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Evidence for an active role of the DnaK chaperone system in the degradation of σ 32
Abstract
Under non-stressed conditions in Escherichia coli, the heat shock transcription factor σ32 is rapidly degraded by the AAA protease FtsH. The DnaK chaperone system is also required for the rapid turnover of σ32 in the cell. It has been hypothesized that the DnaK chaperone system facilitates the degradation of σ32 by sequestering it from RNA polymerase core. This hypothesis predicts that mutant σ32 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 σ32 proteins. Results indicated that the mutant σ32 proteins as similar as authentic σ32 were stabilized in ΔdnaK and ΔdnaJ/ΔcbpA cells. The interaction between σ32 and DnaK/DnaJ/GrpE was not affected by these mutations. These results strongly suggest that the degradation of σ32 requires an unidentified active role of the DnaK chaperone system.
... In addition to LpxC, FtsH degrades some other cytoplasmic key regulators like the heat shock sigma factor σ 32 (RpoH) and the phage λ proteins CII, CIII and Xis (Herman et al ., 1995;Shotland et al ., 1997;Leffers and Gottesman, 1998). Degradation of RpoH is mediated by the chaperones DnaK, DnaJ (Tatsuta et al ., 2000) and GroEL (Guisbert et al ., 2004), whereas degradation of the other cytoplasmic substrates seems to be chaperone-independent. ...
... Processive degradation of misfolded membrane proteins can be initiated from exposed tails at either end of the substrate (Chiba et al., 2002). Turnover of σ 32 requires an internal region around amino acid 50 of the sigma factor (Bertani et al., 2001;Horikoshi et al., 2004;Obrist and Narberhaus, 2005) and the contribution of the chaperones DnaKJ (Tatsuta et al., 2000) and GroEL (Guisbert et al., 2004). On the other hand, Cterminal recognition of the λ CII protein was shown to be Table 2. ...
... As hflD mutants are viable, degradation of LpxC does not require HflD. The association of DnaK and DnaJ with purified LpxC prompted us to ask whether these chaperones are involved in degradation of the deacetylase although preliminary experiments had suggested that it might not be the case (Tatsuta et al., 2000). Our more detailed analysis demonstrated that degradation of LpxC by FtsH is completely independent of DnaKJ. ...
Lipopolysaccharide (LPS) biosynthesis is essential in Gram negative bacteria. LpxC, the key enzyme in LPS formation, catalyses the limiting reaction and controls the ratio between LPS and phospholipids. As overproduction of LPS is toxic, the cellular amount of LpxC must be regulated carefully. The membrane-bound protease FtsH controls the level of LpxC via proteolysis making FtsH the only essential protease of Escherichia coli. We found that the chaperones DnaK and DnaJ co-purified with LpxC. However, degradation of LpxC was DnaK/J-independent in contrast to turnover of the heat shock sigma factor sigma32 (RpoH). The stability of LpxC in a bacterial one-hybrid system suggested that a terminus of LpxC might be important for degradation. Different LpxC truncations and extensions were constructed. Removal of at least five amino acids from the C-terminus abolished degradation by FtsH in vivo. While addition of two aspartic acids to LpxC did not alter its half-life, the exchange of the last two residues against aspartic acids resulted in stabilization. All stable LpxC enzymes were active in vivo as assayed by their high toxicity. Our data demonstrate that the C-terminus of LpxC contains a signal sequence necessary for FtsH-dependent degradation.
... Although σ 32 overaccumulates in the absence of FtsH, the overproduced σ 32 is not fully active as a transcription initiation factor owing presumably to its association with the DnaKJ chaperone system (85,90). The DnaK chaperone may actually have a positive role in the degradation (84), presumably by presenting σ 32 to FtsH. Although FtsH provides a basis for the cellular instability of σ 32, it is not a factor that directly controls the heat shock response of E. coli cells. ...
... It is a peripherally membraneinteracting protein that binds to and inactivates cII while accelerating its in vivo degradation by FtsH (49). On the other hand, the DnaK chaperone system may present σ 32 to FtsH (84), whereas cII degradation is unaffected by DnaK (79). These factors could be categorized as substrate-specific modulators of FtsH, conceptually analogous to the adaptor proteins for the Clp and other AAA+ enzyme systems (27). ...
This chapter covers the structural chemistry and the biological aspects of FtsH protease. FtsH has an ATP-dependent protease activity against a set of soluble regulatory proteins (such as σ32) and uncomplexed subunits (such as SecY) of membrane protein assemblies. Degradation of these substrates seems to proceed processively. The nucleotide sequence predicts that E. coli FtsH is a 70.7 kDa protein with 644 amino acid residues. However, an alternative initiation codon (UUG), which extends the open reading frame (71.0 kDa protein with 647 residues) by three amino acid residues at the N-terminus, was suggested from analyses of the N-terminal amino acid sequences of purified samples. FtsH undergoes self-processing, in which seven amino acid residues are removed from its C-terminus, although both the processed and the unprocessed forms have indistinguishable activities, and hence biological significance of this self-processing is obscure. The heat-shock σ factor (σ32) is unstable in vivo, and its degradation is catalyzed by FtsH, although involvement of other ATP-dependent proteases was also reported.
... However, in this background we found that the expression level of the complex was too low. This could be due to the fact that, in vivo, σ 32 is degraded by the AAA+ protein FtsH, which is a membrane-anchored ATPdependent metalloprotease (Tomoyasu et al., 1998, Tatsuta et al., 2000, and DnaK is necessary for this degradation. We transformed the vector expressing the complex into a ∆dnaK-∆ftsH strain. ...
... The expression level of the complex was too low. In vivo, σ 32 is degraded by the AAA+ protein FtsH (a membrane-anchored ATP dependent metal protease, Tomoyasu et al., 1998, Tatsuta et al., 2000. It has been shown in the past that σ 32 is better expressed in delta ftsH strains. ...
Hsp70 chaperones assist a large variety of protein folding processes in the cell by ATP-controlled cycles of substrate binding and release that are regulated by J-proteins and nucleotide exchange factors. Hsp70 chaperones bind to almost all unfolded proteins but generally do not interact with their native counterparts. However, Hsp70 also recognize certain folded proteins as substrates, like natively folded regulatory proteins, and modulates their activities. Even though the binding to peptide substrates has been extensively studied, it is still unclear how the binding to natively folded substrates occurs. It is also unknown whether Hsp70 proteins keep their substrates in an unfolded conformation in solution or play a more active role by inducing conformational changes on them. The aim of this Thesis was to contribute to a deeper understanding of the Hsp70 interaction with natively folded substrates, studying their conformation and probing possible conformational changes due to the action of Hsp70. I have analyzed the interaction of the E. coli Hsp70 homologue DnaK and its co-chaperone DnaJ with two protein substrates whose activity is regulated by DnaK and DnaJ: the heat-shock transcription factor s32 and the replication initiator protein RepE. Using amide hydrogen exchange experiments combined with mass spectrometry, and deletion and point-mutation constructs, I have identified the DnaK and DnaJ binding sites in s32. I have been able to show that both chaperones influence the conformation of s32 by destabilizing specific regions distant to their binding sites. DnaJ binding to s32 destabilizes a region in close spatial vicinity to the DnaK binding site, thereby explaining the catalytic action of DnaJ in loading s32 onto DnaK and the synergistic stimulation of DnaKs ATPase activity by the simultaneous interaction of DnaJ and s32. DnaK destabilizes a region in the N-terminal domain, the primary target for the FtsH protease, which degrades s32 in vivo. RepE, on the other hand, performs different functions depending on its oligomeric state: as a dimer it represses its own synthesis while as a monomer it promotes replication initiation. Monomerization of RepE is regulated by DnaK. I have characterized the molecular mechanism of this regulation by investigating the conformation of dimeric RepE wild type and the constitutively monomeric variant RepE54 by amide hydrogen exchange experiments. I have been able to map the dimer interface in RepE and to identify the DnaK binding site which, interestingly, is not close to the dimer interface. Chaperone der Hsp70-Familie sind an einer Vielzahl zellulärer Proteinfaltungsvorgänge beteiligt, indem sie über ATP verbrauchende Reaktionszyklen Substrate binden und freisetzen. Diese Reaktionszyklen werden durch J-Proteine sowie Nukleotidaustauschfaktoren reguliert. Hsp70 Chaperone binden überwiegend ungefaltete Polypeptide, interagieren jedoch im allgemeinen nicht mit deren nativ gefalteten Formen. Hsp70 erkennt aber auch hochspezifisch bestimmte nativ gefaltete Proteine, insbesondere regulatorische Proteine, als Substrate und moduliert deren Aktivität. Obwohl die Bindung an Substrate bereits extensiv untersucht wurde, wobei hauptsächlich Modellpeptide zum Einsatz kamen, ist es immer noch weitgehend unverstanden, wie die Bindung an nativ gefaltete Substrate erfolgt. Außerdem ist unklar, ob Hsp70 Proteine ihre Substrate nur in einem ungefalteten Zustand halten können oder eine aktive Rolle übernehmen, indem sie Konformationsänderungen im Substrat auslösen. Das Ziel dieser Arbeit war, zum Verständnis der Interaktion zwischen Hsp70 und nativ gefalteten Substraten beizutragen, indem deren Konformation und durch Hsp70 verursachte Konformationsänderungen untersucht wurden. Ich analysierte dafür die Interaktion zwischen dem Hsp70-Homologen aus E. coli DnaK sowie dessen Co-Chaperon DnaJ mit zwei Proteinsubstraten, deren Aktivität über DnaK und DnaJ reguliert wird: den Hitzeschocktranskriptionsfaktor s32 und das Replikationsinitiatorprotein RepE. Die Bindestellen von DnaK und DnaJ in s32 wurden mittels Amidprotonenaustausch und Massenspektrometrie sowie über Deletions-und Punktmutationskonstrukte identifiziert. Ich konnte zeigen, dass beide Chaperone die Konformation von s32 beeinflussen, indem sie bestimmte Regionen destabilisieren, welche erstaunlicherweise entfernt von der jeweiligen Bindestelle liegen. Die Bindung von DnaJ an s32 destabilisiert einen Bereich nahe der Bindestelle von DnaK, wodurch die katalytische Aktivität von DnaJ erklärt wird, welche darin besteht, das Substrat auf DnaK zu laden und die ATPase-Aktivität von DnaK synergistisch zu stimulieren. DnaK destabilisiert eine Region in der N-terminalen Domäne, dem Hauptangriffspunkt der Protease FtsH, die s32 in vivo abbaut. RepE führt abhängig vom Oligomerzustand verschiedene Funktionen aus: Als Dimer verhindert es seine eigene Synthese, als Monomer begünstigt es die Initiation der Replikation. DnaK reguliert die Monomerisierung von RepE. Ich konnte den molekularen Mechanismus der Monomerisierung aufklären, indem ich die Konformation des dimeren RepE und einer konstitutiv monomeren Varianten, RepE54, mittels Amidprotonenaustausch-Experimenten verglich. Dadurch konnte ich die Dimerisierungsgrenzfläche kartieren und außerdem die Bindestelle von DnaK identifizieren, welche überraschenderweise nicht in der räumlichen Nähe der Dimerisierungsregion liegt.
... Altered RpoH stability might be caused by enhanced or decreased interaction with any of these partners. A role of region C in DnaK binding is disputed (Nagai et al., 1994;McCarty et al., 1996;Joo et al., 1998;Arsène et al., 1999;Tatsuta et al., 2000). Hence, we conducted pull-down experiments with histidine-tagged RpoH-A131E-K134V, RpoH-A131E and RpoH-K134V. ...
... Region C is predicted to serve multiple functions in heat shock gene expression in E. coli mediating interaction with RNAP and with DnaK (Nagai et al., 1994;Nakahigashi et al., 1995;McCarty et al., 1996;Joo et al., 1998;Arsène et al., 1999;Tatsuta et al., 2000). According to structural models (based on crystal structures of s 70 ), residues A131 and K134 of RpoH turn away from the RNAP-binding site (Fig. 6) which is also the case for the RssB-binding motif in RpoS (Fig. 6). ...
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.
... Although σ 32 overaccumulates in the absence of FtsH, the overproduced σ 32 is not fully active as a transcription initiation factor owing presumably to its association with the DnaKJ chaperone system (85,90). The DnaK chaperone may actually have a positive role in the degradation (84), presumably by presenting σ 32 to FtsH. Although FtsH provides a basis for the cellular instability of σ 32, it is not a factor that directly controls the heat shock response of E. coli cells. ...
... It is a peripherally membraneinteracting protein that binds to and inactivates cII while accelerating its in vivo degradation by FtsH (49). On the other hand, the DnaK chaperone system may present σ 32 to FtsH (84), whereas cII degradation is unaffected by DnaK (79). These factors could be categorized as substrate-specific modulators of FtsH, conceptually analogous to the adaptor proteins for the Clp and other AAA+ enzyme systems (27). ...
FtsH is a cytoplasmic membrane protein that has N-terminally located transmembrane segments and a main cytosolic region consisting of AAA-ATPase and Zn2+-metalloprotease domains. It forms a homo-hexamer, which is further complexed with an oligomer of the membrane-bound modulating factor HflKC. FtsH degrades a set of short-lived proteins, enabling cellular regulation at the level of protein stability. FtsH also degrades some misassembled membrane proteins, contributing to their quality maintenance. It is an energy-utilizing and processive endopeptidase with a special ability to dislocate membrane protein substrates out of the membrane, for which its own membrane-embedded nature is essential. We discuss structure-function relationships of this intriguing enzyme, including the way it recognizes the soluble and membrane-integrated substrates differentially, on the basis of the solved structure of the ATPase domain as well as extensive biochemical and genetic information accumulated in the past decade on this enzyme.
... These phenomena can be accounted by the accumulation of RpoH, also known as r 32 or r H , in these mutant strains, which induces expression of DnaK and DnaJ. 35 These results are also consistent with the previous observation. 16 In addition, the level of DjlA was also much higher in the strain DdnaK compared to the wild-type strain via unknown mechanisms. ...
In Escherichia coli, the major bacterial Hsp70 system consists of DnaK, three J-domain proteins (JDPs: DnaJ, CbpA, and DjlA), and nucleotide exchange factor GrpE. JDPs determine substrate specificity for the Hsp70 system; however, knowledge on their specific role in bacterial cellular functions is limited. In this study, we demonstrated the role of JDPs in bacterial survival during heat stress and the DnaK-regulated formation of curli—extracellular amyloid fibers involved in biofilm formation. Genetic analysis demonstrate that only DnaJ is essential for survival at high temperature. On the other hand, either DnaJ or CbpA, but not DjlA, is sufficient to activate DnaK in curli production. Additionally, several DnaK mutants with reduced activity are able to complement the loss of curli production in E. coli ΔdnaK, whereas they do not recover the growth defect of the mutant strain at high temperature. Biochemical analyses reveal that DnaJ and CbpA are involved in the expression of the master regulator CsgD through the solubilization of MlrA, a DNA-binding transcriptional activator for the csgD promoter. Furthermore, DnaJ and CbpA also keep CsgA in a translocation-competent state by preventing its aggregation in the cytoplasm. Our findings support a hierarchical model wherein the role of JDPs in the Hsp70 system differs according to individual cellular functions.
... CheY (Rank 12) is a signal transduction system response regulator, while FtsY (Rank 18) is a signal recognition particle receptor. DNA polymerase III DnaN (Rank 9) is required for initiation and processivity of DNA replication; DnaK (Rank 10) acts as a chaperone protein which provides stability in the transcriptional regulation process [32]; and RpoS (Rank 15) is an RNA polymerase sigma factor which can be used to coordinate transcription factors through protein-protein interactions [33]. ...
Shewanella oneidensis MR-1 can transfer electrons from the intracellular environment to the extracellular space of the cells to reduce the extracellular insoluble electron acceptors (Extracellular Electron Transfer, EET). Benefiting from this EET capability, Shewanella has been widely used in different areas, such as energy production, wastewater treatment, and bioremediation. Genome-wide proteomics data was used to determine the active proteins involved in activating the EET process. We identified 1012 proteins with decreased expression and 811 proteins with increased expression when the EET process changed from inactivation to activation. We then networked these proteins to construct the active protein networks, and identified the top 20 key active proteins by network centralization analysis, including metabolism- and energy-related proteins, signal and transcriptional regulatory proteins, translation-related proteins, and the EET-related proteins. We also constructed the integrated protein interaction and transcriptional regulatory networks for the active proteins, then found three exclusive active network motifs involved in activating the EET process-Bi-feedforward Loop, Regulatory Cascade with a Feedback, and Feedback with a Protein-Protein Interaction (PPI)-and identified the active proteins involved in these motifs. Both enrichment analysis and comparative analysis to the whole-genome data implicated the multiheme c-type cytochromes and multiple signal processing proteins involved in the process. Furthermore, the interactions of these motif-guided active proteins and the involved functional modules were discussed. Collectively, by using network-based methods, this work reported a proteome-wide search for the key active proteins that potentially activate the EET process.
... The function of the AAA + -protease ClpAP is inhibited by ClpS (Guo et al., 2002;Zeth et al., 2002); in contrast ClpXP function is stimulated by SspB Levchenko et al., 2000). Functional stimulation of FtsH is achieved by the DnaK/DnaJ/GroE chaperone system (Tatsuta et al., 2000). No such adaptors are known for bacterial Lon. ...
... Another important function of FtsH is to regulate expression of σ 32 , the heat shock sigma factor required for heat shock or other stress responses in E. coli. Regulation of σ 32 by FtsH is assumed to involve its association with the DnaKJ chaperone system in which the DnaK chaperone is assumed to have a positive role in the degradation by presenting σ 32 to FtsH (Tatsuta et al., 2000;Tatsuta et al., 1998;Tomoyasu et al., 1998). ...
... Yet another regulation of 32 happens on the level of 32 protein stability. Under nonstress conditions, 32 has a very short half-life due to its inactivation with the DnaK chaperone system (24,50), which mediates 32 's degradation by the protease FtsH (26,51). Heat shock conditions lead to the unfolding of cellular proteins, which become substrates of DnaK and in turn titrate the DnaK system. ...
Formation of nonnative disulfide bonds in the cytoplasm, so-called disulfide stress, is an integral component of oxidative
stress. Quantification of the extent of disulfide bond formation in the cytoplasm of Escherichia coli revealed that disulfide stress is associated with oxidative stress caused by hydrogen peroxide, paraquat, and cadmium. To
separate the impact of disulfide bond formation from unrelated effects of these oxidative stressors in subsequent experiments,
we worked with two complementary approaches. We triggered disulfide stress either chemically by diamide treatment of cells
or genetically in a mutant strain lacking the major disulfide-reducing systems TrxB and Gor. Studying the proteomic response
of E. coli exposed to disulfide stress, we found that intracellular disulfide bond formation is a particularly strong inducer of the
heat shock response. Real-time quantitative PCR experiments showed that disulfide stress induces the heat shock response in
E. coli σ32 dependently. However, unlike heat shock treatment, which induces these genes transiently, transcripts of σ32-dependent genes accumulated over time in disulfide stress-treated cells. Analyzing the stability of σ32, we found that this constant induction can be attributed to an increase of the half-life of σ32 upon disulfide stress. This is concomitant with aggregation of E. coli proteins treated with diamide. We conclude that oxidative stress triggers the heat shock response in E. coli σ32 dependently. The component of oxidative stress responsible for the induction of heat shock genes is disulfide stress. Nonnative
disulfide bond formation in the cytoplasm causes protein unfolding. This stabilizes σ32 by preventing its DnaK- and FtsH-dependent degradation.
... Durch diesen Prozess wird die Erkennung von SsrA-markierten Proteinen durch ClpX verstärkt ( Levchenko et al., 2000). Im Gegensatz dazu bindet ClpS an die N-Domänen von ClpA und inhibiert die Proteolyse von SsrA-markierten Proteinen Zeth et al., 2002;Xia et al., 2004 Blaszczak et al., 1999;Tatsuta et al., 2000). ...
ATP-abhängige Proteasen der AAA+-Proteinfamilie vermitteln die energieabhängige Proteolyse zellulärer Proteine. Zwei hochkonservierte AAA-Proteasen in der inneren Mitochondrienmembran üben wichtige Funktionen bei der mitochondrialen Qualitätskontrolle und Biogenese aus. Die zugrunde liegenden Mechanismen der Substraterkennung von AAA-Proteasen sind allerdings kaum verstanden. Zur Identifizierung und funktionalen Charakterisierung von Substratbindestellen sollte daher die i-AAA-Protease Yme1 aus S. cerevisiae untersucht werden. In dieser Arbeit konnten zwei Substratbinderegionen in Yme1 identifiziert werden, die in der Kristallstruktur einer homologen AAA-Protease den hexameren Ringkomplex an der Oberfläche käfigartig umfassen. Die Substitution von konservierten, negativ geladenen Resten innerhalb einer überwiegend alpha-helikalen Region am N-Terminus der AAA-Domäne(NH-Region) führte zu einer gestörten Substratbindung an diese Region und zu einem generellen Defekt der Proteolyse. Eine zweite Bindestelle wurde durch Domänenaustauschexperimente zwischen Yme1 und der orthologen AAA-Protease IAP-1 aus N. crassa identifiziert. Diese Region setzte sich aus konservierten C-terminalen alpha-Helices der proteolytischen Domäne zusammen (CH-Region) und vermittelte die Proteolyse von Substratproteinen in Abhängigkeit von deren Faltungszustand und Membraninsertion sowie einem substratspezifischen Faktor. Des Weiteren wurde die Funktion des konservierten YVG-Motivs, das in der zentralen Pore von hexameren AAA+-Ringstrukturen lokalisiert ist, für den Abbau von Membranproteinen durch Yme1 über Mutationsanalyse untersucht. Eine hydrophile Substitution von Y354 bewirkte dabei substratspezifische Defekte bei der Bindung von Substratproteinen. Diese führen wahrscheinlich auch zu der beobachteten Abhängigkeit der proteolytischen und in vivo-Aktivität der Protease von Aminosäureresten mit hoher Hydrophobizität an dieser Position. Die Ergebnisse dieser Untersuchung legen ein Modell von alternativen Bindungswegen für die Substratinteraktion von AAA-Proteasen nahe, in dem entfaltete Substrate sequentiell mit zwei oberflächenexponierten Bindestellen interagieren können, bevor sie durch die zentrale Pore des AAA-Rings in die proteolytische Kammer transloziert werden. Die Identifizierung von definierten Substratbindestellen erlaubt es nun, in weiteren Experimenten die molekularen Grundlagen der Substraterkennung durch AAA-Proteasen detailliert zu bestimmen. ATP-dependent proteases of the AAA+ protein family mediate the energy-dependent proteolysis of cellular proteins. Two highly conserved AAA proteases anchored to the inner membrane of mitochondria exert crucial functions for mitochondrial protein quality control and biogenesis. However, molecular mechanisms for substrate recognition by AAA proteases are barely understood. In order to define initial steps of substrate engagement by AAA protease the i-AAA protease Yme1 from S. cerevisiae was examined. This study led to the identification of two substrate binding regions. They formed, based on the crystal structure of a homologous AAA protease, a lattice-like structure on the surface of the hexameric ring complex. Substitution of conserved negatively charged residues present in a mainly alpha-helical region at the C-terminal end of the AAA domain (NH-region) caused an impaired substrate binding to this region and led to a complete loss of proteolytic activity. A second region was identified by a domain swapping approach based on Yme1 and the orthologous AAA protease IAP-1 from N. crassa. This region was composed of conserved C-terminal helices within the proteolytic domain (CH-region). Its involvement in proteolysis depended on the folding and membrane insertion of substrates as well as a substrate specific factor. Furthermore, a conserved YVG-motif has been located to the central pore of other hexameric AAA+ ring complexes. Therefore, its role for proteolysis of membrane-bound proteins by Yme1 has been examined by mutational analysis. Hydrophilic substitution of Y354 led to substrate-specific defects on substrate engagement. Consistently, the presence of hydrophobic residues at this position was required for proteolytic and in vivo activity of the protease. Taken together, the mutational analysis suggests alternative pathways for substrate engagement. Misfolded proteins can interact sequentially with surface-exposed binding sites before they are translocated through the central pore into a proteolytic chamber formed by AAA proteases. The identification of substrate binding sites will allow defining molecular determinants of substrate recognition by AAA proteases.
... The proteolysis of r 32 by HflB is very rapid and requires the DnaK-DnaJ-GrpE chaperone machine in vivo [18][19][20]. However, this proteolysis is much slower in vitro, because of a lack of chaperone machinery. ...
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.
... DnaK has also been implicated in directing abnormal proteins to the degradation machinery [56] and in controlling the activity of other regulatory proteins such as RepA, the initiator protein for plasmid replication [57]. Both DnaK and DnaJ have been shown to autoregulate the heat shock response by directly binding to σ 325859, the heat shock transcription factor, and targeting it for degradation by the inner-membraneassociated protease FtsH [60]. ...
Folding of polypeptides in the cell typically requires the assistance of a set of proteins termed molecular chaperones. Chaperones are an essential group of proteins necessary for cell viability under both normal and stress conditions. There are several chaperone systems which carry out a multitude of functions all aimed towards insuring the proper folding of target proteins. Chaperones can assist in the efficient folding of newly-translated proteins as these proteins are being synthesized on the ribosome and can maintain pre-existing proteins in a stable conformation. Chaperones can also promote the disaggregation of preformed protein aggregates. Many of the identified chaperones are also heat shock proteins. The general mechanism by which chaperones carry out their function usually involves multiple rounds of regulated binding and release of an unstable conformer of target polypeptides. The four main chaperone systems in the Escherichia coli cytoplasm are as follows. (1) Ribosome-associated trigger factor that assists in the folding of newly-synthesized nascent chains. (2) The Hsp 70 system consisting of DnaK (Hsp 70), its cofactor DnaJ (Hsp 40), and the nucleotide exchange factor GrpE. This system recognizes polypeptide chains in an extended conformation. (3) The Hsp 60 system, consisting of GroEL (Hsp 60) and its cofactor GroES (Hsp 10), which assists in the folding of compact folding intermediates that expose hydrophobic surfaces. (4) The Clp ATPases which are typically members of the Hsp 100 family of heat shock proteins. These ATPases can unfold proteins and disaggregate preformed protein aggregates to target them for degradation. Several advances have recently been made in characterizing the structure and function of all of these chaperone systems. These advances have provided us with a better understanding of the protein folding process in the cell.
... It has been proposed, based on data from different research groups working with E. coli, that the DnaK chaperone system (DnaK, DnaJ and GrpE) controls s 32 activity by inhibiting its binding to the core RNAP and by sequestering s 32 from RNAP, making it accessible to degradation by the FtsH protease Tomoyasu et al., 1998;Blaszczak et al., 1999). More recently, Tatsuta et al. (2000) have shown that mutant s 32 proteins, which are deficient in RNAP-binding, are still dependent on DnaK chaperone system for degradation, suggesting an active unidentified role of this chaperone machine in that process. ...
Expression of heat shock genes in Gram-negative proteobacteria is positively modulated by the transcriptional regulator RpoH, the sigma(32) subunit of RNA polymerase (RNAP). In this study we investigated the chaperones DnaK/DnaJ and GroES/GroEL as possible modulators of the heat response in Caulobacter crescentus. We have shown that cells overexpressing DnaK show poor induction of heat shock protein (HSP) synthesis, even though sigma(32) levels present a normal transient increase upon heat stress. On the other hand, depletion of DnaK led to higher levels of sigma(32) and increased transcription of HSP genes, at normal growth temperature. In contrast, changes in the amount of GroES/EL had little effect on sigma(32) levels and HSP gene transcription. Despite the strong effect of DnaK levels on the induction phase of the heat shock response, downregulation of HSP synthesis was not affected by changes in the amount this chaperone. Thus, we propose that competition between sigma(32) and sigma(73), the major sigma factor, for the core RNAP could be the most important factor controlling the shut-off of HSP synthesis during recovery phase. In agreement with this hypothesis, we have shown that expression of sigma(73) gene is heat shock inducible.
... In vivo 32 is very unstable with a half-life of approximately 1 min at 30°C (2). Degradation of 32 in vivo requires the AAAϩ protease FtsH and the DnaK chaperone system (3,4). Imme-diately after temperature up-shift the synthesis as well as the half-life of 32 transiently increase by 10-and 8-fold, respectively. ...
Stress conditions such as heat shock alter the transcriptional profile in all organisms. In Escherichia coli the heat shock transcription factor, σ32, out-competes upon temperature up-shift the housekeeping σ-factor, σ70, for binding to core RNA polymerase and initiates heat shock gene transcription. To investigate possible heat-induced conformational
changes in σ32 we performed amide hydrogen (H/D) exchange experiments under optimal growth and heat shock conditions combined with mass
spectrometry. We found a rapid exchange of around 220 of the 294 amide hydrogens at 37 °C, indicating that σ32 adopts a highly flexible structure. At 42 °C we observed a slow correlated exchange of 30 additional amide hydrogens and
localized it to a helix-loop-helix motif within domain σ2 that is responsible for the recognition of the -10 region in heat shock promoters. The correlated exchange is shown to constitute
a reversible unfolding with a half-life of about 30 min due to a temperature-dependent decrease in stabilization energy. We
propose that this gradual decrease in stabilization energy of domain σ2 with increasing temperatures facilitates the unfolding of σ32 by the AAA+ protease FtsH thereby decreasing its half-life. Taken together our data show that the σ2 domain of σ32 can act as a thermosensor, which might be important for the heat shock regulation.
... Stabilization of 32 might therefore result from elevated affinity for core RNA polymerase, since region 2.1 has been thought to be one of the sites involved in binding to core RNA polymerase (21,28,39,51) and 32 bound to polymerase is hardly degraded by proteases in vitro (4,17). To exclude this possibility, we constructed and examined the stability of stable 32 mutants containing the Q80R mutation, which is known to reduce the affinity for core RNA polymerase (13) and not to affect the 32 stability (44). When the Q80R mutation was introduced into two stable 32 mutants, L47Q-L55Q and I54A, the resulting 32 mutants (L47Q-L55Q-Q80R and I54A-Q80R) showed reduced DnaK and GroEL levels compared to wild-type 32 ( Fig. 5A and data not shown), unlike I54A (Fig. 4), presumably as a result of reduced affinity for core RNA polymerase. ...
Escherichia coli heat shock transcription factor σ32 is rapidly degraded in vivo, with a half-life of about 1 min. A set of proteins that includes the DnaK chaperone team (DnaK,
DnaJ, GrpE) and ATP-dependent proteases (FtsH, HslUV, etc.) are involved in degradation of σ32. To gain further insight into the regulation of σ32 stability, we isolated σ32 mutants that were markedly stabilized. Many of the mutants had amino acid substitutions in the N-terminal half (residues
47 to 55) of region 2.1, a region highly conserved among bacterial σ factors. The half-lives ranged from about 2-fold to more
than 10-fold longer than that of the wild-type protein. Besides greater stability, the levels of heat shock proteins, such
as DnaK and GroEL, increased in cells producing stable σ32. Detailed analysis showed that some stable σ32 mutants have higher transcriptional activity than the wild type. These results indicate that the N-terminal half of region
2.1 is required for modulating both metabolic stability and the activity of σ32. The evidence suggests that σ32 stabilization does not result from an elevated affinity for core RNA polymerase. Region 2.1 may, therefore, be involved in
interactions with the proteolytic machinery, including molecular chaperones.
... The predominant group of heat shock proteins is the 70 kDa Hsp70s. In addition to their function in the folding of newly synthesized proteins and the refolding of denatured proteins, they have also been implicated in protein degradation, such as through the lysosomal pathway [3], the specific degradation of the heat shock transcription factor r 32 in Escherichia coli [4], and via the ubiquitination pathway in eukaryotes, for example, in the degradation of polyglutamine repeat containing proteins (reviewed in [5]). All Hsp70 functions are dependent on its chaperone property of maintaining substrate polypeptides in an extended conformation and stabilizing the exposed hydrophobic regions [6]. ...
Molecular chaperones are integral components of the cellular machinery involved in ensuring correct protein folding and the continued maintenance of protein structure. An understanding of these ubiquitous molecules is key to finding cures to protein misfolding diseases such as Alzheimer's and Creutzfeldt-Jacob diseases. In addition, further understanding of chaperones will enhance our comprehension of the way the body copes with the environmental stresses that humans encounter daily. Our laboratory and our collaborators specialize in the production and characterization of chaperones from a wide variety of sources in order to gain a fuller understanding of how chaperones function in the cell. In this review, we primarily use the Hsp70/Hsp40 chaperone pair as an example to discuss recent advances in technology and reductions in cost that lend themselves to chaperone purification from both native and recombinant sources. Common assays to assess purified chaperone activity are also discussed.
Shewanella species are well-known for their extracellular electron transfer (EET) capacity, by which these microorganisms can transfer the electrons from intracellular environment to extracellular space for the reduction of the extracellular insoluble electron acceptors. Using a time-stamped data for the paired protein-mRNA, we investigate the impact of differential translation on the EET process of Shewanella oneidensis MR-1. Firstly, differentially translated proteins when O2 levels are switched from high-O2 to low-O2 are identified by using a soft clustering method, 629 up-regulated translated proteins and 767 down-regulated translated proteins are considered to reflect the changes from inactivated to activated EET process. Then, we showed that the degrees of connectivity of differentially translated proteins were significantly larger than those of non-differentially translated proteins, and thereby these differentially translated proteins will be more important in the protein networks. After that, we networked these differentially translated proteins to construct the differentially translated sub-networks, and discussed the most important proteins that are involved in the EET process with the help of centralization analysis of these differentially translated networks. Furthermore, we also studied the differentially translated operonic genes. Taking together, this work searches key proteins that potentially activated the EET process from a translational efficiency viewpoint.
Shewanella oneidensis MR‐1 shows remarkable respiratory versatility with a large variety of extracellular electron acceptors (termed extracellular electron transfer, EET). To utilize the various electron acceptors, the bacterium must employ complex regulatory mechanisms to elicit the relevant EET pathways. To investigate the relevant mechanisms, we integrated EET genes and related transcriptional factors (TFs) into transcriptional regulatory modules (TRMs) and showed that many bridge proteins in these modules were signal proteins, which generally contained one or more signal processing domains (eg, GGDEF, EAL, PAS, etc.). Since Shewanella has to respond to diverse environmental conditions despite encoding few EET‐relevant TFs, the overabundant signal proteins involved in the TRMs can help decipher the mechanism by which these microbes elicit a wide range of condition‐specific responses. By combining proteomic data and protein bioinformatic analysis, we demonstrated that diverse signal proteins reconciled the different EET pathways, and we discussed the functional roles of signal proteins involved in the well‐known MtrCAB pathway. Additionally, we showed that the signal proteins SO_2145 and SO_1417 played central roles in triggering EET pathways in anaerobic environments. Taken together, our results suggest that signal proteins have a profound impact on the transcriptional regulation of EET genes and thus have potential applications in microbial fuel cells (MFCs). This article is protected by copyright. All rights reserved.
This review portraits FK506 binding protein (FKBP) 51 as “reactivity protein” and collates recent publications to develop the concept of FKBP51 as contributor to different levels of adaptation. Adaptation is a fundamental process that enables unicellular and multicellular organisms to adjust their molecular circuits and structural conditions in reaction to environmental changes threatening their homeostasis. FKBP51 is known as chaperone and co‐chaperone of heat shock protein (HSP) 90, thus involved in processes ensuring correct protein folding in response to proteotoxic stress. In mammals, FKBP51 both shapes the stress response and is calibrated by the stress levels through an ultrashort molecular feedback loop. More recently, it has been linked to several intracellular pathways related to the reactivity to drug exposure and stress. Through its role in autophagy and DNA methylation in particular it influences adaptive pathways, possibly also in a transgenerational fashion.
Also see the video abstract here.
The stability of heat-shock transcription factor σ(32) in Escherichia coli has long been known to be modulated only by its own transcribed chaperone DnaK. Very few reports suggest a role for another heat-shock chaperone, GroEL, for maintenance of cellular σ(32) level. The present study demonstrates in vivo physical association between GroEL and σ(32) in E. coli at physiological temperature. This study further reveals that neither DnaK nor GroEL singly can modulate σ(32) stability in vivo; there is an ordered network between them, where GroEL acts upstream of DnaK.
Systems biology is not new but in recent years it has begun to receive renewed attention. Properties of biological systems such as robustness and fragility emerge as central issues which can be well understood by application of principles of engineering to the biological systems. Besides this automatic control has played a vital role in the advance of engineering and science. Advances in control theory provide the means for obtaining efficient performance of the dynamic systems and improving productivity. In this context some of the recent and important applications of engineering principles particularly control theory in biology are discussed in this review.
heat shock promoter-specific 32 subunit of RNA polymerase by the AAA protease, FtsH. Previous studies implicated the C termini of protein substrates, including 32 , as degradation signals for AAA proteases. We investigated the role of the C terminus of 32 in FtsH-dependent degradation by analysis of C-terminally truncated 32 mutant proteins. Deletion of the 5, 11, 15, and 21 C-terminal residues of 32 did not affect degradation in vivo or in vitro. Furthermore, a peptide comprising the C-terminal 21 residues of 32 was not degraded by FtsH in vitro and thus did not serve as a recognition sequence for the protease, while an unrelated peptide of similar length was efficiently degraded. The truncated 32 mutant proteins remained capable of associating with DnaK and DnaJ in vitro but showed intermediate (5-amino-acid deletion) and strong (11-, 15-, and 21-amino-acid deletions) defects in association with RNA polymerase in vitro and biological activity in vivo. These results indicate an important role for the C terminus of 32 in RNA polymerase binding but no essential role for FtsH-dependent degradation and association of chaperones.
The ability to sense and respond to the environment is essential for the survival of all living organisms. Bacterial pathogens such as Salmonella enterica are of particular interest due to their ability to sense and adapt to the diverse range of conditions they encounter, both in vivo and in environmental reservoirs. During this cycling from host to non-host environments, Salmonella encounter a variety of environmental insults ranging from temperature fluctuations, nutrient availability and changes in osmolarity, to the presence of antimicrobial peptides and reactive oxygen/nitrogen species. Such fluctuating conditions impact on various areas of bacterial physiology including virulence, growth and antimicrobial resistance. A key component of the success of any bacterial pathogen is the ability to recognize and mount a suitable response to the discrete chemical and physical stresses elicited by the host. Such responses occur through a coordinated and complex programme of gene expression and protein activity, involving a range of transcriptional regulators, sigma factors and two component regulatory systems. This review briefly outlines the various stresses encountered throughout the Salmonella life cycle and the repertoire of regulatory responses with which Salmonella counters. In particular, how these Gram-negative bacteria are able to alleviate disruption in periplasmic envelope homeostasis through a group of stress responses, known collectively as the Envelope Stress Responses, alongside the mechanisms used to overcome nitrosative stress, will be examined in more detail.
This chapter demonstrates how measures of dynamic behavior can be used to study cell signaling systems. It analyzes the signaling time and signal duration of linear kinase-phosphatase cascades. It illustrates how quantitative measures can be used to investigate features of signaling cascades. This chapter shows how the quantitative signaling measures can be used to investigate various intrinsic properties of signaling cascades. It presents three commonly used models of mitogen-activated protein kinase (MAPK) cascades, representing different levels of approximation. It suggests that the MAPK pathway has evolved into a structure that favors a fast response of short duration to a stimulus.
Molecular chaperones are involved in the cell's quality control of protein synthesis and maintenance. This article reviews the pros and cons of various techniques for the purification of molecular chaperones, in particular Heat Shock Protein 70 (Hsp70) and Heat Shock Protein 40 (Hsp40), as well as approaches to assessing the capability of chaperones to do their job of folding and refolding proteins.
Introduction“Soluble” Hsp70s/J-proteins Function in General Protein Folding“Tethered” Hsp70s/J-proteins: Roles in Protein Folding on the Ribosome and in Protein TranslocationModulating of Protein Conformation by Hsp70s/J-proteinsCases of a Single Hsp70 Functioning With Multiple J-ProteinsHsp70s/J-proteins — When an Hsp70 Maybe Isn't Really a ChaperoneEmerging Concepts and Unanswered Questions
Via their interaction with client proteins, Hsp70 molecular chaperone machines function in a variety of cellular processes, including protein folding, translocation of proteins across membranes and assembly/disassembly of protein complexes. Such machines are composed of a core Hsp70, as well as a J‐protein and a nucleotide exchange factor as co‐chaperones. These co‐factors regulate the cycle of adenosine triphosphate (ATP) hydrolysis and nucleotide exchange, which is critical for Hsp70's interaction with client proteins. Cellular compartments often contain multiple Hsp70s, J‐proteins and nucleotide exchange factors. The capabilities of Hsp70s to carry out diverse cellular functions can result from either specialisation of an Hsp70 or by interaction of a multifunctional Hsp70 with a suite of J‐protein co‐chaperones. The well‐studied Hsp70 systems of mitochondria provide an example of such modes of diversification and specialisation of Hsp70 machinery, which are applicable to other cellular compartments.
Key Concepts
Fundamental biochemical properties of different Hsp70 systems are very similar, yet very adaptable.
Diversification of Hsp70 function is often due to multiple J‐protein partners.
Although most Hsp70s bind a broad array of peptide sequences, some have become specialised and have a very restricted binding specificity.
This chapter surveys evidence that Hsp40 and Hsp70 act as a molecular machine to disassemble protein complexes. Recent papers
have discussed whether Hsp70 actively unfolds proteins during translocation across membranes (Matouschek et al., 2000), whether it fragments aggregates or extracts polypeptides (Zietkiewicz et al., 2006), and whether it acts a “holdase” or a “foldase” (Slepenkov and Witt, 2002a,b). These are related questions because they ask whether Hsp70 exerts a force on its clients during these processes. The first
two sections of the chapter discuss the structures of Hsp70 and Hsp40 with an eye toward their interactions and conformational
changes, that is, the “moving parts”. Hsp40-Hsp70 chaperone machines have three levels of functional organization: (i) the
articulation of Hsp70 domains, (ii) the interactions with J-domain-containing protein(s), and (iii) interactions with additional
protein factors for targeting (e.g., TPR proteins) and regulation (e.g., GrpE). These additional factors are diverse and probably
not fundamental to a disassembly activity, and thus they generally will not be discussed. Subsequent sections discuss the
machine-like role of Hsp40-Hsp70 in several major classes of biochemical systems: the disassembly of protein complexes, protein
degradation, protein translocation across membranes, and protein folding.
Bacterial cells have limited abilities to modify and choose their dynamic environment. They utilize information processing
systems to monitor their surroundings constantly for important changes. Among the appropriate responses to environmental changes
are alterations in physiology, development, virulence, and location. In most species, highly sophisticated global regulatory
networks modulate the expression of genes. These effects are mediated in large part through the activation or repression of
mRNA transcript initiation by DNA-binding proteins, σ-factors, and corresponding signal transduction systems. This adaptive
response is based on appropriate genetic programmes allowing them to respond rapidly and effectively to environmental changes
that impair growth or even threaten their life. Cellular homeostasis is achieved by a multitude of sensors and transcriptional
regulators, which are able to sense and respond to changes in temperature (heat and cold shock), external pH (alkaline and
acid shock), reactive oxygen species (hydrogen peroxide and superoxide), osmolarity (hyper- and hypoosmotic shock), and nutrient
availability to mention the most important ones. These changes are often called stress factors, and stresses can come at a
sudden (catastrophic stress) or grow and grow (pervasive stress). Each stress factor leads to the induction of a subset of
genes, the stress genes coding for stress proteins. It should be mentioned that challenge to any stress factor will not only
result in induction of genes, but also in repression or even turn off of a subset of genes, but the underlying mechanisms
are largely unknown. While some genes are induced by only one single stress factor, others respond to several. The former
are termed specific stress genes and the latter general stress genes.
Since Ritossa’s seminal discovery in 1962 that the puffing pattern changes of Drosophila salivary gland polytene chromosomes can be induced by heat shock and chemical treatment (Ritossa, 1962), the heat shock response (HSR) has served as an excellent model and paradigm of inducible gene expression. During the ensuing
decades considerable evidence has accumulated, from diverse areas of biology, about the regulation of the stress response
during development, homeostatic maintenance of organs and organisms, and pathophysiological conditions. From bacteria to man,
environmental stress, cell growth, differentiation, and pathophysiological states are all known to induce the rapid and reversible
synthesis of evolutionary conserved set of proteins commonly termed, heat shock proteins (HSPs). HSPs, acting as molecular
chaperones, play essential roles in protein folding, trafficking, higher order assembly and degradation of proteins thereby
ensuring survival under both stressful and extreme physiological conditions (Lindquist, 1986; Lindquist and Craig, 1988; Morimoto, 1998).
The heat shock or stress response has been studied mainly as a cellular response. Most of the data come from bacterial cells,
eukaryotic microorganisms (yeast primarily), and cultured animal cells. Often these cultured cells are tumor cell lines, i.e.,
cells that are functionally eukaryotic microorganisms as a consequence of genetic changes that change their social behavior
and proliferative control. These systems have provided useful information about stress protein function and their roles in
the defensive cellular state of cytoprotection. However, a full understanding of stress response biology in complex multicellular
organisms requires different thinking and different models. This conclusion stems from the paradigm that the basic unit of
function in animals and plants is not the individual cell but the tissue. Therefore stress response biology in these complex
biological systems is primarily about tissue-level protection. Ultimately we would like to know how these responses are deployed
in humans and how these inducible defenses may be used to prevent tissue damage from disease and from surgical intervention.
Most bacteria live in a dynamic environment where temperature, availability of nutrients and the presence of various chemicals vary, which requires rapid adaptation. Many of the adaptive changes are determined by changes in the transcription of global regulatory networks, but this response is slow because most bacterial proteins are stable and their concentration remains high even after transcription slows down. To respond rapidly, an additional level of regulation has evolved: the degradation of key proteins. However, as proteolysis is an irreversible process, it is subject to tight regulation of substrate binding and degradation. Here we review the roles of the proteolytic enzymes in Gram-negative bacteria and how these enzymes can be regulated to target only a subset of proteins.
Molecular chaperones consist of several highly conserved families of proteins, many of which consist of heat shock proteins. The primary function of molecular chaperones is to facilitate the folding or refolding of proteins, and therefore they play an important role in diverse cellular processes including protein synthesis, protein translocation, and the refolding or degradation of proteins after cell stress. Cells are often exposed to different stressors, resulting in protein misfolding and aggregation. It is now well established that the levels of certain molecular chaperones are elevated during stress to provide protection to the cell. The focus of this review is on the impact of molecular chaperones in biology, medicine and protein biotechnology, and thus covers both fundamental and applied aspects of chaperone biology. Attention is paid to the functions and applications of molecular chaperones from bacterial and eukaryotic cells, focusing on the heat shock proteins 90 (Hsp90), 70 (Hsp70) and 40 (Hsp40) classes of chaperones, respectively. The role of these classes of chaperones in human diseases is discussed, as well as the parts played by chaperones produced by the causative agents of malaria and trypanosomiasis. Recent advances have seen the application of chaperones in improving the yields of a particular target protein in recombinant protein production. The prospects for the targeted use of molecular chaperones for the over-production of recombinant proteins is critically reviewed, and current research on these chaperones at Rhodes University is also discussed.
The heat shock response (HSR) is a homeostatic response that maintains the proper protein-folding environment in the cell. This response is universal, and many of its components are well conserved from bacteria to humans. In this review, we focus on the regulation of one of the most well-characterized HSRs, that of Escherichia coli. We show that even for this simple model organism, we still do not fully understand the central component of heat shock regulation, a chaperone-mediated negative feedback loop. In addition, we review other components that contribute to the regulation of the HSR in E. coli and discuss how these additional components contribute to regulation. Finally, we discuss recent genomic experiments that reveal additional functional aspects of the HSR.
The proteolysis of regulatory proteins plays an important role in the control of gene expression. The Escherichia coli heat shock sigma factor RpoH (sigma(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.
The heat shock response in bacteria is a complex phenomenon in which sigma 32 plays the central role. The DnaK/J chaperone system binds and promotes degradation of sigma 32 at lower temperatures. At heat shock temperatures, the DnaK/J-mediated degradation of sigma 32 is largely abolished by a mechanism, which is not yet fully understood. In this article we have shown that interaction of DnaK with sigma 32 is highly temperature-dependent. This interaction is completely abolished at 42 degrees C. To investigate the origin of such strong temperature dependence, we have monitored the structural changes that occur in the sigma 32 protein upon upshift of temperature and attempted to elucidate its functional roles. Upon a shift of temperature from 30 to 42 degrees C, the CD spectrum of sigma 32 becomes significantly more positive without significant change in either tryptophan fluorescence spectra or quenchability to external quenchers. 1,8-Anilinonaphthalene sulfonic acid binding at 42 degrees C is not significantly affected. The equilibrium guanidine hydrochloride denaturation of sigma 32 is biphasic. The first phase shifts to even lower guanidine hydrochloride concentrations at 42 degrees C, whereas the major phase remains largely unchanged. The sigma 32-core interaction remains unchanged as a function of temperature. This suggests that increased temperature destabilizes a structural element. We discuss the possible location of this temperature-sensitive structural element.
FtsH is a cytoplasmic membrane-integrated, ATP-dependent metalloprotease, which processively degrades both cytoplasmic and membrane proteins in concert with unfolding. The FtsH protein is divided into the N-terminal transmembrane region and the larger C-terminal cytoplasmic region, which consists of an ATPase domain and a protease domain. We have determined the crystal structures of the Thermus thermophilus FtsH ATPase domain in the nucleotide-free and AMP-PNP- and ADP-bound states, in addition to the domain with the extra preceding segment. Combined with the mapping of the putative substrate binding region, these structures suggest that FtsH internally forms a hexameric ring structure, in which ATP binding could cause a conformational change to facilitate transport of substrates into the protease domain through the central pore.
The dnaK and dnaJ genes, encoding heat shock proteins, were cloned from a psychrophilic bacterium, Colwellia maris. Significant homology was evident comparing DnaK and DnaJ of the psychrophilile with the counterparts of mesophilic and thermophilic bacteria. In the DnaJ protein, three conserved regions of the Hsp40 family were observed. A putative promoter similar to the sigma32 consensus sequence was found upstream of the dnaK gene. The G+C content in the 5'-untranslated region of the dnaK gene was much lower than that in the corresponding region of mesophilic bacteria. Northern-blot analysis and primer-extension analysis showed that both genes were transcribed separately as monocistronic mRNAs. Following several temperature upshifts from 10 to 26 degrees C, maximum induction of the dnaK and dnaJ mRNAs was detected at 20 degrees C, suggesting that this temperature induces the heat shock response in this bacterium. In addition, the level of the induction of the dnaJ gene was much lower than that of the dnaK gene. These findings together revealed several specific features of the heat shock response at a relatively low temperature in psychrophiles.
Molecular biology studies the cause-and-effect relationships among microscopic processes initiated by individual molecules within a cell and observes their macroscopic phenotypic effects on cells and organisms. These studies provide a wealth of information about the underlying networks and pathways responsible for the basic functionality and robustness of biological systems. At the same time, these studies create exciting opportunities for the development of quantitative and predictive models that connect the mechanism to its phenotype then examine various modular structures and the range of their dynamical behavior. The use of such models enables a deeper understanding of the design principles underlying biological organization and makes their reverse engineering and manipulation both possible and tractable The heat shock response presents an interesting mechanism where such an endeavor is possible. Using a model of heat shock, we extract the design motifs in the system and justify their existence in terms of various performance objectives. We also offer a modular decomposition that parallels that of traditional engineering control architectures.
• control theory
• heat shock stress response
• mathematical modeling
The rpoH gene encoding a heat shock sigma factor, sigma(32), was cloned from the psychrophilic bacterium Colwellia maris. The deduced amino acid sequence of sigma(32) from C. maris is more than 60% homologous to that of sigma(32) from mesophilic bacteria. The RpoH box, a 9-amino-acid sequence region (QRKLFFNLR) specific to sigma(32), and two downstream box sequences complementary to a part of 16S rRNA were identified. Primer extension analysis showed that the C. maris rpoH is expressed from only one sigma(70)-type promoter. Northern blot analysis showed that the level of rpoH mRNA was clearly increased at 20 degrees C, a temperature that induces heat shock in this organism. In the presence of an inhibitor of transcriptional initiation, the degradation of rpoH mRNA was much slower at 20 degrees C than at 10 degrees C. Thus, increased stability of the rpoH mRNA might be responsible for the rpoH mRNA accumulation. The predicted secondary structure of the 5'-region of C. maris rpoH mRNA was different from the conserved patterns reported for most mesophilic bacteria, and the base pairing of the downstream boxes appeared to be less stable than that of Escherichia coli rpoH mRNA. Thus, essential features that ensure the HSP expression at a relatively low temperature are embedded in the rpoH gene of psychrophiles.
The expression of heterologous proteins in the cytoplasm of Escherichia coli is often accompanied by limitations resulting in uncontrollable fermentation processes, increased rates of cell lysis, and thus limited yields of target protein. To deal with these problems, reporter tools are required to improve the folding properties of recombinant protein. In this work, the well-known sigma(32)-dependent promoters ibpAB and fxsA were linked in a tandem promoter (ibpfxs), fused with the luciferase reporter gene lucA to allow enhanced monitoring of the formation of misfolded proteins and their aggregates in E. coli cells. Overexpression of MalE31, a folding-defective variant of the maltose-binding protein, and other partially insoluble heterologous proteins showed that the lucA reporter gene was activated in the presence of these misfolded proteins. Contrary to this, the absence of damaged proteins or overexpression of mostly soluble proteins led to a reduced level of luciferase induction. Through performing expression of aggregation-prone proteins, we were able to demonstrate that the ibpfxs::lucA reporter unit is 2.5-4.5 times stronger than the single reporter units ibp::lucA and fxs::lucA. Data of misfolding studies showed that this reporter system provides an adequate tool for in vivo folding studies in E. coli from microtiter up to fermentation scales.
We adopt a control theory approach to reverse engineer the complexity of a known system--the bacterial heat shock response. Using a computational dynamic model, we explore the organization of the heat shock system and elucidate its various regulation strategies. We show that these strategies are behind much of the complexity of the network. We propose that complexity is a necessary outcome of robustness and performance requirements that are achieved by the heat shock system's exquisite regulation modules. The techniques we use rely on dynamic computational models and principles from the field of control theory.
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 ofN-ethylmaleimide-sensitive fusion protein suggests a plausible mechanism of ATP hydrolysis by the AAA ATPases, which invokes
an intermolecular catalytic role for the SRH.
Escherichia coli FtsH is an essential integral membrane protein that has an AAA-type ATPase domain at its C-terminal cytoplasmic part, which is homologous to at least three ATPase subunits of the eukaryotic 26S proteasome. We report here that FtsH is involved in degradation of the heat-shock transcription factor sigma32, a key element in the regulation of the E.coli heat-shock response. In the temperature-sensitive ftsHJ mutant, the amount of sigma32 at a non-permissive temperature was higher than in the wild-type under certain conditions due to a reduced rate of degradation. In an in vitro system with purified components, FtsH catalyzed ATP-dependent degradation of biologically active histidine-tagged sigma32. FtsH has a zinc-binding motif similar to the active site of zinc-metalloproteases. Protease activity of FtsH for histidine-tagged sigma32 was stimulated by Zn2+ and strongly inhibited by the heavy metal chelating agent o-phenanthroline. We conclude that FtsH is a novel membrane-bound, ATP-dependent metallo-protease with activity for sigma32. These findings indicate a new mechanism of gene regulation in E.coli.
Proteins with short nonpolar carboxyl termini are unstable in Escherichia coli. This proteolytic pathway is used to dispose of polypeptides synthesized from truncated mRNA molecules. Such proteins are tagged with an 11-amino-acid nonpolar destabilizing tail via a mechanism involving the 10Sa (SsrA) stable RNA and then degraded. We show here that the ATP-dependent zinc protease HflB (FtsH) is involved in the degradation of four unstable derivatives of the amino-terminal domain of the lambdacI repressor: three with nonpolar pentapeptide tails (cI104, cI105, cI108) and one with the SsrA tag (cI-SsrA). cI105 and cI-SsrA are also degraded by the ClpP-dependent proteases. Loss of ClpP can be compensated for by overproducing HflB. In an in vitro system, cI108 and cI-SsrA are degraded by HflB in an energy-dependent reaction, indicating that HflB itself recognizes the carboxyl terminus. These results establish a tail-specific pathway for removing abnormal cytoplasmic proteins via the HflB and Clp proteases.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
The Escherichia coli DnaK heat shock protein has been identified previously as a negative regulator of E. coli heat shock gene expression. We report that two other heat shock proteins, DnaJ and GrpE, are also involved in the negative regulation of heat shock gene expression. Strains carrying defective dnaK, dnaJ, or grpE alleles have enhanced synthesis of heat shock proteins at low temperature and fail to shut off the heat shock response after shift to high temperature. These regulatory defects are due to the loss of normal control over the synthesis and stability of sigma 32, the alternate RNA polymerase sigma-factor required for heat shock gene expression. We conclude that DnaK, DnaJ, and GrpE regulate the concentration of sigma 32. We suggest that the synthesis of heat shock proteins is controlled by a homeostatic mechanism linking the function of heat shock proteins to the concentration of sigma 32.
Heat shock proteins in Escherichia coli are relatively abundant and some are essential for growth, but the function that they provide is unknown. The observation that heat shock proteins are induced by some abnormal, rapidly degraded polypeptides, and that strains with mutations in the rpoH gene, the positive regulator of heat shock gene expression, are defective in proteolysis, has led to the proposal that heat shock proteins are required for normal degradation of polypeptides. We have investigated this hypothesis by examining the degradation of polypeptide fragments generated by puromycin and the degradation of a nonsense fragment of beta-galactosidase. Mutations in the dnaK, dnaJ, grpE, and groEL heat shock genes result in defective proteolysis. Furthermore, overproduction of heat shock proteins results in enhanced rates of puromycyl fragment decay. The proteolysis defect of the heat shock gene mutants primarily affects energy-dependent protein degradation. These results indicate that at least one general function of heat shock proteins is to contribute to the ability of the cell to degrade abnormal polypeptides.
Escherichia coli K-12 strain 285c contains a mutation in rpoD, the gene encoding the sigma subunit of RNA polymerase. The 70-kilodalton sigma polypeptide encoded by this allele is unstable, and this instability leads to temperature-sensitive growth. We describe the isolation and characterization of four temperature-resistant pseudorevertants of 285c that can grow at high temperature. Each of these revertants increased the stability of the sigma 70 mutant protein. The map position of the suppressor mutations was close to that of the rpoH (htpR) gene. A multicopy plasmid containing the intact rpoH gene restored the temperature-sensitive phenotype. Marker rescue experiments established the positions of three of the alleles within the rpoH gene. One mutation has been sequenced and causes a leucine-to-tryptophan change 7 amino acids from the carboxyl terminus of the rpoH gene product.
The heat shock response in Escherichia coli is governed by the concentration of the highly unstable sigma factor sigma 32. The essential protein HflB (FtsH), known to control proteolysis of the phage lambda cII protein, also governs sigma 32 degradation: an HflB-depleted strain accumulated sigma 32 and induced the heat shock response, and the half-life of sigma 32 increased by a factor up to 12 in mutants with reduced HflB function and decreased by a factor of 1.8 in a strain overexpressing HflB. The hflB gene is in the ftsJ-hflB operon, one promoter of which is positively regulated by heat shock and sigma 32. The lambda cIII protein, which stabilizes sigma 32 and lambda cII, appears to inhibit the HflB-governed protease. The E. coli HflB protein controls the stability of two master regulators, lambda cII and sigma 32, responsible for the lysis-lysogeny decision of phage lambda and the heat shock response of the host.
When secY is overexpressed over secE or secE is underexpressed, a fraction of SecY protein is rapidly degraded in vivo. This proteolysis was unaffected in previously described protease-defective mutants examined. We found, however, that some mutations in ftsH, encoding a membrane protein that belongs to the AAA (ATPase associated with a variety of cellular activities) family, stabilized oversynthesized SecY. This stabilization was due to a loss of FtsH function, and overproduction of the wild-type FtsH protein accelerated the degradation. The ftsH mutations also suppressed, by alleviating proteolysis of an altered form of SecY, the temperature sensitivity of the secY24 mutation, which alters SecY such that its interaction with SecE is weakened and it is destabilized at 42 degrees C. We were able to isolate a number of additional mutants with decreased ftsH expression or with an altered form of FtsH using selection/screening based on suppression of secY24 and stabilization of oversynthesized SecY. These results indicate that FtsH is required for degradation of SecY. Overproduction of SecY in the ftsH mutant cells proved to deleteriously affect cell growth and protein export, suggesting that elimination of uncomplexed SecY is important for optimum protein translocation and for the integrity of the membrane. The primary role of FtsH is discussed in light of the quite pleiotropic mutational effects, which now include stabilization of uncomplexed SecY.
Accumulation of abnormal proteins in cells of bacteria or eukaryotes can induce synthesis of a set of heat shock proteins. We examined such induction following addition of azetidine (a proline analog) or synthesis of a heterologous protein (human prourokinase) in Escherichia coli. Synthesis of heat shock proteins under these conditions increased almost immediately and continued with increasing rates until it reached a maximum after 30 to 60 min at 30 degrees C. The induction was closely accompanied by an increase in the cellular level of sigma 32 specifically required for transcription of heat shock genes. The increase in sigma 32 initially coincided with increased synthesis of heat shock proteins but then exceeded the latter, particularly following accumulation of prourokinase. The sigma 32 level increase upon either treatment was found to result solely from stabilization of sigma 32, which is ordinarily very unstable, and not from increased synthesis of sigma 32. This is in contrast to what had been found when cells were exposed to a higher temperature, at which both increased synthesis and stabilization of sigma 32 contributed to the increased sigma 32 level. On the basis of these and other findings, we propose that abnormal proteins stabilize sigma 32 by a pathway or a mechanism distinct from that used for the induction of sigma 32 synthesis known to occur at the level of translation. Evidence further suggests that the DnaK chaperone plays a crucial regulatory role in induction of the heat shock response by abnormal proteins.
To identify cellular factors that assist in membrane protein biogenesis, we looked for mutants affected in the "stop transfer" anchoring process. Using a SecY-PhoA fusion protein in which alkaline phosphatase (PhoA) mature sequence is attached to the last cytoplasmic domain following the 10th transmembrane segment of SecY, we isolated a mutation (std101) that allowed significant export of the PhoA moiety across the membrane. The mutation did not cause nonspecific leakage of cytoplasmic proteins. The mutation was identified as a single base change in the ftsH gene, causing an amino acid substitution in the proposed periplasmic region of FtsH, a putative membrane-bound ATPase. In addition, the ftsH1 temperature-sensitive mutation caused a similar phenotype. Disruption of the chromosomal ftsH in combination with a lac promoter-controlled copy of ftsH on a plasmid rendered the cell viability dependent on lac induction. Repression of this system resulted in a strong Std phenotype as well as significant export defects of beta-lactamase and OmpA. Thus, the loss of ftsH function enhances translocation of normally anchored protein segments and retards that of normally translocated proteins. These results suggest that FtsH participates in assembly of proteins into and through the membrane. It is needed for the cell to assure efficient stop-transfer of some transmembrane proteins.
The novel transcription system of bacteriophage T7 was used to express Escherichia coli genes preferentially with a new low-copy-number plasmid vector, pFN476, to minimize toxic gene effects. Selected E. coli chromosomal fragments from an ordered genomic library (Y. Kohara, K. Ikiyama, and K. Isono, Cell 50:495-508, 1987) were recloned into this vector, and their genes were preferentially expressed in vivo utilizing its T7 promoter. The protein products were analyzed by two-dimensional gel electrophoresis. By using DNA sequence information, the gel migration was predicted for the protein products of open reading frames from these segments, and this information was used to identify gene products visualized as spots on two-dimensional gels. Even in the absence of DNA sequence information, this approach offers the opportunity to identify all gene products of E. coli and map their genes to within 10 kb on the E. coli genome; with sequence information, this approach can produce a definitive expression map of the E. coli genome.
The ftsH gene is essential for cell viability in Escherichia coli. We cloned and sequenced the wild-type ftsH gene and the temperature-sensitive ftsH1(Ts) gene. It was suggested that FtsH protein was an integral membrane protein of 70.7 kDa (644 amino acid residues) with a putative ATP-binding domain. The ftsH1(Ts) gene was found to have two base substitutions within the coding sequence corresponding to the amino acid substitutions Glu-463 by Lys and Pro-587 by Ala. Homology search revealed that an approximately 200-amino-acid domain, including the putative ATP-binding sequence, is highly homologous (35 to 48% identical) to the domain found in members of a novel, eukaryotic family of putative ATPases, e.g., Sec18p, Pas1p, CDC48p, and TBP-1, which function in protein transport pathways, peroxisome assembly, cell division cycle, and gene expression, respectively. Possible implications of these observations are discussed.
We have investigated the role of DnaJ in protein degradation by examining the degradation of intrinsically unstable proteins
by Lon protease in vivo. In Escherichia coli, Lon protease is responsible for the rate-limiting step in degradation of highly unstable proteins such as SulA, RcsA, and
λN protein, as well as for about 50% of the rapid degradation of abnormal proteins such as canavanine-containing proteins.
We found that Lon-dependent degradation of both SulA and λN protein was unaffected in cells lacking functional DnaJ, whereas
Lon-dependent turnover of canavanine-containing proteins was slower in dnaJ mutant cells. DnaJ also affected the slow SulA degradation seen in the absence of Lon. The rate of degradation of RcsA was
reduced in dnaJ mutants, but both Lon-dependent and Lon-independent degradation was affected; abnormal, canavanine-containing proteins were
similarly affected. Both the RcsA that accumulated in dnaJ mutant cells and the SulA that accumulated in lon dnaJ mutant cells was aggregated. The abnormal proteins that partitioned to the insoluble pellet became solubilized over time
in dnaJ+ cells but not in dnaJ− cells. Therefore, the co-chaperone DnaJ is not essential for Lon-dependent degradation and may act in protein turnover only
as an accessory factor helping to maintain substrates in a soluble form. Such an accessory factor is apparently necessary
for abnormal proteins and for RcsA. The relative rates of degradation and aggregation of specific protein targets may determine
the importance of the chaperone systems in turnover of a given protein.
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.
B. B. thanks members of his lab and J. Reinstein for critical reading of the manuscript and C. Gassler, T. Laufen, and S. Rudiger for figure preparation. A. H. thanks Wayne Fenton for critical reading and Zhaohui Xu for figure preparation. A. H. dedicates this work to Guenter Brueckner, always an inspiration.
We have analyzed the core RNA polymerase (RNAP) binding activity of the purified products of nine defective alleles of the rpoH gene, which encodes sigma32 in Escherichia coli. All mutations studied here lie outside of the putative core RNAP binding regions 2.1 and 2.2. Based on the estimated K(s)s for the mutant sigma and core RNAP interaction determined by in vitro transcription and by glycerol gradient sedimentation, we have divided the mutants into three classes. The class III mutants showed greatly decreased affinity for core RNAP, whereas the class II mutants' effect on core RNAP interaction was only clearly seen in the presence of sigma70 competitor. The class I mutant behaved nearly identically to the wild type in core RNAP binding. Two point mutations in class III altered residues that were distant from one another. One was found in conserved region 4.2, and the other was in a region conserved only among heat shock sigma factors. These data suggest that there is more than one core RNAP binding region in sigma32 and that differences in contact sites probably exist among sigma factors.
Expression of heat shock genes is controlled in Escherichia coli by the antagonistic action of the sigma32 subunit of RNA polymerase and the DnaK chaperone system, which inactivates sigma32 by stress-dependent association and mediates sigma32 degradation by the FtsH protease. A stretch of 23 residues (R122 to Q144) conserved among sigma32 homologs, termed region C, was proposed to play a role in sigma32 degradation, and peptide analysis identified two potential DnaK binding sites central and peripheral to region C. Region C is thus a prime candidate for mediating stress control of sigma32, a hypothesis that we tested in the present study. A peptide comprising the central DnaK binding site was an excellent substrate for FtsH, while a peptide comprising the peripheral DnaK binding site was a poor substrate. Replacement of a single hydrophobic residue in each DnaK binding site by negatively charged residues (I123D and F137E) strongly decreased the binding of the peptides to DnaK and the degradation by FtsH. However, introduction of these and additional region C alterations into the sigma32 protein did not affect sigma32 degradation in vivo and in vitro or DnaK binding in vitro. These findings do not support a role for region C in sigma32 control by DnaK and FtsH. Instead, the sigma32 mutants had reduced affinities for RNA polymerase and decreased transcriptional activities in vitro and in vivo. Furthermore, cysteines inserted into region C allowed cysteine-specific cross-linking of sigma32 to RNA polymerase. Region C thus confers on sigma32 a competitive advantage over other sigma factors to bind RNA polymerase and thereby contributes to the rapidity of the heat shock response.
The heat shock response is a universally conserved mechanism protecting cells from external stress. Here we present an overall picture of the heat shock response of E. coli. Under steady state conditions, the DnaK chaperone system downregulates the activity, synthesis and stability of the heat shock transcription factor σ32, which plays a central role in the synthesis of heat shock proteins. Upon heat shock, the downregulation by the DnaK system is overcome and σ32 is induced, leading to rapid and transient induction of heat shock proteins. Degradation of σ32 is mainly achieved by the membrane-bound ATP-dependent protease FtsH.
The Escherichia coliσ32 transcriptional regulator has been shown to be degraded both in vivo and in vitro by the FtsH (HflB) protease, a member of the AAA protein family. In our attempts to study this process in detail, we found that two σ32 mutants lacking 15–20 C-terminal amino acids had substantially increased half-lives in vivo or in vitro, compared with wild-type σ32. A truncated version of σ32, σ32CΔ, was purified to homogeneity and shown to be resistant to FtsH-dependent degradation in vitro, suggesting that FtsH initiates σ32 degradation from its extreme C-terminal region. Purified σ32CΔ interacted with the DnaK and DnaJ chaperone proteins in a fashion similar to that of wild-type σ32. However, in contrast to wild-type σ32, σ32CΔ was largely deficient in its in vivo and in vitro interaction with core RNA polymerase. As a consequence, the truncated σ32 protein was completely non-functional in vivo, even when overproduced. Furthermore, it is shown that wild-type σ32 is protected from degradation by FtsH when complexed to the RNA polymerase core, but sensitive to proteolysis when in complex with the DnaK chaperone machine. Our results are in agreement with the proposal that the capacity of the DnaK chaperone machine to autoregulate its own synthesis negatively is simply the result of its ability to sequester σ32 from RNA polymerase, thus making it accessible to degradation by the FtsH protease.
The heat shock response of Escherichia coli is regulated by the cellular level and the activity of σ32, an alternative sigma factor for heat shock promoters. FtsH, a membrane-bound AAA-type metalloprotease, degrades σ32 and has a central role in the control of the σ32 level. The ftsH null mutant was isolated, and establishment of the ΔftsH mutant allowed us to investigate control mechanisms of the stability and the activity of σ32 separately in vivo. Loss of the FtsH function caused marked stabilization and consequent accumulation of σ32 (≈20-fold of the wild type), leading to the impaired downregulation of the level of σ32. Surprisingly, however, ΔftsH cells express heat shock proteins only two- to threefold higher than wild-type cells, and they also show almost normal heat shock response upon temperature upshift. These results indicate the presence of a control mechanism that downregulates the activity of σ32 when it is accumulated. Overproduction of DnaK/J reduces the activity of σ32 in ΔftsH cells without any detectable changes in the level of σ32, indicating that the DnaK chaperone system is responsible for the activity control of σ32in vivo. In addition, CbpA, an analogue of DnaJ, was demonstrated to have overlapping functions with DnaJ in both the activity and the stability control of σ32.
Lambda Xis, which is required for site-specific excision of phage lambda from the bacterial chromosome, has a much shorter functional half-life than Int, which is required for both integration and excision (R. A. Weisberg and M. E. Gottesman, p. 489-500, in A. D. Hershey, ed., The Bacteriophage Lambda, 1971). We found that Xis is degraded in vivo by two ATP-dependent proteases, Lon and FtsH (HflB). Xis was stabilized two- to threefold more than in the wild type in a lon mutant and as much as sixfold more in a lon ftsH double mutant at the nonpermissive temperature for the ftsH mutation. Integration of lambda into the bacterial chromosome was delayed in the lon ftsH background, suggesting that accumulation of Xis in vivo interferes with integration. Overexpression of Xis in wild-type cells from a multicopy plasmid inhibited integration of lambda and promoted curing of established lysogens, confirming that accumulation of Xis interferes with the ability of Int to establish and maintain an integrated prophage.
Genetic evidence indicates central roles for Hsp70 chaperones in the regulation of heat shock gene expression. This regulatory function has been postulated for Escherichia coli to rely on the direct association of DnaK (Hsp70) with the heat shock transcription factor sigma 32. This report presents evidence for the physical association of DnaK, DnaJ, and GrpE chaperones with sigma 32 in vivo. Surprisingly, an interaction of DnaJ with sigma 32 exists that is distinguishable from an interaction of DnaK and GrpE with sigma 32: addition of ATP disrupts the association of DnaK and GrpE with sigma 32, but not the association of DnaJ with sigma 32. Furthermore, DnaJ-sigma 32 and DnaK-sigma 32 associations occur independent of DnaK and DnaJ, respectively. These results suggest distinct regulatory functions of DnaJ and DnaK/GrpE.
Cells respond to an increase in temperature by inducing the synthesis of the heat shock proteins, which are a small set of evolutionarily conserved proteins. We review the evidence leading us to suggest that the free pool of one of these proteins, hsp70, serves as a cellular thermometer that regulates the expression of all heat shock proteins.
Cells subjected to a heat shock, or a variety of other stresses increase the synthesis of a set of proteins, known as heat shock proteins. This response is apparently universal, occurring in the entire range from bacterial to mammalian cells. In Escherichia coli heat shock protein synthesis transiently increases following a shift from 30 degrees C to 42 degrees C as a result of changes in transcription initiation at heat shock promoters. Heat shock promoters are recognized by RNA polymerase containing a sigma factor of relative molecular mass (Mr) 32,000 (32K) E sigma 32 and not E sigma 70, the major form of RNA polymerase holoenzyme. To determine whether changes in the concentration of sigma 32 regulate this response, we measured the amount of sigma 32 before and after shift to high temperature and found that it increased transiently during heat shock as a result of changes in sigma 32 synthesis and stability. Our results indicate that sigma 32 is directly responsible for regulation of the heat shock response.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
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 FtsH is a membrane-bound ATPase with a proteolytic activity against the SecY subunit of protein translocase. We now report that subunit a of the membrane-embedded Fo part of H+-ATPase is another substrate of FtsH. Pulse-chase experiments showed that subunit a is unstable when it alone (without Fo subunits b and c) was oversynthesized and that it is stabilized in the ftsH mutants. Selective and ATP-dependent degradation of subunit a by purified FtsH protein was demonstrated in vitro. These results suggest that FtsH serves as a quality-control mechanism to avoid potentially harmful accumulation of free subunit a in the membrane.
Proteolysis in Escherichia coli serves to rid the cell of abnormal and misfolded proteins and to limit the time and amounts of availability of critical regulatory proteins. Most intracellular proteolysis is initiated by energy-dependent proteases, including Lon, ClpXP, and HflB; HflB is the only essential E. coli protease. The ATPase domains of these proteases mediate substrate recognition. Recognition elements in target are not well defined, but are probably not specific amino acid sequences. Naturally unstable protein substrates include the regulatory sigma factors for heat shock and stationary phase gene expression, sigma 32 and RpoS. Other cellular proteins serve as environmental sensors that modulate the availability of the unstable proteins to the proteases, resulting in rapid changes in sigma factor levels and therefore in gene transcription. Many of the specific proteases found in E. coli are well-conserved in both prokaryotes and eukaryotes, and serve critical functions in developmental systems.
sigma32, the product of the rpoH gene in Escherichia coli, provides promoter specificity by interacting with core RNAP. Amino acid sequence alignment of sigma32 with other sigma factors in the sigma70 family has revealed regions of sequence homology. We have investigated the function of the most highly conserved region, 2.2, using purified products of various rpoH alleles. Core RNAP binding analysis by glycerol gradient sedimentation has revealed reduced core RNAP affinity for one of the mutant sigma32 proteins, Q80R. This reduced core interaction is exacerbated in the presence of sigma70, which competes with sigma32 for binding of core RNAP. When a different but more conserved amino acid was introduced at this position by site-directed mutagenesis (Q80N), this mutant sigma factor still displayed a significant reduction in its core RNAP affinity. Based on these results, we conclude that at least one specific amino acid in region 2.2 is involved in core RNAP interaction.
Rapid proteolysis plays an important role in regulation of gene expression. Proteolysis of the phage lambda CII transcriptional activator plays a key role in the lysis-lysogeny decision by phage lambda. Here we demonstrate that the E. coli ATP-dependent protease FtsH, the product of the host ftsH/hflB gene, is responsible for the rapid proteolysis of the CII protein. FtsH was found previously to degrade the heat-shock transcription factor sigma32. Proteolysis of sigma32 requires, in vivo, the presence of the DnaK-DnaJ-GrpE chaperone machine. Neither DnaK-DnaJ-GrpE nor GroEL-GroES chaperone machines are required for proteolysis of CII in vivo. Purified FtsH carries out specific ATP-dependent proteolysis of CII in vitro. The degradation of CII is at least 10-fold faster than that of sigma32. Electron microscopy revealed that purified FtsH forms ring-shaped structures with a diameter of 6-7 nm.
Escherichia coli FtsH (HflB) is a membrane-bound and ATP-dependent zinc-metalloproteinase, which forms a complex with a pair of periplasmically exposed membrane proteins, HflK and HflC. It is the protease that degrades uncomplexed forms of the SecY subunit of protein translocase. Here, we characterized a new class of SecY-stabilizing mutation on the E. coli chromosome. The mutation (yccA11) is an internal deletion within a gene (yccA) known as an open reading frame for a hydrophobic protein with putative seven transmembrane segments. The YccA protein was found to be degraded in an FtsH-dependent manner in vivo and in vitro, whereas the YccA11 mutant protein, lacking eight amino acid residues within the amino-terminal cytoplasmic domain, was refractory to the degradation. The yccA11 mutation exhibited partial dominance when overexpressed. Cross-linking, co-immunoprecipitation, and histidine tagging experiments showed that YccA11 as well as YccA can associate with both the FtsH and the HflKC proteins. Thus, the mutant YccA protein appeared to compete with SecY for recognition by the FtsH proteolytic system and the residues deleted by the yccA mutation are required for the initiation of proteolysis by FtsH. Interestingly, the inhibitory action of YccA11 was mediated by HflKC, since the deletion of hflK-hflC suppressed the yccA11 phenotype. The yccA11 mutation stabilized subunit a of the proton ATPase F0 segment as well, but not the CII protein of bacteriophage lambda or the sigma 32 protein. From these results we suggest that there are at least two pathways for FtsH-dependent protein degradation, only one of which (probably for membrane proteins) is subject to the HflKC-dependent interference by the YccA11 mutant substrate.
The expression of heat shock genes in Escherichia coli is regulated by the antagonistic action of the transcriptional activator, the sigma32 subunit of RNA polymerase, and negative modulators. Modulators are the DnaK chaperone system, which inactivates and destabilizes sigma32, and the FtsH protease, which is largely responsible for sigma32 degradation. A yet unproven hypothesis is that the degree of sequestration of the modulators through binding to misfolded proteins determines the level of heat shock gene transcription. This hypothesis was tested by altering the modulator concentration in cells expressing dnaK, dnaJ and ftsH from IPTG and arabinose-controlled promoters. Small increases in levels of DnaK and the DnaJ co-chaperone (< 1.5-fold of wild type) resulted in decreased level and activity of sigma32 at intermediate temperature and faster shut-off of the heat shock response. Small decreases in their levels caused inverse effects and, furthermore, reduced the refolding efficiency of heat-denatured protein and growth at heat shock temperatures. Fewer than 1500 molecules of a substrate of the DnaK system, structurally unstable firefly luciferase, resulted in elevated levels of heat shock proteins and a prolonged shut-off phase of the heat shock response. In contrast, a decrease in FtsH levels increased the sigma32 levels, but the accumulated sigma32 was inactive, indicating that sequestration of FtsH alone cannot induce the heat shock response efficiently. DnaK and DnaJ thus constitute the primary stress-sensing and transducing system of the E. coli heat shock response, which detects protein misfolding with high sensitivity.
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.
Current models of both heat induction and the chaperone-mediated feedback control of the sigma32 regulon in Escherichia coli have been further substantiated, and the extent of conservation among Gram-negative bacteria has been assessed. Analyses of the 'CIRCE' and other regulons or operons in Gram-positive and Gram-negative bacteria have provided new insights into their significance and regulatory mechanisms.
- D M Joo
- A Nolte
- R Calendar
- Y N Zhou
- D J Jin
Joo, D.M., Nolte, A., Calendar, R., Zhou, Y.N. and Jin, D.J.
(1998) J. Bacteriol. 180, 1095^1102.
- K Nakahigashi
- H Yanagi
- T Yura
Nakahigashi, K., Yanagi, H. and Yura, T. (1995) Nucleic Acids
Res. 23, 4383^4390.
- F Arsene
- T Tomoyasu
- A Mogk
- C Schirra
- A Schulze-Specking
- B Bukau
Arsene, F., Tomoyasu, T., Mogk, A., Schirra, C., Schulze-Specking, A. and Bukau, B. (1999) J. Bacteriol. 181, 3552^3561.
- T Yura
- K Nakahigashi
Yura, T. and Nakahigashi, K. (1999) Curr. Opin. Microbiol. 2,
153^158.
- M Lonetto
- M Gribskov
- C A Gross
Lonetto, M., Gribskov, M. and Gross, C.A. (1992) J. Bacteriol.
174, 3843^3849.
- T Tatsuta
- T Tomoyasu
- B Bukau
- M Kitagawa
- H Mori
- K Karata
- T Ogura
Tatsuta, T., Tomoyasu, T., Bukau, B., Kitagawa, M., Mori, H.,
Karata, K. and Ogura, T. (1998) Mol. Microbiol. 30, 583^593.
- U K Laemmli
Laemmli, U.K. (1970) Nature 227, 7850^7854.
- D B Straus
- W A Walter
- C A Gross
Straus, D.B., Walter, W.A. and Gross, C.A. (1988) Genes Dev. 2,
1851^1858.
- A Blaszczak
- C Georgopoulos
- K Liberek
Blaszczak, A., Georgopoulos, C. and Liberek, K. (1999) Mol.
Microbiol. 31, 157^166.
- T Tomoyasu
- T Ogura
- T Tatsuta
- B Bukau
[20] Tomoyasu, T., Ogura, T., Tatsuta, T. and Bukau, B. (1998) Mol.
Microbiol. 30, 567^581.
- A Kihara
- Y Akiyama
- K Ito
Kihara, A., Akiyama, Y. and Ito, K. (1998) J. Mol. Biol. 279,
175^188.
- T Ogura
- K Inoue
- T Tatsuta
- T Suzaki
- K Karata
- K Young
- L H Su
- C A Fierke
- J E Jackman
- C R Raetz
- J Coleman
- T Tomoyasu
- H Matsuzawa
Ogura, T., Inoue, K., Tatsuta, T., Suzaki, T., Karata, K., Young,
K., Su, L.H., Fierke, C.A., Jackman, J.E., Raetz, C.R., Coleman,
J., Tomoyasu, T. and Matsuzawa, H. (1999) Mol. Microbiol. 31,
833^844.
- D Straus
- W Walter
- C A Gross
Straus, D., Walter, W. and Gross, C.A. (1990) Genes Dev. 4,
2202^2209.