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B Bukau

Publications and source records attributed to B Bukau.

At least 55 records · Page 3Linked to original sources
PubMed1998

Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of sigma32 in vivo.

The heat shock response of Escherichia coli is regulated by the cellular level and the activity of sigma32, an alternative sigma factor for heat shock promoters. FtsH, a membrane-bound AAA-type metalloprotease, degrades sigma32 and has a central role in the control of the sigma32 level. The ftsH null mutant was isolated, and establishment of the DeltaftsH mutant allowed us to investigate control mechanisms of the stability and the activity of sigma32 separately in vivo. Loss of the FtsH function caused marked stabilization and consequent accumulation of sigma32 ( approximately 20-fold of the wild type), leading to the impaired downregulation of the level of sigma32. Surprisingly, however, DeltaftsH 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 sigma32 when it is accumulated. Overproduction of DnaK/J reduces the activity of sigma32 in DeltaftsH cells without any detectable changes in the level of sigma32, indicating that the DnaK chaperone system is responsible for the activity control of sigma32 in 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 sigma32.

ATP-Dependent Proteases↗
PubMed1998

Hsp70 chaperone systems: diversity of cellular functions and mechanism of action.

Hsp70 chaperone systems play an essential role in the life cycle of many proteins not only in an hostile environment but also under normal growth conditions. In the course of evolution the diversification of functions was accompanied by an amplification of components of the Hsp70 system. Here strategies are reviewed how different Hsp70 systems work independently or cooperate with each other in a functional network to perform their housekeeping tasks even under stress conditions. We further discuss how co-chaperones which act as targeting factors regulate the cycle of substrate binding and release upon which the Hsp70 chaperone activity depends.

HSP70 Heat-Shock Proteins↗
PubMed1997

The human DnaJ homologue dj2 facilitates mitochondrial protein import and luciferase refolding.

DnaJ homologues function in cooperation with hsp70 family members in various cellular processes including intracellular protein trafficking and folding. Three human DnaJ homologues present in the cytosol have been identified: dj1 (hsp40/hdj-1), dj2 (HSDJ/hdj-2), and neuronal tissue-specific hsj1. dj1 is thought to be engaged in folding of nascent polypeptides, whereas functions of the other DnaJ homologues remain to be elucidated. To investigate roles of dj2 and dj1, we developed a system of chaperone depletion from and readdition to rabbit reticulocyte lysates. Using this system, we found that heat shock cognate 70 protein (hsc70) and dj2, but not dj1, are involved in mitochondrial import of preornithine transcarbamylase. Bacterial DnaJ could replace mammalian dj2 in mitochondrial protein import. We also tested the effects of these DnaJ homologues on folding of guanidine-denatured firefly luciferase. Unexpectedly, dj2, but not dj1, together with hsc70 refolded the protein efficiently. We propose that dj2 is the functional partner DnaJ homologue of hsc70 in the mammalian cytosol. Bacterial DnaJ protein could replace mammalian dj2 in the refolding of luciferase. Thus, the cytosolic chaperone system for mitochondrial protein import and for protein folding is highly conserved, involving DnaK and DnaJ in bacteria, Ssa1-4p and Ydj1p in yeast, and hsc70 and dj2 in mammals.

Biological Transport↗
PubMed1997

The amino-terminal 118 amino acids of Escherichia coli trigger factor constitute a domain that is necessary and sufficient for binding to ribosomes.

Escherichia coli trigger factor has prolyl-isomerase and chaperone activities and associates with nascent polypeptide chains. Trigger factor has a binding site on ribosomes, which is a prerequisite for its efficient association with nascent chains and its proposed function as a cotranslational folding catalyst. We set out to identify the domain of trigger factor that mediates ribosome binding. Of a series of recombinant fragments, the amino-terminal fragments, TF (1-144) and TF (1-247), cofractionated with ribosomes from cell extracts and rebound to isolated ribosomes in vitro. They competed efficiently with full-length trigger factor for stoichiometric binding to a single site on the large ribosomal subunit. However, TF (1-144) and TF (1-247) differed from full-length trigger factor in that their association with ribosomes was not strengthened by the presence of nascent chains, indicating a role for carboxyl-terminal trigger factor segment in sensing the translational status. The domain responsible for ribosome binding was further investigated by limited proteolysis of recombinant fragments. A stable domain comprising the amino-terminal 118 residues was identified that was still capable of ribosome binding and thus represents a novel structural and functional element of trigger factor.

Amino Acid Isomerases↗
PubMed1997

Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries.

Hsp70 chaperones assist protein folding by ATP-dependent association with linear peptide segments of a large variety of folding intermediates. The molecular basis for this ability to differentiate between native and non-native conformers was investigated for the DnaK homolog of Escherichia coli. We identified binding sites and the recognition motif in substrates by screening 4360 cellulose-bound peptides scanning the sequences of 37 biologically relevant proteins. DnaK binding sites in protein sequences occurred statistically every 36 residues. In the folded proteins these sites are mostly buried and in the majority found in beta-sheet elements. The binding motif consists of a hydrophobic core of four to five residues enriched particularly in Leu, but also in Ile, Val, Phe and Tyr, and two flanking regions enriched in basic residues. Acidic residues are excluded from the core and disfavored in flanking regions. The energetic contribution of all 20 amino acids for DnaK binding was determined. On the basis of these data an algorithm was established that predicts DnaK binding sites in protein sequences with high accuracy.

Algorithms↗
PubMed1997

GrpE accelerates nucleotide exchange of the molecular chaperone DnaK with an associative displacement mechanism.

The ATP hydrolysis and protein-binding and release cycle of the molecular chaperone DnaK is regulated by the accessory proteins GrpE and DnaJ. Here we describe a study of the formation of complexes between the molecular chaperone DnaK, its nucleotide exchange factor GrpE, and the fluorescent ADP analog N8-[4-[(N'-methylanthraniloyl)amino]butyl]-8-aminoadenosine 5'-diphosphate (MABA-ADP) by equilibrium and stopped flow kinetic experiments. The catalytic cycle of the GrpE-stimulated nucleotide exchange involves a ternary DnaK x GrpE x ADP complex as well as the binary DnaK x GrpE and DnaK x ADP complexes. The equilibrium data of the interaction of GrpE with DnaK x ADP and the nucleotide-free DnaK can be described by a simple equilibrium system where GrpE reduces the affinity of ADP for DnaK 200-fold. However, transient kinetic studies revealed that the functional cycle of GrpE in addition includes at least two distinct ternary DnaK x GrpE x ADP complexes. Our data indicate that the initial weak binding of GrpE to DnaK x ADP is followed by an isomerization of the ternary complex which leads to weakening of nucleotide binding and finally to its rapid dissociation. The maximal stimulation for nucleotide exchange brought about by GrpE was found to be 5000-fold. We propose that this kinetically observed isomerization represents a structural change (opening) of the nucleotide binding pocket of DnaK that allows for fast nucleotide exchange.

Adenosine Diphosphate↗
PubMed1997

Interaction of Hsp70 chaperones with substrates.

Determination of the structure of the substrate binding domain of the Escherichia coli Hsp70 chaperone, DnaK, and the biochemical characterisation of the motif it recognizes within substrates provide insights into the principles governing Hsp70 interaction with polypeptide chains. DnaK recognizes extended peptide strands composed of up to five consecutive hydrophobic residues within and positively charged residues outside the substrate binding cavity.

Binding Sites↗
PubMed1997

Proteolysis of the phage lambda CII regulatory protein by FtsH (HflB) of Escherichia coli.

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.

ATP-Dependent Proteases↗
PubMed1996

The second step of ATP binding to DnaK induces peptide release.

The interaction of the nucleotide-free molecular chaperone DnaK (Hsp70) from Escherichia coli with nucleotides was studied under equilibrium and transient kinetic conditions. These studies used the intrinsic fluorescence signal of the single tryptophan residue (Trp102) of DnaK, or of novel fluorescent nucleotide analogs of ADP and ATP, N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine 5'-di- or triphosphate (MABA-ADP and MABA-ATP) as spectroscopic probes. Titration of MABA-ADP with DnaK resulted in a 2.3-fold increase of the fluorescence signal, from which a binding stoichiometry of 1:1, and a dissociation constant (Kd) of 0.09 microM were derived. The intrinsic rate constant of hydrolysis of ATP or MABA-ATP in single turnover experiments was found to be 1.5 x 10(-3) s-1 and 1.6 x 10(-3) s-1, identical with the catalytic rate constant of 1.5(+/- 0.17) x 10(-3) s-1 obtained under steady-state conditions. The dissociation rate constant of ADP was measured to be 35(+/- 7) x 10(-3) s-1 in the absence or 15(+/- 5) x 10(-3) in the presence of 2 mM inorganic phosphate (Pi) and is therefore 10 to 20 times faster than the rate of hydrolysis. These results demonstrated that processes governing ATP hydrolysis are rate-limiting in the DnaK ATPase reaction cycle. The three observed different fluorescent states of the single tryptophan residue were investigated. The binding of ATP gave a decrease of 15% in fluorescence intensity compared with the nucleotide-free state. Subsequent ATP hydrolysis, or the simultaneous addition of ADP and Pi, increased the fluorescence 7% above the fluorescence intensity of the nucleotide-free protein. Changes in the tryptophan fluorescence could not be detected when ADP, Pi or the non-hydrolyzable nucleotide analogs AMPPNP (Kd = 1.62(+/- 0.1) microM) or ATP gamma S (Kd = 0.044(+/- 0.003) microM) were added. These data suggested that DnaK exists in at least three different conformational states, depending on nucleotide site occupancy. The fluorescence increase of DnaK upon ATP binding was resolved into two steps; a rapid first step (Kd 1 = 7.3 microM) is followed by a second slow step (k+2 = 1.5 s-1 and k-2 < or = 1.5 x 10(-3) s-1) that causes the decrease in the tryptophan fluorescence signal. The addition of ATP also resulted in the release of DnaK-bound peptide substrate with koff = 3.8 s-1, comparable with the rate of the second step of nucleotide binding. AMPPNP or ATP gamma S were not able to change the fluorescence signal nor to release the peptide. We therefore conclude that the second step of ATP binding, and not the 1000-fold slower ATP hydrolysis is coupled to peptide release.

Adenosine Diphosphate↗
PubMed1996

Substrate shuttling between the DnaK and GroEL systems indicates a chaperone network promoting protein folding.

GroEL and DnaK with their cofactors constitute the major chaperone systems promoting protein folding in the Escherichia coli cytosol. The ability of GroEL to bind and promote folding of a substrate released from DnaK led to the proposal that the DnaK and GroEL systems act successively along a protein folding pathway. Here we have investigated the role of both systems in preventing aggregation and assisting refolding of firefly luciferase denatured by guanidinium chloride and heat. We find that DnaK and GroEL compete with each other for binding to non-native luciferase. Addition of ATP and co-operating proteins results in release of luciferase from either chaperone in a non-native conformation. Only a small fraction of luciferase molecules released from GroEL can reach the native state. Instead, the released luciferase must bind repeatedly to the DnaK system, and only then is it able to fold to the native state. Thus, during a folding reaction, DnaK and GroEL do not obligatorily act in succession by promoting earlier and later protein folding steps, respectively. Rather, the two chaperone systems and perhaps others can form a lateral network of co-operating proteins. This chaperone network is proposed to be of particular importance for the assisted refolding of proteins that are unfolded by stress treatment such as heat shock and whose size is too large to allow folding inside the substrate binding cavity of the GroEL ring underneath GroES.

Bacterial Proteins↗
PubMed1996

The Escherichia coli trigger factor.

E. coli trigger factor is an abundant cytosolic protein originally identified by its ability to maintain the precursor of a secretory protein in a translocation competent form. Recent studies shed new light on the function of this protein. Trigger factor was found to be a peptidyl-prolyl-cisltrans-isomerase capable of catalysing protein folding in vitro, to associate with nascent cytosolic and secretory polypeptide chains, and to cooperate with the GroEL chaperone in promoting proteolysis of an unstable polypeptide in vivo. These findings suggest roles for trigger factor in various folding processes of secretory as well as cytosolic proteins.

Amino Acid Isomerases↗
PubMed1996

Escherichia coli trigger factor is a prolyl isomerase that associates with nascent polypeptide chains.

Correct folding of newly synthesized proteins is proposed to be assisted by molecular chaperones and folding catalysts. To identify cellular factors involved in the initial stages of this process we searched for proteins associated with nascent polypeptide chains. In an Escherichia coli transcription/translation system synthesizing beta-galactosidase we identified a 58-kDa protein which associated with translating ribosomes but dissociated from these ribosomes upon release of nascent beta-galactosidase. N-terminal sequencing identified it as trigger factor, previously implicated in protein secretion. Direct evidence for association of trigger factor with nascent polypeptide chains was obtained by crosslinking. In a wheat germ translation system complemented with E. coli lysates, epsilon-4-(3-trifluoromethyldiazirino)benzoic acid-lysine residues were incorporated into nascent secretory preprolactin and a nonsecretory preprolactin mutant. Trigger factor crosslinked to both types of nascent chains, provided they were ribosome bound. Trigger factor contains key residues of the substrate-binding pocket of FK506-binding protein-type peptidyl-prolyl-cis/trans-isomerases and has prolyl isomerase activity in vitro. We propose that trigger factor is a folding catalyst acting cotranslationally.

Amino Acid Isomerases↗
PubMed1996

Identification of the prolyl isomerase domain of Escherichia coli trigger factor.

E. coli trigger factor is a protein of 48 kDa which was recently identified as a ribosome-bound peptidyl-prolyl-cis/transisomerase (PPIase) capable of catalysing protein folding in vitro. We found trigger factor in association with nascent polypeptide chains, suggesting a function in the co-translational folding of proteins. Sequence comparisons revealed a homology of a segment of trigger factor with PPIases of the FK506 binding protein (FKBP) family. Here, we report on the purification of trigger factor and a domain assignment of its polypeptide chain by microsequencing and mass spectroscopy of proteolytic fragments. Two proteases generated fragments of 12-13 kDa molecular weight that encompass the predicted FKBP domain and possess PPIase activity in vitro. Sequence alignment of the known trigger factor proteins demonstrates a high degree of conservation within this central functional domain of the protein.

Amino Acid Isomerases↗
PubMed1996

Regulatory region C of the E. coli heat shock transcription factor, sigma32, constitutes a DnaK binding site and is conserved among eubacteria.

The E. coli heat shock response is regulated at the transcriptional level through stress-dependent controls of the heat shock promoter-specific sigma32 subunit of RNA polymerase. A key aspect of this regulation, the sensing of stress and transmission of this information to sigma32, involves the chaperone system formed by the DnaK, DnaJ and GrpE heat shock proteins. This system mediates stress- dependent controls of levels and activity of sigma32 which rely, at least in part, on direct association of DnaK and DnaJ with sigma32. We identified DnaK binding sites within the sigma32 sequence by probing a cellulose-bound peptide library scanning sigma32. Two sites with high affinity for DnaK, containing the motifs RKLFFNLR and LRNWRIVK, were located centrally and peripherally, respectively, to the region C of sigma32, previously implicated genetically in chaperone-dependent control of sigma32 levels. Cloning and sequencing of rpoH homologs from five Gram-negative proteobacteria revealed that region C, including the DnaK binding motif central to it, is highly conserved among sigma32 homologs but missing in the other sigma factors. We propose that binding of DnaK to region C is central to a conserved regulatory mechanism allowing the sensing of stress by the heat shock gene transcription machinery.

Amino Acid Sequence↗
PubMed1996

A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the Escherichia coli heat shock transcription factor sigma32.

The chaperone system formed by DnaK, DnaJ and GrpE mediates stress-dependent negative modulation of the Escherichia coli heat shock response, probably through association with the heat shock promoter-specific sigma32 subunit of RNA polymerase. Interactions of the DnaK system with sigma32 were analysed. DnaJ and DnaK bind free, but not RNA polymerase-bound, sigma32 with dissociation constants of 20 nM and 5 muM respectively. Association and dissociation rates of DnaJ-sigma32 complexes are 5900- and 20-fold higher respectively than those of DnaK-sigma32 complexes in the absence of ATP. ATP destabilizes DnaK-sigma32 interactions. DnaJ, through rapid association with sigma32 and stimulation of hydrolysis of DnaK-bound ATP, mediates efficient binding of DnaK to sigma32 in the presence of ATP, resulting in DnaK-DnaJ-sigma32 complexes containing ADP. GrpE binding to these complexes stimulates nucleotide release and subsequent complex dissociation by ATP. We propose that the principles of this cycle also operate in other chaperone activities of the DnaK system. DnaK and DnaJ cooperatively inhibit sigma32 activity in heat shock gene transcription and GrpE partially reverses this inhibition. These data indicate that reversible inhibition of sigma32 activity through transient association of DnaK and DnaJ is a central regulatory element of the heat shock response.

Adenosine Diphosphate↗
PubMed1996

Growing up in a dangerous environment: a network of multiple targeting and folding pathways for nascent polypeptides in the cytosol.

The first events in the lives of proteins are the most hazardous. Starting at the ribosome, nascent polypeptides undergo complex folding processes endangered by aggregation reactions. Proteins with organellar destinations require correct targeting to the translocation machineries and prevention from premature folding. The high precision and speed of these processes is ensured by a cystosolic system consisting of molecular chaperones, folding catalysts and targeting factors. This review focuses on the interactions of this system with nascent polypeptides and discusses new concepts for protein folding in the cytosol. It is proposed that folding and targeting are promoted by a flexible network of multiple unassisted and assisted pathways.

Journal Article↗
PubMed1995

Conserved ATPase and luciferase refolding activities between bacteria and yeast Hsp70 chaperones and modulators.

We have reconstituted an ATP-dependent protein folding machinery using purified yeast cytosolic proteins. The S. cerevisiae Hsp70 Ssa1p and the DnaJ homolog Ydj1p refolded denatured firefly luciferase. In E. coli, efficient refolding of luciferase requires the Hsp70 DnaK and two modulators, DnaJ and GrpE, that synergistically stimulate its ATPase activity. Exchanging DnaJ homologs between the S. cerevisiae and E. coli systems revealed that their ability to stimulate Hsp70 ATPase activity was conserved. In contrast, GrpE further stimulated only DnaK's ATPase activity. Efficient refolding of luciferase by Ssa1p and DnaJ, but not by DnaK and Ydj1p, suggests that a compatible Hsp70/DnaJ homolog pair can act as a protein folding machinery.

Adenosine Triphosphatases↗