Aggregation of heat-shock-denatured, endogenous proteins and distribution of the IbpA/B and Fda marker-proteins in Escherichia coli WT and grpE280 cells

Ewa Laskowska1, Jerzy Bohdanowicz2, Dorota Kuczynska-Wisnik1, Ewelina Matuszewska1, Sabina Kdzierska1 and Alina Taylor3

1 Department of Biochemistry, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland
2 Department of Genetics, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland
3 Department of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland

Correspondence
Alina Taylor
ataylor{at}biotech.univ.gda.pl


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Submission of wild-type Escherichia coli to heat shock causes an aggregation of cellular proteins. The aggregates (S fraction) are separable from membrane fractions by ultracentrifugation in a sucrose density gradient. In contrast, no protein aggregation was detectable in an E. coli grpE280 mutant either by this technique or by electron microscopy. In search of an explanation for this observation at a molecular level, two kinds of marker proteins were used: Fda (fructose-1,6-biphosphate aldolase), the previously identified S fraction component, and IbpA/B, small heat-shock proteins abundantly associated with the S fraction proteins. Both types of marker proteins, normally never found in the outer-membrane (OM) fraction of WT cells, were present in the OM fraction from grpE cells after heat shock. This pointed to the presence of aggregates smaller than those in WT cells that cosedimented with the OM fraction. The OM fraction was enlarged in grpE cells. Although not proven directly, the presence of still smaller aggregates, not exceeding the solubility level and containing inactive Fda, was noted in the soluble CP fraction containing the cytoplasmic and periplasmic proteins. Therefore, aggregation occurred in both strains, but in a different way. The autoregulation of the heat-shock response causes a greater increase of DnaK/DnaJ and IbpAB levels in grpE cells than in WT after temperature elevation. This may explain the prevalence of the small-sized aggregates in the grpE cells. Estimation of total Fda protein before and after heat shock did not show any loss. This indicated that renaturation rather than proteolysis underlies the final disappearance of the aggregates. Though surprising at first, this is not contradictory with the participation of heat-shock proteases in removal of protein components of the S fraction as shown before, since proteins that are irreversibly denatured are probably substrates for the proteases.


Abbreviations: CP, cytoplasmic and periplasmic; Fda, fructose-1,6-biphosphate aldolase; IM, inner-membrane; OM, outer-membrane


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cellular proteins of Escherichia coli that aggregate upon heat shock are separable from the outer- and inner-membrane (OM and IM, respectively) fractions as a distinct S fraction by centrifugation in a sucrose density gradient. This technique allows for observation of the accumulation of aggregated proteins in vivo in different genetic backgrounds and under various experimental conditions. The S fraction appears 15 min after a change in temperature from 30 to 45 °C in E. coli WT strains. It lasts for only about 10 min after the temperature shift to 37 °C. In contrast, the S fraction is enlarged and stable in various E. coli mutants, e.g. rpoH (Kucharczyk et al., 1991), dnaK and dnaJ (Kdzierska et al., 1999). The DnaK, DnaJ (Kucharczyk et al., 1991) and IbpA/B (Laskowska et al., 1996b) proteins have been found associated with S fraction proteins, but GrpE, GroEL and GroES were present in the soluble (CP, cytoplasmic and periplasmic) fraction (Kucharczyk et al., 1991).

We were surprised by the lack of an S fraction in grpE mutant cells submitted to heat shock. The aim of this work was to explain this observation at a molecular level.

There are several aspects of the roles and interactions of DnaK, DnaJ and GrpE in the heat-shock response.

(i) Mutations in the dnaK, dnaJ or grpE gene result in similar, pleiotropic phenotypes when phage {lambda} DNA replication or bacterial physiology is considered (Georgopoulos et al., 1990; Liberek et al., 1995). Mutations in these genes affect cell division, protein export, proteolysis (Straus et al., 1988; Sherman & Goldberg, 1992) and cause inhibition of DNA replication and RNA synthesis at elevated temperatures (Ang et al., 1986). These effects can be explained at the molecular level, reflecting the cooperation of the DnaK, DnaJ and GrpE proteins as a system (Bukau & Horwich, 1998) participating in crucial cellular processes.

(ii) GrpE, a protein devoid of ATPase and chaperone activity, acts as a co-chaperone in the DnaK/DnaJ/GrpE system. The crystal structure of the GrpE-DnaK ATPase domain complex has been determined (Harrison et al., 1997). GrpE is active as a dimer. It facilitates an exchange of ADP bound to DnaK for ATP, with concomitant conformational changes in DnaK which determine its affinity for substrate proteins. Grimshaw et al. (2003) proposed that the GrpE dimer acts as a thermosensor and modulates temperature-dependent DnaK-substrate interaction. Renaturation of an unfolded protein may require multiple ATP-dependent cycles of DnaK binding and release (Liberek et al., 1991; Laufen et al., 1999; Mally & Witt, 2001). In the process, weak ATPase activity of the DnaK-ATP complex is stimulated by association with DnaJ bound to a substrate protein and GrpE. ATP undergoes hydrolysis and the DnaK-ADP-substrate complex is resolved. Thus, GrpE facilitates DnaK recycling (Bukau & Horwich, 1998). It was found recently that GrpE also stimulates the ATPase activity of Hsc62, one of the Hsp70 E. coli homologues (Yoshimune et al., 2002).

(iii) Folding and refolding proteins to their native conformations in many cases requires the cooperation of the DnaK system with other proteins of the heat-shock response. The GrpE protein mediates transfer of a substrate protein from the DnaK system to GroEL/GroES (Bukau & Horwich, 1998). The DnaK system may also cooperate with ClpB93/ClpB79, HtpG (Goloubinoff et al., 1999; Mogk et al., 1999; Thomas & Baneyx, 2000; Schlieker et al., 2002), the small heat-shock proteins IbpA/IbpB (Thomas & Baneyx, 1998; Veinger et al., 1998; Shearstone & Baneyx, 1999) or trigger factor, a peptidyl-prolyl isomerase having a chaperone activity (Hesterkamp et al., 1996; Teter et al., 1999). A possible role for GrpE in the interaction of the DnaK/DnaJ and ClpB systems emerges from the experiments of Zolkiewski (1999) on suppression of luciferase aggregation in vitro.

(iv) DnaK, DnaJ and GrpE proteins are negative autoregulators of the heat-shock response (Liberek et al., 1995; Gamer et al., 1996; Kanemori et al., 1997; Blaszczak et al., 1999). Hence, a mutation inactivating the grpE gene results in higher levels of the products of the heat-shock regulon controlled by {sigma}32.

The similarity of the phenotypes of dnaK, dnaJ and grpE mutants suggests that the grpE280 mutation, like those of dnaK756 and dnaJ259, might result in stabilization and enlargement of the S fraction. However, preliminary experiments with the grpE mutant have shown that the S fraction does not appear at the usual position in the density gradient. It was assumed that denatured proteins could form aggregates with different physical properties and thus cosediment with the OM fraction, or that formation of the aggregates might have been prevented, or that proteolysis of the aggregates was accelerated.

Considering these data one could expect that: (i) a slowed down recycling of DnaK in grpE cells might prolong DnaK/DnaJ association with the substrate, thus targeting it to degradation (Sherman & Goldberg, 1992; Huang et al., 2001), or (ii) raised levels of DnaK and other heat-shock proteins might effectively counteract the aggregation or dissolve the emerging aggregates (Kdzierska et al., 1999). In these cases, the S fraction would not appear. However, it was difficult to anticipate (iii) the result of the lack of GrpE function in the case of substrates that required transfer to another chaperone system, GroEL/GroES for example. Supposedly, in such cases the partially folded substrates might be slowly released to form a fraction of proteins denatured to conformations different from those in WT cells, thus giving rise to an S fraction with changed physical properties. This would explain the difficulty in its separation from the membrane fractions.

Delaney (1990) showed that the grpE mutant of E. coli was more resistant to heat exposure (50 °C) than the WT strain. This added interest to the observation concerning the properties of the putative S fraction in the grpE mutant.

To establish the presence or absence of the S fraction, WT and grpE cell fractionation in a density gradient and electron microscopy were performed. Afterwards, WT and grpE strains bearing the pKEN8 plasmid (Henderson et al., 1994) that overexpresses the endogenous E. coli Fda (a thermostable II class fructose-1,6-biphosphate aldolase) identified as a component of the S fraction (Kdzierska et al., 2001), were investigated. It was expected that the comparison of the levels of activity of the enzyme with the Fda total protein level (estimated by immunoblotting and densitometry) should provide more information on the mechanisms underlying the absence of the standard S fraction in the grpE cells submitted to heat shock. The IbpA/B proteins (sHSPs) were used as an indicator of aggregation. These proteins are undetectable in WT cells growing under physiological conditions. However, the ibpA/B operon is quickly and very strongly induced by heat shock (Chuang & Blattner, 1993). IbpA/B associate abundantly with the S fraction (Laskowska et al., 1996b).

Our data show that protein aggregation after heat shock proceeds differently in WT and grpE cells. The absence of the regular S fraction in grpE cells may be explained by protein renaturation (due to an elevated DnaK/DnaJ level resulting from autoregulation) rather than massive degradation. Evidence is provided that the small protein aggregates in grpE(pKEN8) cells cosediment with the OM fraction.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
Escherichia coli B178 WT (W3101 galE sup+), E. coli B178 grpE280 [bearing the point mutation Gly122 to Asp (Wu et al., 1994)] and E. coli CG459 rpoH-165 [the amber mutant, carrying a temperature-sensitive suppressor, supCts (Yura et al., 1984)], E. coli C600 thr leu supE and E. coli C600 grpE280 (Ang et al., 1986) were grown in LB. LB was supplemented with ampicillin (50 µg ml-1) for B178(pKEN8) and B178 grpE280(pKEN8) (Kdzierska et al., 2001). The bacteria were grown at 30 °C, with aeration to an OD600 of 0·25. The cultures were then transferred to 45 °C for 15 min and afterwards incubated at 37 or 45 °C (as indicated). Plasmid pKEN8 (ampR) bearing the fda gene under the control of the tac promoter and overproducing Fda (Henderson et al., 1994) was purchased from the American Type Culture Collection (ATCC 77472; Manassas, VA, USA). Expression of the fda gene was induced by addition of IPTG (1 mM).

Cell fractionation.
Cell fractionation by ultracentrifugation in sucrose density gradients was performed according to Kucharczyk et al. (1991). Samples (100 ml) of B178 WT, B178 grpE and CG459 rpoH165 cultures grown at 30 °C were collected at time 0 (before the temperature shift to 45 °C), after 15 min at 45 °C and later at 15 min intervals during growth at 37 °C and submitted to cell fractionation. The samples were quickly chilled by pouring them onto 100 ml frozen 10 mM Tris/HCl, pH 7·2. Cells were harvested by centrifugation for 10 min at 7000 g and resuspended in 200 mM Tris/HCl, pH 8·0, to obtain an OD600 of 28 U ml-1, then they were subjected to spheroplastization. The suspension was diluted with an equal volume of 1 M sucrose in 200 mM Tris/HCl, pH 8·0, supplemented with egg-white lysozyme solution (12 mg ml-1 in 100 mM EDTA, pH 7·6) to a final concentration of 60 µg ml-1. After 4 min on ice, an equal volume of ice-cold distilled water was added. After the next 10 min on ice, the spheroplasts were sonicated and centrifuged at 1200 g for 15 min. The supernatant was used for membrane fractionation as described by Kucharczyk et al. (1991). The supernatant was layered on a two-step SG0 gradient, composed of 1 ml 55 % and 6 ml 17 % (w/w) sucrose in 3 mM EDTA solution, pH 7·6. It was centrifuged for 90 min in a Beckman SW41 Ti rotor at 240 000 g. Four CP subfractions (1 ml) were collected from the top of the gradient. IM, OM and S fractions containing the insoluble proteins cosedimented in the SG0 gradient. These fractions were collected jointly (the crude membrane fraction) and submitted to further fractionation on a six-step SGI gradient: 55 (1·4 ml), 50, 45, 40 (2·3 ml of each), 35 (1·4 ml) and 30 % (0·8 ml) (w/w) sucrose in 3 mM EDTA solution, pH 7·6. After centrifugation in a Beckman SW41 Ti rotor at 240 000 g for 16 h, 30 subfractions were collected from the bottom (total volume 12 ml). Aliquots were taken from SGI subfractions for determination of the protein concentration. The subfractions corresponding to discernible fractions were pooled and denoted S (buoyant density 1·26 g ml-1), OM (1·22 g ml-1) and IM (1·14 g ml-1).

Electron microscopy.
Bacteria were grown in LB at 30 °C to an OD600 of 0·25, then transferred to 45 °C. Samples withdrawn from the bacterial cultures at time 0 (before the temperature change) and after 15 min at 45 °C were immediately mixed with an equal volume of 10 mM Tris/HCl buffer, pH 7·5. The bacteria were sedimented at 5000 g for 5 min. Cells were fixed for 2 h in cacodylate buffer (50 mM sodium cacodylate, pH 7·0) containing 2·5 % glutaraldehyde and 2·5 % paraformaldehyde. After washing with the cacodylate buffer, the cells were post-fixed in 1 % OsO4 in the same buffer for 1 h at room temperature, stained in 1 % uranyl acetate for 1 h, dehydrated with acetone and embedded in a low-viscosity resin (Spurr, 1969). Ultrathin sections were stained on grids with uranyl acetate and lead citrate, and examined under a Tesla BS 500 transmission electron microscope. A random series of sections were photographed and used for morphometric analysis. Circumferences and surfaces of the cell sections, and of the protein aggregates of the S fraction visible on the sections were measured with a digitizer linked to a computer (Weibel & Bolender, 1973). For each strain, 30–45 negatives taken at x10 000 or x18 000 magnification were analysed. The results were expressed as a percentage of the cellular section surface occupied by the protein aggregates.

Immunodetection and estimation of proteins: DnaK, DnaJ, IbpAB and Fda.
The bacteria (B178 and B178 grpE) were grown in LB at 30 °C to an OD600 of 0·3, transferred to 45 °C and further grown at the same temperature. Samples containing equal amounts of bacteria were lysed. The bacterial proteins were separated by 0·1 % SDS-10 % PAGE. For immunodetection the proteins were electrotransferred to NC2 nitrocellulose membrane (Serva) (Sambrook et al., 1989). Polyclonal rabbit antisera against DnaJ, DnaK, IbpAB and Fda were used and the reactions were developed using the goat-anti-rabbit IgG-HRP (Sigma) and 4-chloro-1-naphthol and H2O2 as substrates. The amount of protein loaded on the gels was adjusted to obtain an intensity of bands in the linear range of measurements by densitometry (SigmaGel program) and expressed in arbitrary units (AU).

Analytical methods.
Determination of Fda activity in vivo was described previously (Kdzierska et al., 2001). Briefly, WT(pKEN8) and grpE(pKEN8) strains were grown in LB medium supplemented with ampicillin (100 µg ml-1) at 30 °C to an OD600 of 0·25, then IPTG to 1 mM final concentration was added for 1 or 2 h of further growth, as indicated. Subsequently, the cultures were submitted to heat shock (45 °C for 15 min; inactivation period) in the presence of kanamycin (100 µg ml-1) and tetracycline (25 µg ml-1), then transferred to 37 °C (reactivation period). These antibiotics were added to prevent de novo protein synthesis (Kdzierska et al., 2001) which would obscure measurement of Fda renaturation. Samples were collected at time 0 (immediately before heat shock), 15 min after the transfer to 45 °C and later at 10 min intervals during growth at 37 °C. The cells were lysed in a BioBlock Sonifier Vibra Cell and the coupled enzyme assay for the fructose-1,6-bisphosphate cleavage activity of Fda was carried out (Sigma Quality Test procedure). The decrease in A340 at 25 °C was measured.

The Laemmli (1970) method was used for 0·1 % SDS-10 % PAGE and the Bradford (1976) method was used for total protein determination.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Protein aggregation upon heat shock in E. coli B178 WT, B178 grpE and CG459 rpoH165
The S fraction reproducibly reached its maximum (13–17 % of total insoluble proteins) after heat shock in all the E. coli strains tested bearing the wild-type genes of the heat-shock response (Laskowska et al., 1996a; Kucharczyk et al., 1991; Kdzierska et al., 1999). The timing and the temperature regime were adopted from these previous experiments.

Size and stability of the S fraction from the WT (control) and the grpE strains was compared (Fig. 1). The S fraction (~15 % of the insoluble protein) reached its maximum at 15 min at 45 °C (Fig. 1b) and after the bacteria were transferred to 37 °C it disappeared over 15 min (Fig. 1c). However, ~66 % of the S fraction remained intact when the bacteria were incubated at 45 °C for 30 min (Fig. 1d). Apparently, the prolonged high temperature treatment affects the capability of the cellular protein protection/rescue system. The rpoH165 strain, unable to induce the heat-shock response because of the lack of an active {sigma}32 protein, the rpoH gene product, was a negative control. A large increase in the S fraction (~44 % of insoluble proteins) at 15 min at 45 °C (Fig. 1k) and its persistence for 30 min after transfer to 37 °C (Fig. 1l) was observed. In contrast, the S fraction in the grpE cells was not detectable under either set of conditions (Fig. 1e–h). The OM fraction, however, was enlarged in the grpE cells after heat shock (Fig. 1f–h) in comparison to that in WT cells. This might mean that the putative S fraction cosedimented with the OM. A deformation of the symmetry of the OM peaks (on the left side) supported this assumption.



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Fig. 1. Rise and decay of the fraction of aggregated proteins (S fraction) in E. coli B178 WT, B178 grpE and CG459 rpoH after heat shock. The S fraction is shaded. The S, OM and IM peaks are marked at the top of the figure. The percentage values given over the S peaks (b, k) are related to the total insoluble protein from the SG0 gradient (introduced later onto the SGI gradient). The percentage values marked underneath the S peaks are related to the S fraction (d, l). One hundred percent of the total protein of the S fraction corresponded to its maximal amount, i.e. 15 min after the temperature change from 30 to 45 °C. The time of removal of the samples for analyses is marked on the right. Time 0 was immediately before the temperature shift to 45 °C. Temperature changes are indicated.

 
Since E. coli strains carrying inactivating mutations in the genes encoding their major chaperone proteins readily accumulate extragenic suppressor mutations, the effect ascribed to the grpE mutation was checked on another pair of E. coli strains, C600 and C600 grpE280. Exactly the same result, the lack of a regular S fraction in the grpE bacteria, was observed. Thus coherence of the grpE mutation and the lack of the S fraction was confirmed (results not shown).

Electron microscopy was used to verify the presence or absence of protein aggregates. Representative thin sections of WT, grpE and rpoH cells are shown in Fig. 2. To estimate the size of the aggregates in the cells, morphometric analysis was applied (Weibel & Bolender, 1973). In WT and grpE cells grown at 30 °C (Fig. 2) no aggregated protein was visible, but in rpoH cells they amounted to ~2·3 % of the section surface. Fifteen minutes after changing the temperature to 45 °C the aggregates appeared in WT (~2·9 %) cells, while they were undetectable in grpE cells and the amount in rpoH cells reached ~7·3 % of the surface. The grpE mutants were left at 45 °C for 60 min to determine if the aggregates would appear under such challenging conditions. The amount of aggregates reached only ~1·1 % (not shown).



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Fig. 2. Electron microscopy of thin sections of E. coli B178 WT, B178 grpE and CG459 rpoH before and after heat shock. Samples of bacterial cultures were collected at time 0 and at the time of maximal development of the S fraction (as described in the legend to Fig. 1). The thin sections were stained as described in Methods. Arrows indicate the aggregated proteins of the S fraction. Bar, 1 µm.

 
The results of electron microscopy agree with those of density-gradient centrifugation. Hence, the absence of the aggregates in grpE cells was documented by both techniques. This could mean that in grpE cells a mechanism exists which prevents the accumulation of protein aggregates resembling those from WT cells or favours efficient degradation of the denatured proteins. Another possibility was that due to a difference in the properties of the protein aggregates they could escape detection by these techniques. The importance of the DnaK and DnaJ proteins for S fraction removal was shown previously (Kdzierska et al., 1999) by use of dnaK or dnaJ mutant cells. Eighty percent of the S fraction was stabilized in these mutants. On the other hand, a fourfold overproduction of DnaK/DnaJ in vivo prevented the appearance of the S fraction after heat shock in the WT strain (Kdzierska et al., 1999). Therefore, DnaK and DnaJ levels were estimated in WT and grpE cells.

Estimation of DnaK and DnaJ levels by immunoblotting
The concentration of the DnaK protein at 30 °C (27 µM) and 42 °C (54 µM) was determined by Mogk et al. (1999). Our data are in good agreement with these results showing a twofold increase in the DnaK level after heat shock (15 min, 45 °C) in the WT strain.

The DnaK level in grpE cells amounted to about 2·5 times that in WT cells, whereas the DnaJ level was ~30 % higher in grpE cells than in WT cells (Fig. 3).



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Fig. 3. Estimation of DnaK and DnaJ levels in E. coli B178 WT and B178 grpE cells by immunoblotting and scanning densitometry. SDS (0·1 %)-PAGE (10 %) and immunodetection was performed as described in Methods. Samples were collected at 30 °C (time 0) and at 45 °C (15 min). The arbitrary densitometric units (AU) are shown at the bottom.

 
The question was raised whether the increase in the DnaK and DnaJ levels might be sufficient for protection/renaturation of the heat-denaturated proteins, enough to prevent the appearance of the S fraction in the grpE mutant cells.

Sherman & Goldberg (1992), who used the PhoA61 protein as a model, proposed that proteins associated with the DnaK/DnaJ/GrpE system might be targeted to proteolysis if they could not be released from this system. It is not known, in molecular terms, what is to be expected if the GrpE protein alone, normally participating in the release, was absent. To learn more, Fda (Henderson et al., 1994) was used as a model. WT and the mutant strains were transformed with pKEN8 because overexpression of Fda was necessary for reliable measurements of its activity in vivo. Overproduction of Fda could be regulated by the length of the growth period in the presence of IPTG (1 mM). Degradation would be indicated by a coupled decrease in the activity and the protein level.

Protein aggregation in WT(pKEN8) and grpE(pKEN8) cells overproducing Fda
IPTG was added for 1 or 2 h growth at 30 °C for Fda overproduction. Analysis of whole-cell extracts by 0·1 % SDS-12 % PAGE revealed that the Fda level was approximately 2·4- and fourfold higher than in the control WT strain after 1 and 2 h induction, respectively (Fig. 4). It was intended to keep a low excess of Fda to avoid intensive aggregation of the overproduced protein. To examine to what extent Fda overproduction might alter the S fraction size and its persistence, cell fractionation was performed (Fig. 5).



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Fig. 4. Estimation of Fda protein overproduction in WT(pKEN8) and grpE(pKEN8) cells. The cultures were grown in LB at 30 °C to an OD595 of 0·2, then 1 mM IPTG was added to induce overproduction of Fda from pKEN8. After 1 or 2 h of further incubation at 30 °C, samples, equivalent to 20 µg protein, were withdrawn for 0·1 % SDS-12 % PAGE. The gel was stained with Coomassie brilliant blue. Densitometry was performed with the SigmaGel program. The arbitrary units (AU) are shown at the bottom. Purified Fda (2 µg) was used as a marker.

 


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Fig. 5. The increase in the fraction of aggregated proteins (the S fraction) in E. coli WT(pKEN8) and grpE(pKEN8) after heat shock. The bacteria were grown, submitted to heat shock (30–45 °C, 15 min) and fractionated in SG0 and SGI gradients as described in Methods. Panels: (a)–(j), Fda expression was induced by addition of IPTG (1 mM) 1 h before the temperature shift; (k)–(u), IPTG was added 2 h before the temperature shift. The S fraction containing the Fda protein is shaded. The S, OM and IM peaks are marked on top of the panels. The percentage values marked over or underneath the peaks are calculated as described in the legend to Fig. 1. Time of sample collection and the temperature changes during growth are indicated on the right.

 
The 2·4-fold overproduction of Fda (1 h IPTG induction) in the WT(pKEN8) and grpE(pKEN8) cells grown at 30 °C did not cause an increase in the S fraction (Fig. 5a, f). After heat shock the size of the S fraction in WT(pKEN8) cells corresponded to ~33 % of cellular insoluble protein (Fig. 5b), while in non-transformed WT cells it reached ~15 % (Fig. 1b and Kdzierska et al., 1999). The disappearance of the S fraction from WT(pKEN8) cells was delayed – ~21 % was present at 35 min (Fig. 5d) of the experiment. But at 45 min (approx. one generation time) it was already undetectable (Fig. 5e).

The regular S fraction did not appear in grpE(pKEN8) cells after the transfer to 45 °C (Fig. 5g) and later to 37 °C (Fig. 5h–j). However, enlargement and deformation of the OM peak suggested that the SgrpE fraction might have cosedimented with OM because the physical properties of the aggregates could differ from those of the S fraction.

The fourfold excess of Fda in cells induced during 2 h growth with IPTG (Fig. 5l–u) caused formation of a large S fraction (Fig. 5l, ~46 % of insoluble proteins) in the WT(pKEN8) cells submitted to heat shock. The fraction was larger than in the WT(pKEN8) strain producing a 2·4-fold excess of Fda (Fig. 5b). The S fraction was quite stable; ~41 % was still present at 45 min of the experiment (Fig. 5o). The fractionation pattern was different in grpE(pKEN8) cells. The S fraction was nearly half the size of that present in WT(pKEN8) (Fig. 5r), although it was also quite stable, with ~68 % still remaining at 45 min. These results meant that even a 2·4-fold (Fig. 5b–d) overproduction of a homologous protein in WT or grpE cells overburdened the mechanisms controlling folding or degradation of proteins. Nevertheless, the difference between the WT and grpE cells in their capability for removing the S fraction was analogous to that observed in non-transformed cells.

SDS-PAGE analysis of the aggregated proteins isolated from WT(pKEN8) cells before and after Fda induction showed that the enlargement of the S fraction did not result from an increased level of aggregated Fda, but rather from enhanced aggregation of other proteins detectable in the S fraction (not shown). We comment on this observation in the Discussion.

In the following experiments aimed at elucidating whether the SgrpE fraction cosedimented with OM in the grpE cells, a 1 h induction was applied.

Estimation of Fda activity in WT(pKEN8) and grpE(pKEN8) cells submitted to heat shock
The total Fda protein level, equal in the two strains, did not change after heat shock (Fig. 6a). Estimation of total Fda activity in vivo (Fig. 6b) in the strains grown at 30 °C also showed comparable levels [approx. 0·25 unit (mg protein)-1 was accepted as 100 % activity]. After heat shock (at 15 min), the Fda activity dropped to ~88 % of its starting level in WT, and to ~81 % in the grpE strain. The activity continued to drop during the period of growth at 37 °C (25 and 35 min), reaching the lowest points of ~78 % in the WT strain and ~68 % in the grpE strain. Then, recovery of activity began in the WT cells and attained ~92 % at 45 min. In the grpE cells, only a slight tendency (if any) toward reactivation was noticeable, reaching ~69 % of the starting level. In these experiments Fda synthesis de novo was blocked by the appropriate antibiotics (Methods), therefore the results illustrated the reactivation.



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Fig. 6. Estimation of the Fda protein and Fda activity levels in WT(pKEN8) and grpE(pKEN8) cells after heat shock and during reactivation at 37 °C. (a) Bacterial cultures were grown in LB at 30 °C to an OD595 of 0·2, then IPTG (1 mM) was added. Heat shock was applied (45 °C, 15 min) 1 h postinduction. Samples were collected at time 0 and at 25 min into the experiment (10 min after the shift to 37 °C). E. coli proteins were separated by 0·1 % SDS-15 % PAGE and immunodetected using Fda antiserum (Kdzierska et al., 2001). The membrane was scanned and analysed with the SigmaGel program. The arbitrary densitometric units (AU) are shown at the bottom. (b) Fda activity was estimated in the soluble CP fraction from the SG0 sucrose gradient (Kdzierska et al., 2001). No Fda activity was found in the insoluble fractions (not shown). Samples for cell fractionation were collected at time 0, after the shift to 45 °C (15 min) and later, during growth at 37 °C, at the indicated time intervals [WT(pKEN8), grey columns; grpE(pKEN8), white columns]. The experiment was repeated three times. The reactions were carried out twice. The deviation in the values is marked.

 
Localization and estimation of the Fda and IbpA/B proteins in cellular fractions of WT(pKEN8) and grpE(pKEN8) cells
Fda was previously identified as the S fraction component, although its bulk remained in CP in WT strains after heat shock (Kdzierska et al., 2001). However, Fda was never found in OM before or after heat shock in the WT bacteria.

Cells were grown at 30 °C, induced by the addition of IPTG (1 h), submitted to heat shock and fractionated (Methods). Samples of the S, OM, IM and CP fractions were collected at 25 min of the experiment (i.e. 10 min after the transfer to 37 °C for reactivation; Fig. 5c, h). The proteins of each fraction were separated by electrophoresis. Fda was immunodetected and estimated densitometrically (Fig. 7). It occurred in fractions S (~2 %) and CP (~98 %). Some traces were also detectable in IM from WT(pKEN8) cells. In grpE(pKEN8) cells, no distinct S fraction was visible. However, Fda was present in fractions OM (~6 %) and CP (~94 %). Traces of Fda were also present in IM. Detection of Fda in OM from the grpE strain revealed that the hypothetical SgrpE fraction might indeed have cosedimented with OM.



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Fig. 7. Cellular localization and estimation of the Fda protein in the soluble (CP) and insoluble (S or OM) fractions from WT(pKEN8) and grpE(pKEN8) cells. The bacterial culture was induced with IPTG (1 h), then submitted to heat shock and incubated further at 37 °C for 10 min. Fractionation was performed as shown in Fig. 5(c, h). Proteins of the S (5 µg), OM (5 µg), IM (5 µg) and CP (10 µg) fractions were separated by 0·1 % SDS-12 % PAGE. Fda antiserum was used for Fda immunodetection and estimation by densitometry as described in the legend to Fig. 6. The results are shown under the figure in AU. The approximate percentage of the Fda protein in cellular fractions was calculated taking into account that the SWT and OMgrpE fractions contain 2 and 6 % total protein, respectively, while the soluble CP fraction contains 87 % total cellular proteins (Bradford, 1976; not shown). The amount of protein loaded onto the gel was also taken into account.

 
A comparison of aldolase activity (Fig. 6b, 25 min) and Fda protein levels (Fig. 7) in WT(pKEN8) cellular fractions showed that in CP only ~85 % of the Fda activity corresponded to ~98 % of the Fda protein after heat shock. This indicated that about 13 % of the Fda in CP was in a soluble but inactive form after heat shock.

In grpE(pKEN8) cells, the CP fraction contained ~74 % Fda activity (Fig. 6, 25 min) but ~94 % of the Fda protein (Fig. 7). Thus, the quantity of the soluble, inactive enzyme (~20 %) was evidently higher than in WT cells. Even if the results are prone to error (cell fractionation and densitometry of blots are not quantitative methods), the considerable difference must be meaningful.

To confirm the notion that the SgrpE fraction cosediments with OM, the small heat-shock protein, IbpAB, was used as a marker. IbpA/B associates with the proteins of the S fraction (Laskowska et al., 1996b) and generally with the aggregated proteins (Allen et al., 1992). The IbpA/B proteins appear in a cell only after induction of the ibpA/B operon by heat shock.

The expression of the ibpA/B operon is regulated by the {sigma}32 subunit of the RNA polymerase [additionally, the ibpB gene is also regulated by the {sigma}54 subunit (Kuczynska-Wisnik et al., 2001)]. It was expected that after heat shock the content of IbpA/B might be higher in grpE cells than in the WT (due to negative autoregulation by GrpE), as in the case of DnaK and DnaJ. The IbpA/B levels in grpE(pKEN8), WT(pKEN8) and WT cells were compared by electrophoresis of total bacterial protein, immunodetection and densitometry (Fig. 8a). It appeared that the WT(pKEN8) cells contained an approximately sevenfold higher level of IbpA/B, and the grpE(pKEN8) an ~11-fold higher level than the WT non-transformed cells at 25 min of the experiment. These data are in agreement with results presented by Allen et al. (1992). The authors found that IbpA/B were induced in E. coli cells overproducing heterologous proteins which formed inclusion bodies. Our results indicate that overproduction of a homologous protein may also result in an increased IbpA/B level.



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Fig. 8. Estimation and cellular localization of the IbpA/B proteins in WT(pKEN8) and grpE(pKEN8) cells after Fda gene induction and heat shock. (a) Comparison of the total IbpA/B protein level in the WT, WT(pKEN8) and grpE(pKEN8) bacteria. Conditions for bacterial growth, induction with IPTG, cell fractionation, IbpA/B immunodetection (with IbpA/B antiserum) and densitometric estimation were as described in the legend to Fig. 6. Results are shown in AU under the panel. (b) Distribution and estimation of IbpA/B proteins in cellular fractions of WT(pKEN8) and grpE(pKEN8) cells. The following amounts of each subcellular fraction were used for analysis: 5 µg S, 5 µg OM, 5 µg IM and 10 µg CP from WT(pKEN8), and 1·5 µg OM, 5 µg IM and 25 µg CP from grpE(pKEN8). Densitometric AU are shown under the panel. The approximate percentage of the IbpA/B protein in cellular fractions was calculated taking into account that the SWT and OMgrpE fractions represent ~2 and ~6 % of total protein, respectively, while the soluble CP fraction constitutes ~87 % of total cellular proteins (Bradford, 1976). The amount of protein loaded on the gel was also taken into account.

 
For localization and estimation of the IbpAB proteins in cellular fractions of WT(pKEN8) and grpE(pKEN8), the bacteria were heat-shocked and fractionated as described above. Samples for fractionation were collected at 25 min of the experiment.

IbpA/B protein levels were estimated by the use of immunodetection and densitometry. Distribution of the IbpA/B proteins among the four cellular fractions is shown as a percentage of the total IbpA/B protein in each strain (Fig. 8b). In WT(pKEN8) cells the majority of IbpA/B, ~96 %, was found in CP and only ~4 % in the S fraction (Fig. 8b). The IbpA/B proteins were practically absent from OM in WT(pKEN8) cells. In contrast, the enlarged OM fraction from the grpE(pKEN8) cells (shown in Fig. 5h) contained ~81 % of IbpA/B (Fig. 8b). The remaining ~19 % were found in CP and ~1 % in IM at 25 min of the experiment. There is a discrepancy between these and our previous results (Laskowska et al., 1996b) showing the presence of a 16 kDa protein, taken as IbpA/B, in the OM fraction. It was noticed later, however, that the IbpA/B-immunogen preparation was contaminated by 16 kDa membrane protein. We found that separation of this protein from IbpA/B by one-dimensional electrophoresis could be achieved using a Tricine-containing buffer (Strom et al., 1993). The newly prepared serum does not recognize the 16 kDa membrane protein and does not react with components of the OM fraction.

The results taken together show that both kinds of marker proteins, Fda (its insoluble part) and IbpAB, were co-localized with the OM fraction in the grpE(pKEN8) cells, contrary to their localization in the distinct S fraction in the WT or WT(pKEN8) cells. The OM fraction sediments at a lower sucrose density in the gradient than the S fraction, hence the protein aggregates cosedimenting with OM must be smaller than those forming the S fraction. There is also an indication that still smaller, soluble aggregates, containing inactive Fda are present in the CP fraction.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The results presented here demonstrate that the aggregation of endogenous proteins in vivo proceeds differently upon heat shock in WT and grpE cells. Experiments on separating the aggregated protein as the S fraction (Fig. 1) and electron microscopy of thin cell sections (Fig. 2) pointed to the absence of the regular S fraction in grpE cells. However, the use of two proteins as markers, Fda, one of the S fraction components (Kdzierska et al., 2001), and IbpA/B, the small heat-shock proteins abundantly associating with proteins of the S fraction (Laskowska et al., 1996b) or with the aggregated heterologous proteins (Allen et al., 1992), revealed an alternative possibility.

Estimation of enzymic activity of Fda versus total Fda protein (by immunoblotting and densitometry) allowed us to distinguish between two possible causes of the absence of the S fraction: renaturation or proteolysis. It was determined that the total Fda content in the WT(pKEN8) and grpE(pKEN8) strains was almost identical before or after heat shock (Fig. 6a). The loss of 25 % of Fda activity in heat-treated grpE(pKEN8) cells had no counterpart in the loss of Fda protein (Fig. 6a, 25 min). This spoke against massive proteolysis of the aggregated Fda protein in the grpE cells. The finding does not contradict our previous results demonstrating that the S fraction also encompasses substrates for the heat-shock proteolytic system (Laskowska et al., 1996a), since it may be directed against irreversibly damaged proteins.

Fda is resistant to denaturation in vitro at 45 °C for 15 min, and a temperature of 55 °C had to be applied for enzyme inactivation (Henderson et al., 1994; Kdzierska et al., 2001). Nevertheless, in cells submitted to heat shock at 45 °C for 15 min it was denatured and aggregated. It was supposed that the denaturation of the thermostable Fda in vivo results rather from a temporary and transient deficit of heat-shock proteins, rather than from the direct heat effect in WT cells (Kdzierska et al., 2001). This notion is in agreement with the observation that fourfold overexpression of DnaK/DnaJ proteins before heat shock resulted in a total lack of the S fraction in the E. coli HB101 WT strain (Kdzierska et al., 1999).

In the WT cells submitted to heat shock, ~2 % of Fda protein was found in the S fraction and it was absent from the OM fraction (Fig. 7). In the grpE cells no regular S fraction was observed but ~6 % of total Fda protein cosedimented with OM. Fda present in the insoluble fractions was devoid of activity (Kdzierska et al., 2001). Diamant et al. (2000), in their in vitro studies on glucose-6-phosphate dehydrogenase (G6PDH), proved that the DnaK system recognized not only nascent proteins as substrates, but also previously aggregated proteins. The efficiency of disaggregation and renaturation of G6PDH decreased with the increase in the aggregate size. Controlled ranges of aggregate size were obtained by the use of different conditions for G6PDH denaturation. In our experiments, buoyant density of the protein aggregates was an indicator of their size. The S fraction formed a band at 1·26 g sucrose ml-1 in the gradient. It corresponded to the aggregates which were effectively processed in WT cells in ~15 min (Fig. 1) by the cooperative, natural chaperone systems. The OM fraction was localized at 1·22 g ml-1 (Kucharczyk et al., 1991). Hence, the protein aggregates which cosedimented with the OM fraction had a smaller size than those from the S fraction from WT cells. This observation may be interpreted as reflecting a less advanced process of denaturation/aggregation in the grpE cells due to protection by the elevated levels of DnaK/DnaJ and IbpA/B (Fig. 3 and Fig. 8a, respectively). Predictably, other members of the {sigma}32 heat-shock regulon were also up-regulated in the grpE mutant cells (Connolly et al., 1999; Arsène et al., 2000). However, it is also possible that the small aggregates are products of disaggregation of larger aggregates by the DnaK/DnaJ and ClpB systems (Diamant et al., 2000).

In both strains tested the bulk of Fda remained in the CP fraction: ~98 % in WT and ~94 % in grpE. The occurrence of the majority of the Fda protein in the soluble form (CP) after heat shock was consistent with the notion that only newly synthesized proteins are subject to aggregation (leading to inclusion body formation), and those with a native conformation are resistant (Mitraki & King, 1989). However, a comparison of the distribution of total Fda protein versus Fda activity among cellular fractions revealed that ~13 % of cellular Fda in WT(pKEN8) cells and ~20 % in grpE(pKEN8) cells was present in the CP fraction, in a soluble but inactive form (Figs 6 and 7). Coincident was the presence of IbpA/B in CP (Fig. 8b), suggesting that an inactive fraction of Fda (and presumably of other proteins) formed small aggregates that did not exceed the solubility level. IbpA/B proteins are not detectable in bacteria growing under physiological conditions when protein aggregation does not occur. The ibpAB operon is immediately and very efficiently inducible by heat shock (Allen et al., 1992; Chuang & Blattner, 1993; Mogk et al., 1999). The IbpA/B proteins associate with aggregated proteins (Allen et al., 1992; Laskowska et al., 1996b) and protect them from irreversible denaturation and extensive proteolysis. In other words, the IbpA/B proteins preserve denaturating proteins as a pool of substrates competent for refolding and reactivation by the ATP-dependent chaperone systems (Veinger et al., 1998; Shearstone & Baneyx, 1999; Kuczynska-Wisnik et al., 2002; Kitagawa et al., 2002). This fits in with our observations. Noticeably, the higher level of the soluble, inactive Fda in the grpE cells than in the WT cells coincided with the higher level of IbpAB (Figs 7 and 8b).

Maintenance of various denatured proteins in the form of small, soluble aggregates may be required for recognition by the GroEL/GroES system acting for the final renaturation. The transfer from DnaK/DnaJ to GroEL/GroES is GrpE- (exchange factor) and ATP-dependent, although the molecular mechanism of substrate transfer remains obscure. The lack of GrpE, abolishing or slowing down the cooperation of DnaK/DnaJ with GroEL/GroES (and possibly with other systems), might be responsible for the increased size of the soluble aggregate fraction in grpE cells. One should add here that GroEL/GroES proteins were localized to the CP fraction (Kucharczyk et al., 1991) and were never found in the S fraction. We now assume that they bind with the postulated soluble aggregate fraction for their final renaturation.

The experimental system used here differs slightly from the natural system because of the overproduction of Fda. The presence of a 2·4-fold excess of one kind of molecule, the Fda protein, probably created a deficit of DnaK/DnaJ required not only for their refolding, but also for other, newly synthesized proteins (Kdzierska et al., 2001). This explains the increased size and persistence of the S fraction (Fig. 5b–d) in WT(pKEN8) cells. We propose that in transformed cells the denaturation of cellular proteins might be caused not only by high temperature, but also by a temporary and transient insufficient supply of the appropriate chaperone proteins. We are not aware if any of these types of denaturation may be more troublesome from the point of view of renaturation. Apparently, the deficit of chaperones in grpE(pKEN8) cells is not entirely covered by the 2·4-fold overproduction of DnaK/DnaJ resulting from autoregulation. But the extent of aggregation (visible only as a deformation of the S fraction peak) is smaller in grpE(pKEN8) cells than in WT(pKEN8) (Fig. 5a–j). This demonstrates the limits of autoregulation. The effect, strengthened by fourfold overproduction of Fda, is shown in the Fig. 5(k–u).

In summary, we propose that the elevated level (due to the negative autoregulation of the heat-shock regulon) of the chaperone proteins in the grpE mutant are responsible for the absence of the regular S fraction after heat shock. Instead two fractions of small agregates are formed. One insoluble fraction cosediments with OM, the other soluble fraction is found in CP. Both are represented by inactive Fda protein. Supposedly, the IbpA/B proteins found in OM and CP protect the small aggregates from further aggregation or proteolysis. This aggregation pattern may reflect the lack of the functional GrpE protein that, among other functions, mediates transfer of substrate proteins from the DnaK/DnaJ system to GroEL/GroES.


   ACKNOWLEDGEMENTS
 
We thank K. Krzewski and J. Osipiuk for anti-DnaK and anti-DnaJ sera and J. Potrykus for reading the manuscript. This work was supported by grants: DS/1160-4-0115-03 and DS/1190-4-0114-03 from University of Gdansk, and 0461/P04/2001/20 from the Polish Committee for Scientific Research.


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Received 8 May 2003; revised 18 September 2003; accepted 18 September 2003.



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