutions in CIII that abolish its activity. We characterize a dominant negative effect of a CIII mutant. Thus, we suggest that CIII oligomrization is required for its function. Real-time analysis of CII activity demonstrates that the effect of CIII is not seen in the absence of either FtsH or HflKC. When CIII is provided ectopically, CII activity increases linearly as a function of the multiplicity of infection, suggesting that CIII enhances CII stability and the lysogenic response. FtsH function is essential for cellular viability as it regulates the balance in the synthesis of phospholipids and lipopolysaccharides. Genetic experiments confirmed that the CIII bacteriostatic effects are due to inhibition of FtsH. Thus, the early presence of CIII following infection stimulates the lysogenic response, while its degradation at later times ensures the reactivation of FtsH allowing the growth of the established lysogenic cell.

GTP/GDP binding stabilizes bacterial cell division protein FtsZ against degradation by FtsH protease in vitro

Factors contributing to the stability of bacterial cell division protein FtsZ remain unknown. In order to identify FtsZ-stabilizing factor(s), we exploited FtsH protease-based in vitro FtsZ degradation assay system. Whole cell lysate from an ftsH-null strain of Escherichia coli inhibited degradation of FtsZ by FtsH in vitro. However, activated charcoal-treated lysate did not inhibit degradation. The loss of ability of the activated charcoal-treated lysate to inhibit degradation of FtsZ was restored when it was replenished with GTP, but not when replenished with other NTPs or dNTPs. The lysate did not protect either FtsZ deletion mutants, which do not bind GTP, or FtsH substrates, σ32 and cI-108 proteins, against FtsH. GDP and GTPγS also stabilized FtsZ against FtsH. Neither GTP nor GDP inhibited proteolytic activity of FtsH per se. These observations demonstrate that binding of GTP/GDP ligands is responsible for the proteolytic stability of FtsZ against FtsH.

The Thylakoid Lumen Protease Deg1 Is Involved in the Repair of Photosystem II from Photoinhibition in Arabidopsis

Deg1 is a Ser protease peripherally attached to the lumenal side of the thylakoid membrane. Its physiological function is unknown, but its localization makes it a suitable candidate for participation in photoinhibition repair by degradation of the photosystem II reaction center protein D1. We transformed Arabidopsis thaliana with an RNA interference construct and obtained plants with reduced levels of Deg1. These plants were smaller than wild-type plants, flowered earlier, were more sensitive to photoinhibition, and accumulated more of the D1 protein, probably in an inactive form. Two C-terminal degradation products of the D1 protein, of 16 and 5.2 kD, accumulated at lower levels compared with the wild type. Moreover, addition of recombinant Deg1 to inside-out thylakoid membranes isolated from the mutant could induce the formation of the 5.2-kD D1 C-terminal fragment, whereas the unrelated proteases trypsin and thermolysin could not. Immunoblot analysis revealed that mutants containing less Deg1 also contain less FtsH protease, and FtsH mutants contain less Deg1. These results suggest that Deg1 cooperates with the stroma-exposed proteases FtsH and Deg2 in degrading D1 protein during repair from photoinhibition by cleaving lumen-exposed regions of the protein. In addition, they suggest that accumulation of Deg1 and FtsH proteases may be coordinated.

A proteomic study of Corynebacterium glutamicumAAA+ protease FtsH

BackgroundThe influence of the membrane-bound AAA+ protease FtsH on membrane and cytoplasmic proteins of Corynebacterium glutamicum was investigated in this study. For the analysis of the membrane fraction, anion exchange chromatography was combined with SDS-PAGE, while the cytoplasmic protein fraction was studied by conventional two-dimensional gel electrophoresis.ResultsIn contrast to the situation in other bacteria, deletion of C. glutamicum ftsH has no significant effect on growth in standard minimal medium or response to heat or osmotic stress. On the proteome level, deletion of the ftsH gene resulted in a strong increase of ten cytoplasmic and membrane proteins, namely biotin carboxylase/biotin carboxyl carrier protein (accBC), glyceraldehyde-3-phosphate dehydrogenase (gap), homocysteine methyltransferase (metE), malate synthase (aceB), isocitrate lyase (aceA), a conserved hypothetical protein (NCgl1985), succinate dehydrogenase A (sdhA), succinate dehydrogenase B (sdhB), succinate dehydrogenase CD (sdhCD), and glutamate binding protein (gluB), while 38 cytoplasmic and membrane-associated proteins showed a decreased abundance. The decreasing amount of succinate dehydrogenase A (sdhA) in the cytoplasmic fraction of the ftsH mutant compared to the wild type and its increasing abundance in the membrane fraction indicates that FtsH might be involved in the cleavage of a membrane anchor of this membrane-associated protein and by this changes its localization.ConclusionThe data obtained hint to an involvement of C. glutamicum FtsH protease mainly in regulation of energy and carbon metabolism, while the protease is not involved in stress response, as found in other bacteria.

Proteomic analysis of leaf proteins during dehydration of the resurrection plant Xerophyta viscosa

The desiccation-tolerant phenotype of angiosperm resurrection plants is thought to rely on the induction of protective mechanisms that maintain cellular integrity during water loss. Two-dimensional (2D) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the Xerophyta viscosa Baker proteome was carried out during dehydration to identify proteins that may play a role in such mechanisms. Quantitative analysis revealed a greater number of changes in protein expression levels at 35% than at 65% relative water content (RWC) compared to fully hydrated plants, and 17 dehydration-responsive proteins were identified by tandem mass spectrometry (MS). Proteins showing increased abundance during drying included an RNA-binding protein, chloroplast FtsH protease, glycolytic enzymes and antioxidants. A number of photosynthetic proteins declined sharply in abundance in X. viscosa at RWC below 65%, including four components of photosystem II (PSII), and Western blot analysis confirmed that two of these (psbP and Lhcb2) were not detectable at 30% RWC. These data confirm that poikilochlorophylly in X. viscosa involves the breakdown of photosynthetic proteins during dismantling of the thylakoid membranes. In contrast, levels of these photosynthetic proteins were largely maintained during dehydration in the homoiochlorophyllous species Craterostigma plantagineum Hochst, which does not dismantle thylakoid membranes on drying.

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a ΔFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in ΔDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in ΔFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the ΔDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.

Protease gene families in Populus and Arabidopsis

BackgroundProteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution.ResultsWe have performed a comparative analysis of protease genes in the two sequenced dicot genomes, Arabidopsis thaliana and Populus trichocarpa by using genes coding for proteases in the MEROPS database [1] for Arabidopsis to identify homologous sequences in Populus. A multigene-based phylogenetic analysis was performed. Most protease families were found to be larger in Populus than in Arabidopsis, reflecting recent genome duplication. Detailed studies on e.g. the DegP, Clp, FtsH, Lon, rhomboid and papain-Like protease families showed the pattern of gene family expansion and gene loss was complex. We finally show that different Populus tissues express unique suites of protease genes and that the mRNA levels of different classes of proteases change along a developmental gradient.ConclusionRecent gene family expansion and contractions have made the Arabidopsis and Populus complements of proteases different and this, together with expression patterns, gives indications about the roles of the individual gene products or groups of proteases.

The significance of Arabidopsis AAA proteases for activity and assembly/stability of mitochondrial OXPHOS complexes

The Arabidopsis genome encodes four mitochondrially localized adenosine 5′‐triphosphate‐dependent metalloproteases called FtsH or AAA proteases. All of them span the inner mitochondrial membrane but the catalytic site of two of them (AtFtsH4 and AtFtsH11) faces the intermembrane space, while AtFtsH3 and AtFtsH10 face the matrix. We used a combination of blue‐native polyacrylamide gel electrophoresis and histochemical staining to reveal the consequences of the loss of one of mitochondrial FtsHs on the efficiency of the oxidative phosphorylation system in Arabidopsis mitochondria. To address this issue, we have selected homozygous lines of respective transferred DNA (T‐DNA) insertional mutants. A decrease in the activity of complexes I and V but not complex IV was observed in the ftsh mutants, except for the mutant lacking functional FtsH11. The reduced activity of complexes I and V was well correlated with a decreased protein level of these complexes. Western blots experiments using specific antibodies against complex V subunits showed a significant reduction of these subunits only in the ftsh4 mutant. Taken together, our results reveal a role of FtsH3, FtsH4 and FtsH10 proteases in the biogenesis of a plant oxidative phosphorylation system.