FtsH Protease Research Articles

Role of FtsH2 in the repair of Photosystem II in mutants of the cyanobacterium Synechocystis PCC 6803 with impaired assembly or stability of the CaMn 4 cluster

The FtsH2 protease, encoded by the slr0228 gene, plays a key role in the selective degradation of photodamaged D1 protein during the repair of Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. To test whether additional proteases might be involved in D1 degradation during high rates of photodamage, we have studied the synthesis and degradation of the D1 protein in ΔPsbO and ΔPsbV mutants, in which the CaMn 4 cluster catalyzing oxygen evolution is less stable, and in the D1 processing mutants, D1-S345P and ΔCtpA, which are unable to assemble a functional cluster. All four mutants exhibited a dramatically increased rate of D1 degradation in high light compared to the wild-type. Additional inactivation of the ftsH2 gene slowed the rate of D1 degradation dramatically and increased the level of PSII complexes. We conclude that FtsH2 plays a major role in the degradation of both precursor and mature forms of D1 following donor-side photoinhibition. However, this conclusion concerned only D1 assembled into larger complexes containing at least D2 and CP47. In the Δ psbEFLJ deletion mutant blocked at an early stage in PSII assembly, unassembled D1 protein was efficiently degraded in the absence of FtsH2 pointing to the involvement of other protease(s). Significantly, the ΔPsbO mutant displayed unusually low levels of cellular chlorophyll at extremely low-light intensities. The possibilities that PSII repair may limit the availability of chlorophyll for the biogenesis of other chlorophyll-binding proteins and that PsbO might have a regulatory role in PSII repair are discussed.

Mitochondrial protease AtFtsH4 protects ageing Arabidopsis rosettes against oxidative damage under short-day photoperiod

Mitochondrial AtFtsH4 protease, whose catalytic site is exposed to the intermembrane space, is one of four inner membrane-bound FtsH proteases in Arabidopsis. We found that the loss of AtFtsH4 altered Arabidopsis leaf morphology at the late stage of rosette growth under short-day photoperiod, while such changes were not observed in ftsh4 mutants grown under long days. These morphological changes were correlated with elevated levels of both reactive oxygen species (ROS) and carbonylated proteins, which strongly suggested that ageing ftsh4 plants experienced oxidative stress. This view was supported by the accumulation of electron-dense material, presumably containing aggregated oxidized proteins, in mitochondria of ftsh4 plants with the most strongly malformed leaf blades. Taken together, our data published in the May issue of Plant J 1 suggest a link between the lack of AtFtsH4 protease, oxidative stress and altered leaf morphology at the late rosette stage under short days. Here, we present evidence that the onset of altered leaf morphology in ftsh4 correlates with an increase in the abundance of AtFtsH4 transcript observed in wild-type Arabidopsis growing under the same conditions. We also discuss how the lack of AtFtsH4 may cause oxidative stress towards the end of the vegetative growth in short days.

Phosphorylation of Photosystem II Controls Functional Macroscopic Folding of Photosynthetic Membranes inArabidopsis

Photosynthetic thylakoid membranes in plants contain highly folded membrane layers enriched in photosystem II, which uses light energy to oxidize water and produce oxygen. The sunlight also causes quantitative phosphorylation of major photosystem II proteins. Analysis of the Arabidopsis thaliana stn7xstn8 double mutant deficient in thylakoid protein kinases STN7 and STN8 revealed light-independent phosphorylation of PsbH protein and greatly reduced N-terminal phosphorylation of D2 protein. The stn7xstn8 and stn8 mutants deficient in light-induced phosphorylation of photosystem II had increased thylakoid membrane folding compared with wild-type and stn7 plants. Significant enhancement in the size of stacked thylakoid membranes in stn7xstn8 and stn8 accelerated gravity-driven sedimentation of isolated thylakoids and was observed directly in plant leaves by transmission electron microscopy. Increased membrane folding, caused by the loss of light-induced protein phosphorylation, obstructed lateral migration of the photosystem II reaction center protein D1 and of processing protease FtsH between the stacked and unstacked membrane domains, suppressing turnover of damaged D1 in the leaves exposed to high light. These findings show that the high level of photosystem II phosphorylation in plants is required for adjustment of macroscopic folding of large photosynthetic membranes modulating lateral mobility of membrane proteins and sustained photosynthetic activity.

Proteases in bacterial pathogenesis

Bacterial pathogens rely on proteolysis for protein quality control under adverse conditions experienced in the host, as well as for the timely degradation of central virulence regulators. We have focused on the contribution of the conserved Lon, Clp, HtrA and FtsH proteases to pathogenesis and have highlighted common biological processes for which their activities are important for virulence.

Stress responses in Prochlorococcus MIT9313 vs. SS120 involve differential expression of genes encoding proteases ClpP, FtsH and Lon

Prochlorococcus is a marine cyanobacterium responsible for a significant part of global primary production as well as being one of the most abundant organisms on Earth. Protein turnover is an essential and poorly understood aspect of the cyanobacterial response to environmental stresses. In the present work, cultures of the SS120 and MIT9313 strains were subjected to several conditions, and quantitative real time RT-PCR was used to measure changes in the expression of genes encoding three representative ATP-dependent proteases. We found common responses to conditions such as aging. However, the expression pattern under nutrient starvation was strikingly different in the two strains, probably reflecting the different regulatory backgrounds of the two ecotypes here studied.

Identification and characterization of two members of the FtsH gene family in maize (Zea mays L.)

Two full-length cDNAs, designated as ZmFtsH2A and ZmFtsH2B, were isolated from maize (Zea mays L.) by suppression subtractive hybridization coupled with in silico cloning approach. The predicted proteins of ZmFtsH2A and ZmFtsH2B both consisted of 677 amino acid residues and displayed high similarity to FtsH2 protease of Arabidopsis thaliana. DNA gel blotting analysis indicated that AtFtsH2-like genes exist as two copies in maize genome. The genomic sequences of ZmFtsH2A and ZmFtsH2B were cloned and the main difference was that the first intron of ZmFtsH2B was much longer than that of ZmFtsH2A. RT-PCR analysis revealed that both genes were constitutively expressed in all examined tissues and the expression level of ZmFtsH2B transcripts was higher than that of ZmFtsH2A. The responses of the two genes in maize seedlings to PEG, cold, high salt, and ABA treatments were compared, and the results showed that ZmFtsH2B transcription in leaves was markedly up-regulated by water deficit stress and ABA treatments while ZmFtsH2A constitutively expressed both in leaves and roots under all tested stressful conditions. Drought tolerance of transgenic tobaccos overexpressing ZmFtsH2A and ZmFtsH2B weren't improved compared to wild-type controls, which indicated that two genes might not be directly involved in plant drought tolerance or the number of functional FtsH heterocomplex might not be increased in this condition. Our current study provides fundamental information for the further investigation of the maize FtsH proteins.

Effects of Antibiotics and a Proto-Oncogene Homolog on Destruction of Protein Translocator SecY

Protein secretion occurs via translocation by the evolutionarily conserved Sec complex. LacZ hybrid proteins have long been used to study translocation in Escherichia coli. Some LacZ hybrids were thought to block secretion by physically jamming the Sec complex, leading to cell death. We found that jammed Sec complexes caused the degradation of essential translocator components by the protease FtsH. Increasing the amounts or the stability of the membrane protein YccA, a known inhibitor of FtsH, counteracted this destruction. Antibiotics that inhibit translation elongation also jammed the translocator and caused the degradation of translocator components, which may contribute to their effectiveness. Intriguingly, YccA is a functional homolog of the proto-oncogene product Bax Inhibitor-1, which may share a similar mechanism of action in regulating apoptosis upon prolonged secretion stress.

Large Scale Comparative Proteomics of a Chloroplast Clp Protease Mutant Reveals Folding Stress, Altered Protein Homeostasis, and Feedback Regulation of Metabolism

The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate-limiting photosynthetic electron transport. We discuss the quantitative proteomics data and the role of Clp proteolysis using a "systems view" of chloroplast homeostasis and metabolism and provide testable hypotheses and putative substrates to further determine the significance of Clp-driven proteolysis.

Stress responses to heterologous membrane protein expression in Escherichia coli

The stress response of E. coli to the expression of two recombinant membrane proteins, the E. coli AAA+ protease FtsH and the human G-protein coupled receptor CB1, was examined using several members of a promoter-GFP library. Several genes from the heat-shock and envelope stress regulons (rpoH, clpP, lon, and ftsH) were strongly induced by expression of either membrane protein. Flow cytometry was used to monitor the real-time dynamics of the transcription of these reporter genes in response to membrane protein expression. Co-expression of CB1 and FtsH led to an additive response in these four reporter genes suggesting that the stresses may be occurring via different physiological mechanisms.

ATP-dependent Proteases Differ Substantially in Their Ability to Unfold Globular Proteins

ATP-dependent proteases control the concentrations of hundreds of regulatory proteins and remove damaged or misfolded proteins from cells. They select their substrates primarily by recognizing sequence motifs or covalent modifications. Once a substrate is bound to the protease, it has to be unfolded and translocated into the proteolytic chamber to be degraded. Some proteases appear to be promiscuous, degrading substrates with poorly defined targeting signals, which suggests that selectivity may be controlled at additional levels. Here we compare the abilities of representatives from all classes of ATP-dependent proteases to unfold a model substrate protein and find that the unfolding abilities range over more than 2 orders of magnitude. We propose that these differences in unfolding abilities contribute to the fates of substrate proteins and may act as a further layer of selectivity during protein destruction.

Protein Quality Control in Chloroplasts: A Current Model of D1 Protein Degradation in the Photosystem II Repair Cycle

The chloroplast originated from endosymbiosis of photosynthetic bacteria. Thus, mechanisms essential for chloroplast biogenesis/homeostasis (protein synthesis, import from cytosol, assembly, and degradation) are predominantly governed by prokaryotic systems. Among these, the quality control system is crucial, because light energy constantly damages photosynthetic proteins and excessive light often limits plant growth by irreversibly inactivating the photosynthetic apparatuses. Here, we overview prokaryotic proteases (FtsH and Deg) which are two enzymes that play critical roles in this system. We particularly focus on Photosystem II (PSII) in thylakoid membranes, which is composed of more than 20 subunits. Among the subunits is one of the intrinsic reaction centre proteins (D1) which is considered to be the target of photodamage. Its rapid and specific turnover suggests that photodamaged D1 is degraded by these proteases and replaced with a de novo synthesized one in a system which is termed the PSII repair cycle. We discuss a current model of D1 degradation which is executed by a concerted action of particular FtsH and Deg isoforms.

ClpXP Degrades SsrA-Tagged Proteins inStreptococcus pneumoniae

Bacterial proteins that are abnormally truncated due to incomplete mRNA or the presence of rare codons are extended by an SsrA tag during ribosome rescue in a trans-translation process important for maintaining protein quality. In Escherichia coli, the SsrA-tagged proteins become the target of the Tsp, Lon, FtsH, ClpXP, and ClpAP proteases. Here we show that degradation of model SsrA-tagged proteins in Streptococcus pneumoniae depends primarily or exclusively on ClpXP in vivo. In addition, we show the E. coli SsrA tag is also a target of S. pneumoniae ClpXP in vivo, even though the N-terminal portions of the tags differ significantly between the two species, suggesting there may be no adaptor protein for SsrA in S. pneumoniae.

Mechanism of protonophores-mediated induction of heat-shock response in Escherichia coli

BackgroundProtonophores are the agents that dissipate the proton-motive-force (PMF) across E. coli plasma membrane. As the PMF is known to be an energy source for the translocation of membrane and periplasmic proteins after their initial syntheses in cell cytoplasm, protonophores therefore inhibit the translocation phenomenon. In addition, protonophores also induce heat-shock-like stress response in E. coli cell. In this study, our motivation was to investigate that how the protonophores-mediated phenomena like inhibition of protein translocation and induction of heat-shock proteins in E. coli were correlated.ResultsInduction of heat-shock-like response in E. coli attained the maximum level after about 20 minutes of cell growth in the presence of a protonophore like carbonyl cyanide m-chloro phenylhydrazone (CCCP) or 2, 4-dinitrophenol (DNP). With induction, cellular level of the heat-shock regulator protein sigma-32 also increased. The increase in sigma-32 level was resulted solely from its stabilization, not from its increased synthesis. On the other hand, the protonophores inhibited the translocation of the periplasmic protein alkaline phosphatase (AP), resulting its accumulation in cell cytosol partly in aggregated and partly in dispersed form. On further cell growth, after withdrawal of the protonophores, the previously accumulated AP could not be translocated out; instead the AP-aggregate had been degraded perhaps by an induced heat-shock protease ClpP. Moreover, the non-translocated AP formed binary complex with the induced heat-shock chaperone DnaK and the excess cellular concentration of DnaK disallowed the induction of heat-shock response by the protonophores.ConclusionOur experimental results suggested that the protonophores-mediated accumulation and aggregation of membrane proteins (like AP) in cell cytosol had signaled the induction of heat-shock proteins in E. coli and the non-translocated protein aggregates were possibly degraded by an induced heat-shock protease ClpP. Moreover, the induction of heat-shock response occurred by the stabilization of sigma-32. As, normally the DnaK-bound sigma-32 was known to be degraded by the heat-shock protease FtsH, our experimental results further suggested that the engagement of DnaK with the non-translocated proteins (like AP) had made the sigma-32 free and stable.

Evolution of prokaryotic SPFH proteins.

BackgroundThe SPFH protein superfamily is a diverse family of proteins whose eukaryotic members are involved in the scaffolding of detergent-resistant microdomains. Recently the origin of the SPFH proteins has been questioned. Instead, convergent evolution has been proposed. However, an independent, convergent evolution of three large prokaryotic and three eukaryotic families is highly unlikely, especially when other mechanisms such as lateral gene transfer which could also explain their distribution pattern have not yet been considered.To gain better insight into this very diverse protein family, we have analyzed the genomes of 497 microorganisms and investigated the pattern of occurrence as well as the genomic vicinity of the prokaryotic SPFH members.ResultsAccording to sequence and operon structure, a clear division into 12 subfamilies was evident. Three subfamilies (SPFH1, SPFH2 and SPFH5) show a conserved operon structure and two additional subfamilies are linked to those three through functional aspects (SPFH1, SPFH3, SPFH4: interaction with FtsH protease). Therefore these subgroups most likely share common ancestry. The complex pattern of occurrence among the different phyla is indicative of lateral gene transfer. Organisms that do not possess a single SPFH protein are almost exclusively endosymbionts or endoparasites.ConclusionThe conserved operon structure and functional similarities suggest that at least 5 subfamilies that encompass almost 75% of all prokaryotic SPFH members share a common origin. Their similarity to the different eukaryotic SPFH families, as well as functional similarities, suggests that the eukaryotic SPFH families originated from different prokaryotic SPFH families rather than one. This explains the difficulties in obtaining a consistent phylogenetic tree of the eukaryotic SPFH members. Phylogenetic evidence points towards lateral gene transfer as one source of the very diverse patterns of occurrence in bacterial species.

光化学系IIのquality control：光・熱損傷D1タンパク質の分解に関与するFtsHプロテアーゼの分布と存在状態

光化学系IIのquality control：光・熱損傷D1タンパク質の分解に関与するFtsHプロテアーゼの分布と存在状態

Region C of the Escherichia coli heat shock sigma factor RpoH (σ32) contains a turnover element for proteolysis by the FtsH protease

Transcription of most heat shock genes in Escherichia coli is initiated by the alternative sigma factor sigma(32) (RpoH). At physiological temperatures, RpoH is rapidly degraded by chaperone-mediated FtsH-dependent proteolysis. Several RpoH residues critical for degradation are located in the highly conserved region 2.1. However, additional residues were predicted to be involved in this process. We introduced mutations in region C of RpoH and found that a double mutation (A131E, K134V) significantly stabilized RpoH against degradation by the FtsH protease. Single-point mutations at these positions only showed a slight effect on RpoH stability. Both double and single amino acid substitutions did not impair sigma factor activity as demonstrated by a groE-lacZ reporter gene fusion, Western blot analysis of heat shock gene expression and increased heat tolerance in the presence of these proteins. Combined mutations in regions 2.1 and C further stabilized RpoH. We also demonstrate that an RpoH fragment composed of residues 37-147 (including regions 2.1 and C) is degraded in an FtsH-dependent manner. We conclude that in addition to the previously described turnover element in region 2.1, a previously postulated second region important for proteolysis of RpoH by FtsH lies in region C of the sigma factor.

Molecular Basis for Regulation of the Heat Shock Transcription Factor σ32 by the DnaK and DnaJ Chaperones

Central to the transcriptional control of the Escherichia coli heat shock regulon is the stress-dependent inhibition of the sigma(32) subunit of RNA polymerase by reversible association with the DnaK chaperone, mediated by the DnaJ cochaperone. Here we identified two distinct sites in sigma(32) as binding sites for DnaK and DnaJ. DnaJ binding destabilizes a distant region of sigma(32) in close spatial vicinity of the DnaK-binding site, and DnaK destabilizes a region in the N-terminal domain, the primary target for the FtsH protease, which degrades sigma(32) in vivo. Our findings suggest a molecular mechanism for the DnaK- and DnaJ-mediated inactivation of sigma(32) as part of the heat shock response. They furthermore demonstrate that DnaK and DnaJ binding can induce conformational changes in a native protein substrate even at distant sites, a feature that we propose to be of general relevance for the action of Hsp70 chaperone systems.

Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli.

G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.

Resolving Individual Steps in the Operation of ATP-Dependent Proteolytic Molecular Machines: From Conformational Changes to Substrate Translocation and Processivity

Clp, Lon, and FtsH proteases are proteolytic molecular machines that use the free energy of ATP hydrolysis to unfold protein substrates and processively present them to protease active sites. Here we review recent biochemical and structural studies relevant to the mechanism of ATP-dependent processive proteolysis. Despite the significant structural differences among the Clp, Lon, and FtsH proteases, these enzymes share important mechanistic features. In these systems, mechanistic studies have provided evidence for ATP binding and hydrolysis-driven conformational changes that drive translocation of substrates, which has significant implications for the processive mechanism of proteolysis. These studies indicate that the nucleotide (ATP, ADP, or nonhydrolyzable ATP analogues) occupancy of the ATPase binding sites can influence the binding mode and/or binding affinity for protein substrates. A general mechanism is proposed in which the communication between ATPase active sites and protein substrate binding regions coordinates a processive cycle of substrate binding, translocation, proteolysis, and product release.

Amos Oppenheim (31 October 1934 to 24 September 2006)

Even when relaxing, Amos was not above discussing experiments. We remember our colleague and friend Amos Oppenheim who passed away last year. His many students, colleagues and friends will sorely miss him. His work in Israel and the legions of collaborations he had around the world were not only made possible by the love and intensity he brought to his scientific studies, but equally by his humour and conviviality. Amos was much respected and admired for his many original contributions to the studies of regulation of gene expression in bacteria and, in particular, to the study of phage Lambda. Understanding Lambda was his passion. His most recent work was directed towards extending the lambda paradigm as a model system for understanding regulatory networks. Indeed much of his most recent work on Systems Biology of Lambda is still in the process of being published by his co-workers. He also made significant contributions to the areas of the structure–function of macromolecules, gene expression in bacteria and its application to biotechnology, and the biological control of plant pathogens. In 1966, Amos earned his PhD from the University of California at Davis, where he also received his undergraduate education, and soon after joined the Hebrew University Medical School, where he was a Professor and a mentor of many generations of scientists and medical students. He was a visiting research fellow with Francois Jacob at the Institut Pasteur, Paris for 2-month periods between 1968 and 1970, a visiting professor at the Institute of Cancer Research, Columbia University, New York (Summers 1986, 1990), and for over 30 years a frequent and a very fruitful Visiting Scientist at the National Cancer Institute, National Institutes of Health, Maryland, the last time during 2005–2006. He was an organizer and invited participant at many international meetings. Among his other activities, Amos was the Head of Israeli National Node of European Molecular Biology Data Network Steering Committee, Head of Authority for Graduate Studies at the Hebrew University and Head of the Biotechnology Teaching Program at the Hebrew University. His contributions to the Lambda field were many. In particular, his long-time studies on the action of the CII and CIII proteins, how they control the decision between lysis and lysogeny, and how FtsH and other proteases modulate the levels of CII and other phage proteins, are seminal contributions to our understanding of Lambda regulation. These studies, along with his connected interest in bacterial proteins including HU and IHF that affect λ transcription control, represent his major outstanding contributions to the field. All who knew Amos will miss his probing intellect, love of experiments and, without a doubt, his infectious laugh and ebullient personality that always lit up the rooms he entered. Everyone loved working with Amos. He was always ready to sit down at the bench and start new experiments, and his enthusiastic hard work rubbed off on everyone. Collaborations were never a problem because his collaborators knew Amos would do more than his fair share of the experimental work and much of the thinking involved in designing the project and interpreting the results. Amos' interests went beyond science; he loved classical music and was an accomplished amateur artist. Just as he strove to be at the top of his field in science, Amos also was driven in his outside pursuits to aim for the peak. Always an outdoors person, in his later years he enjoyed going on treks, not in his locale, but to the Himalayas and the Andes where he was tested on the highest peaks. Amos' last day was spent swimming and enjoying the seaside with his grandchildren. Amos has been a loving husband, friend and colleague of 53 years to Ariella Oppenheim, also a scientist at the Hebrew University. Three sons Arik, Dudik, Yuval, eight grandchildren, and a brother, Danni Oppenheim, also survive Amos.