Toshifumi Tomoyasu

Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB

We systematically analyzed the capability of the major cytosolic chaperones of Escherichia coli to cope with protein misfolding and aggregation during heat stress in vivo and in cell extracts. Under physiological heat stress conditions, only the DnaK system efficiently prevented the aggregation of thermolabile proteins, a surprisingly high number of 150–200 species, corresponding to 15–25% of detected proteins. Identification of thermolabile DnaK substrates by mass spectrometry revealed that they comprise 80% of the large (≥90 kDa) but only 18% of the small (≤30 kDa) cytosolic proteins and include essential proteins. The DnaK system in addition acts with ClpB to form a bi‐chaperone system that quantitatively solubilizes aggregates of most of these proteins. Efficient solubilization also occurred in an in vivo order‐of‐addition experiment in which aggregates were formed prior to induction of synthesis of the bi‐chaperone system. Our data indicate that large‐sized proteins are most vulnerable to thermal unfolding and aggregation, and that the DnaK system has central, dual protective roles for these proteins by preventing their aggregation and, cooperatively with ClpB, mediating their disaggregation.

Trigger factor and DnaK cooperate in folding of newly synthesized proteins

The role of molecular chaperones in assisting the folding of newly synthesized proteins in the cytosol is poorly understood. In Escherichia coli, GroEL assists folding of only a minority of proteins and the Hsp70 homologue DnaK is not essential for protein folding or cell viability at intermediate growth temperatures. The major protein associated with nascent polypeptides is ribosome-bound trigger factor,, which displays chaperone and prolyl isomerase activities in vitro,,. Here we show that Δtig::kan mutants lacking trigger factor have no defects in growth or protein folding. However, combined Δtig::kan and ΔdnaK mutations cause synthetic lethality. Depletion of DnaK in the Δtig::kan mutant results in massive aggregation of cytosolic proteins. In Δtig::kan cells, an increased amount of newly synthesized proteins associated transiently with DnaK. These findings show in vivo activity for a ribosome-associated chaperone, trigger factor, in general protein folding, and functional cooperation of this protein with a cytosolic Hsp70. Trigger factor and DnaK cooperate to promote proper folding of a variety of E. coli proteins, but neither is essential for folding and viability at intermediate growth temperatures.

ESCHERICHIA COLI FTSH IS A MEMBRANE-BOUND, ATP-DEPENDENT PROTEASE WHICH DEGRADES THE HEAT-SHOCK TRANSCRIPTION FACTOR SIGMA 32

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 sigma 32, a key element in the regulation of the E. coli heat‐shock response. In the temperature‐sensitive ftsH1 mutant, the amount of sigma 32 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 sigma 32. FtsH has a zinc‐binding motif similar to the active site of zinc‐metalloproteases. Protease activity of FtsH for histidine‐tagged sigma 32 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 metalloprotease with activity for sigma 32. These findings indicate a new mechanism of gene regulation in E. coli.

The heat shock response of Escherichia coli

A large variety of stress conditions including physicochemical factors induce the synthesis of more than 20 heat shock proteins (HSPs). In E. coli, the heat shock response to temperature upshift from 30 to 42 degrees C consists of the rapid induction of these HSPs, followed by an adaptation period where the rate of HSP synthesis decreases to reach a new steady-state level. Major HSPs are molecular chaperones, including DnaK, DnaJ and GrpE, and GroEL and GroES, and proteases. They constitute the two major chaperone systems of E. coli (15-20% of total protein at 46 degrees C). They are important for cell survival, since they play a role in preventing aggregation and refolding proteins. The E. coli heat shock response is positively controlled at the transcriptional level by the product of the rpoH gene, the heat shock promoter-specific sigma32 subunit of RNA polymerase. Because of its rapid turn-over, the cellular concentration of sigma32 is very low under steady-state conditions (10-30 copies/cell at 30 degrees C) and is limiting for heat shock gene transcription. The heat shock response is induced as a consequence of a rapid increase in sigma32 levels and stimulation of sigma32 activity. The shut off of the response occurs as a consequence of declining sigma32 levels and inhibition of sigma32 activity. Stress-dependent changes in heat shock gene expression are mediated by the antagonistic action of sigma32 and negative modulators which act upon sigma32. These modulators are the DnaK chaperone system which inactivate sigma32 by direct association and mediate its degradation by proteases. Degradation of sigma32 is mediated mainly by FtsH (HflB), an ATP-dependent metallo-protease associated with the inner membrane. There is increasing evidence that the sequestration of the DnaK chaperone system through binding to misfolded proteins is a direct determinant of the modulation of the heat shock genes expression. A central open question is the identity of the binding sites within sigma32 for DnaK, DnaJ, FtsH and the RNA polymerase, and the functional interplay between these sites. We have studied the role of two distinct regions of sigma32 in its activity and stability control: region C and the C-terminal part. Both regions are involved in RNA polymerase binding.

Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol

We investigated the roles of chaperones and proteases in quality control of proteins in the Escherichia coli cytosol. In ΔrpoH mutants, which lack the heat shock transcription factor and therefore have low levels of all major cytosolic proteases and chaperones except GroEL and trigger factor, 5–10% and 20–30% of total protein aggregated at 30°C and 42°C respectively. The aggregates contained 350–400 protein species, of which 93 were identified by mass spectrometry. The aggregated protein species were similar at both temperatures, indicating that thermolabile proteins require folding assistance by chaperones already at 30°C, and showed strong overlap with previously identified DnaK substrates. Overproduction of the DnaK system, or low‐level production of the DnaK system and ClpB, prevented aggregation and provided thermotolerance to ΔrpoH mutants, indicating key roles for these chaperones in protein quality control and stress survival. In rpoH+ cells, DnaK depletion did not lead to protein aggregation at 30°C, which is probably the result of high levels of proteases and thus suggests that DnaK is not a prerequisite for proteolysis of misfolded proteins. Lon was the most efficient protease in degrading misfolded proteins in DnaK‐depleted cells. At 42°C, ClpXP and Lon became essential for viability of cells with low DnaK levels, indicating synergistic action of proteases and the DnaK system, which is essential for cell growth at 42°C.

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.

Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli

BackgroundThe overproduction of recombinant proteins in host cells often leads to their misfolding and aggregation. Previous attempts to increase the solubility of recombinant proteins by co-overproduction of individual chaperones were only partially successful. We now assessed the effects of combined overproduction of the functionally cooperating chaperone network of the E. coli cytosol on the solubility of recombinant proteins.ResultsA two-step procedure was found to show the strongest enhancement of solubility. In a first step, the four chaperone systems GroEL/GroES, DnaK/DnaJ/GrpE, ClpB and the small HSPs IbpA/IbpB, were coordinately co-overproduced with recombinant proteins to optimize de novo folding. In a second step, protein biosynthesis was inhibited to permit chaperone mediated refolding of misfolded and aggregated proteins in vivo. This novel strategy increased the solubility of 70% of 64 different heterologous proteins tested up to 42-fold.ConclusionThe engineered E. coli strains and the two-step procedure presented here led to a remarkable increase in the solubility of a various recombinant proteins and should be applicable to a wide range of target proteins produced in biotechnology.

Levels of DnaK and DnaJ provide tight control of heat shock gene expression and protein repair in Escherichia coli

The expression of heat shock genes in Escherichia coli is regulated by the antagonistic action of the transcriptional activator, the σ32 subunit of RNA polymerase, and negative modulators. Modulators are the DnaK chaperone system, which inactivates and destabilizes σ32, and the FtsH protease, which is largely responsible for σ32 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 σ32 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 σ32 levels, but the accumulated σ32 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.

Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli

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°C. This was accompanied by a dramatic increase in the amount of UDP‐3‐O‐(R‐3‐hydroxymyristoyl)‐N‐acetylglucosamine deacetylase [the lpxC (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.

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 λ CII transcriptional activator plays a key role in the lysis‐lysogeny decision by phage λ. 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 σ32. Proteolysis of σ32 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 σ32. Electron microscopy revealed that purified FtsH forms ring‐shaped structures with a diameter of 6–7 nm.