Small Heat Shock Proteins, Clpb and the Dnak System Form a Functional Triade in Reversing Protein Aggregation

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology 1365-2958Blackwell Publishing Ltd, 2003502585595Original ArticlesHsps, ClpB and DnaK form a functional triadeA. Mogk et al.

Molecular Microbiology (2003) 50(2), 585–595 doi:10.1046/j.1365-2958.2003.03710.x

Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation

Axel Mogk,1,2* Elke Deuerling,1 DnaK chaperone systems prevent the aggregation of ther- Sonja Vorderwülbecke,1 Elizabeth Vierling3 and molabile proteins and support their refolding to the native Bernd Bukau1* state (Langer et al., 1992; Viitanen et al., 1992; Horwich 1ZMBH, Universität Heidelberg, Im Neuenheimer Feld et al., 1993; Schröder et al., 1993). The DnaK system 282, Heidelberg D-69120, Germany. in addition disaggregates small aggregates of heat- 2Institut für Biochemie und Molekularbiologie, denatured proteins (Schröder et al., 1993; Skowyra et al., Hermann-Herder-Str. 7, D-79104 Freiburg, Germany. 1990; Mogk et al., 1999; Diamant et al., 2000). The 3Department of Biochemistry and Molecular Biophysics, AAA+ chaperone ClpB allows the DnaK system to effi- University of Arizona, 1007 E. Lowell St., Tucson AZ ciently disaggregate even large aggregates of misfolded 85721, USA. proteins (Goloubinoff et al., 1999; Mogk et al., 1999; Zolk- iewski, 1999), similar to the homologous Hsp104 and Hsp70 chaperone systems of Saccharomyces cerevisiae Summary (Glover and Lindquist, 1998). Non-native proteins may Small heat shock proteins (sHsps) can efficiently pre- alternatively be degraded by proteases. Lon, ClpAP, vent the aggregation of unfolded proteins in vitro. ClpXP and HslUV represent the major ATP-dependent However, how this in vitro activity translates to func- proteolytic systems of the E. coli cytosol and act syner- tion in vivo is poorly understood. We demonstrate that gistically in vivo in the degradation of abnormal proteins sHsps of Escherichia coli, IbpA and IbpB, co-operate (Kanemori et al., 1997). Chaperones and proteases also with ClpB and the DnaK system in vitro and in vivo, exhibit synergistic and overlapping functions in the protein forming a functional triade of chaperones. IbpA/IbpB quality control system. Importantly, ClpXP and Lon and ClpB support independently and co-operatively become essential for viability at high temperatures under the DnaK system in reversing protein aggregation. conditions of limiting amounts of the DnaK system (Tomo- A DibpAB DclpB double mutant exhibits strongly yasu et al., 2001). Depletion of the DnaK system (KJE) in increased protein aggregation at 42∞C compared with clpXP and lon mutant cells caused strong protein aggre- the single mutants. sHsp and ClpB function become gation, indicating overlapping functions of proteases and essential for cell viability at 37∞C if DnaK levels are KJE in preventing protein aggregation during heat stress. reduced. The DnaK requirement for growth is increas- The role of small heat shock proteins (sHsps) in the ingly higher for DibpAB, DclpB, and the double Dib- protein quality control network is poorly understood. The pAB DclpB mutant cells, establishing the positions of lack of strong phenotypes of mutants lacking sHsp activity sHsps and ClpB in this chaperone triade. has prevented a clear assignment of sHsp function in the chaperone network. While overproduction of sHsps in different cells including E. coli can increase stress tolerance, Introduction sHsp function is not essential for thermotolerance in S. The ensemble of molecular chaperones and proteases cerevisiae or E. coli (Petko and Lindquist, 1986; Thomas constitutes the cellular system for de novo folding and and Baneyx, 1998; Kitagawa et al., 2000). Temperature- quality control of proteins (Hartl, 1996; Gottesman et al., sensitive growth phenotypes, caused by missing sHsp 1997; Wickner et al., 1999; Dougan et al., 2002). This function, have only been reported for Neurospora crassa system is operative under regular growth conditions but (Plesofsky-Vig and Brambl, 1995) and Synechocystis sp. becomes particularly important under stress conditions, PCC6803 (Giese and Vierling, 2002). The behaviour of such as heat shock. The functions of the major cytosolic sHsps in vivo also appears to contrast with their reported chaperone and protease systems during heat stress are in vitro chaperone activity. In vitro sHsps can efficiently best understood in Escherichia coli. The GroE and the prevent the aggregation of heat denatured proteins (Hor- witz, 1992; Jakob et al., 1993; Lee et al., 1997). In vivo, Accepted 7 July, 2003. *For correspondence. E-mail [email protected]; Tel. (+49) 6221 546863; Fax (+49) however, sHsps frequently localize to insoluble protein 6221 545894. fractions of heat stressed cells. Even at normal growth

586 A. Mogk et al. temperatures in the presence of high levels of unfolded proteins sHsps will become insoluble. Indeed they were first defined in E. coli as ‘inclusion body binding proteins’ (IbpA and IbpB), accumulating in association with over- produced heterologous proteins in inclusion bodies (Allen et al., 1992; Laskowska et al., 1996). Because solubilization of stress-induced aggregated proteins is still possible in DibpAB mutant cells, the functional importance of the proposed chaperone activity of sHsps in E. coli has remained in question (Mogk et al., 1999; Kuczynska- Wisnik et al., 2002). Here we link the function of sHsps to the chaperone network of E. coli both in vitro and in vivo. We demonstrate that sHsps co-operate with ClpB and the DnaK system in reversing protein aggregation. In vitro refolding of substrates bound to IbpB is mediated most efficiently by ClpB and KJE. Presence of IbpA/IbpB in insoluble protein aggregates accelerated the ClpB/KJE mediated disaggregation reaction in vivo. Functions of sHsps and ClpB become essential for cell viability and protein resolubilization at elevated temperatures under conditions of limiting DnaK levels. These data provide direct evidence for a functional triade of sHsp, ClpB and DnaK in reversing protein aggregation in vivo.

Fig. 1. ClpB stimulates the KJE-dependent refolding of substrates Results bound to IbpB. A. 2 mM malate dehydrogenase (MDH) was heat denatured at 47∞C ClpB stimulates the KJE-dependent refolding of in presence of 16 mM IbpB resulting in the formation of soluble IbpB/ substrates bound to IbpB MDH complexes. MDH refolding was started by diluting the IbpB/ MDH complexes 1:1 with the indicated cytosolic Escherichia coli sHsps are found associated with aggregated proteins in chaperones at 30∞C (KJE: 1 mM DnaK, 0.2 mM DnaJ, 0.1 mM GrpE; heat stressed E. coli cells (Allen et al., 1992; Laskowska ELS: 4 mM GroEL, 4 mM GroES; ClpB: 1.5 mM ClpB) and MDH activities were determined at the indicated time points. The enzymatic et al., 1996; Mogk et al., 1999). We therefore tested activity of native MDH was set at 100%. whether the chaperone ClpB, which acts to dissociate B. 0.2 mM luciferase was heat-denatured at 43∞C in the presence of protein aggregates, can affect in vitro the refolding of 1.6 mM IbpB, resulting in the formation of soluble IbpB/luciferase complexes. Luciferase refolding was initiated by diluting the IbpB/ substrates from sHsp/substrate complexes using thermo- luciferase complexes 1:1 with the indicated chaperones (KJE: 0.5 mM labile malate dehydrogenase (MDH) and firefly luciferase DnaK, 0.1 mM DnaJ, 0.05 mM GrpE; ELS: 2 mM GroEL, 2 mM GroES; as model substrates (Fig. 1). In these studies, we did not ClpB: 1 mM ClpB) and luciferase activities were determined at the indicated time points. The enzymatic activity of native luciferase was include IbpA because this protein could not be overpro- set at 100%. duced and purified in a soluble state, as observed earlier (Shearstone and Baneyx, 1999). MDH (2 mM) was protected from heat-induced aggregation at 47∞C by IbpB reactivation of MDH bound to IbpB. The refolding rate (16 mM). Reactivation of MDH from soluble MDH/IbpB increased to 14.5 nM MDH min-1 and complete refolding complexes was only observed in the presence of the of sHsp-bound MDH was observed within 60 min. These DnaK system (KJE), similar to published results (Veinger results indicate that ESL and ClpB act during different et al., 1998). Denaturation of MDH in the presence of stages of the MDH reactivation process. sHsps was a prerequisite for KJE activity, as aggregated Similar data were obtained when luciferase was used MDH was no longer refolded by KJE (data not shown). as an alternative substrate (Fig. 1B). Heat denaturation of The GroE system (ELS) and ClpB, alone or in combina- luciferase (0.2 mM) in the presence of IbpB (1.6 mM) tion, did not exhibit refolding activities. However, addition allowed substrate refolding only by KJE but not by ELS or of either ELS or ClpB accelerated the KJE-dependent ClpB alone. Protection of luciferase from aggregation by refolding of MDH, increasing the refolding rate by 1.7-fold IbpB was again essential for the subsequent KJE-depen- to 3.9 nM MDH min-1 (Fig. 1A). Importantly, together ELS dent refolding as KJE did not support luciferase reactiva- and ClpB co-operatively stimulated the KJE-mediated tion from aggregates (data not shown). In the case of

sHsps, ClpB and DnaK form a functional triade 587 IbpB/luciferase complexes ELS did not influence the lacked the activities of KJE and ClpB, and the processes KJE-dependent refolding process, indicating that the which lead to dissociation of sHsps from aggregated pro- beneficial effects of ELS on MDH reactivation are specific teins in vivo are unknown. We therefore followed the fate for this substrate. In contrast ClpB accelerated the KJE- of sHsps upon heat treatment in E. coli wild-type and dependent refolding of luciferase increasing the refold- DclpB and DdnaK52 null mutant cells by quantitative ing rates by 2.9-fold (0.59 nM luciferase min-1). These immunoblotting. After heat shock to 45∞C, more than 50% data demonstrate that, in general, ClpB co-operates of IbpA/IbpB was present in the insoluble protein fraction with KJE in the reactivation of sHsp-bound substrates of wild-type cells (Fig. 3A–C). This value was increased in vitro. to over 80% of total IbpA/IbpB in DclpB and DdnaK52 mutant cells, which are affected in preventing and reversing protein aggregation (Mogk et al., 1999). Interestingly, DibpAB mutant cells exhibit delayed resolubilization of increased amounts of IbpA/IbpB, observed in DclpB and protein aggregates DdnaK52 mutant cells after heat stress, could not prevent heat-induced protein aggregation. Our in vitro data suggest that sHsp/substrate complexes Disappearance of IbpA/IbpB from the insoluble cell frac- are substrates for the ClpB/KJE bi-chaperone system. To tion during the recovery phase at 30∞C occurred in wild- obtain evidence that sHsps also facilitate the action of type cells with the same kinetics as the solubilization of ClpB/KJE in vivo, we followed the kinetics of protein dis- protein aggregates (data not shown). Such solubilization aggregation after severe heat shock treatment (45∞C for of sHsps from protein aggregates was slowed down by 30 min) in E. coli wild-type and DibpAB mutant cells. While threefold in DclpB and nearly abolished in DdnaK52 the total level of heat-aggregated proteins was not mutant cells (Fig. 3D). Identical results were obtained affected in the DibpAB mutant, the subsequent solubiliza- when the fate of [35S]-methionine pulse-labelled IbpA/IbpB tion of protein aggregates was reproducibly delayed com- was followed in wild-type and chaperone mutant cells pared with wild-type cells (Fig. 2). The defect of DibpAB (data not shown). These in vivo findings parallel those mutant cells was mild compared with DclpB and DdnaK52 obtained in vitro in that dissociation of sHsp/substrate mutant cells which showed almost no protein disaggrega- complexes was strictly dependent on KJE and acceler- tion after heat-stress to 45∞C (see Fig. 3; Mogk et al., ated by the additional presence of ClpB. 1999). We conclude that presence of sHsps in protein aggregates is not essential for protein disaggregation in vivo but can accelerate the ClpB/KJE-mediated disaggre- Analysis of DibpAB DclpB double mutants gation process. Despite the noticed co-operation of IbpA/IbpB and ClpB in KJE-mediated protein disaggregation, DibpAB and DclpB mutants are viable at 42∞C (Thomas and Baneyx, Fate of sHsp/substrate complexes in vivo 1998). To determine whether both chaperone systems act The precise distribution of sHsps in soluble and insoluble synergistically in vivo, we constructed a DibpAB DclpB protein fractions of heat-treated cells, which contained or double mutant. Double mutants were characterized with

Fig. 2. Escherichia coli cells lacking sHsp function exhibit delayed resolubilization of protein aggregates. Cultures of E. coli wild type and DibpAB mutant strains were grown in LB medium at 30∞C to logarithmic phase, then shifted to 45∞C for 30 min, followed by incubation at 30∞C for 60 min. Aggregated proteins were isolated at the indicated time points and analysed by SDS–PAGE followed by staining with Coomassie brilliant blue. The amount of aggregated proteins was quantiﬁed by Bradford assay and calculated in relation to the total protein content (set at 100%). Asterisk indicates the position of IbpA/IbpB in the wild-type proteins samples.

Fig. 3. Fate of E. coli sHsps (IbpA/IbpB) during heat stress. A. Cultures of wild-type, DclpB and DdnaK mutant strains were grown in LB medium at 30∞C to logarithmic phase, then shifted to 45∞C for 30 min, followed by incubation at 30∞C for 2 h. Aggregated proteins were isolated at the indicated time points and analysed by SDS– PAGE followed by staining with Coomassie brilliant blue. The amount of aggregated proteins was quantiﬁed by Bradford assay and calculated in relation to the total protein content (set at 100%). B–C. The distribution of IbpA/IbpB in soluble (white bars) and insoluble (black bars) cell fractions of E. coli wild-type, DclpB and DdnaK mutant strains was determined by quantitative immunoblot analysis using IbpB-speciﬁc antibodies. D. IbpA/IbpB levels of pellet fractions were calculated in relation to the sHsp amount, determined directly after heat-shock to 45∞C (set at 100% for each strain).

respect to growth at high temperatures, formation of protein cases, DibpAB mutants reproducibly exhibited a 1.5-fold aggregates after heat shock to 42∞C and the development reduced survival rate, indicating a contribution of IbpA/ of thermotolerance. Growth of DibpAB DclpB double IbpB in the development of thermotolerance. The defects mutant cells was still unaffected at 42∞C (data not shown). of DibpAB mutant cells were mild compared with DclpB However, the absence of IbpA/IbpB and ClpB function mutant cells, which revealed a ﬁve- to 10-fold reduced caused increased protein aggregation at 42∞C, which was survival after heat-shock to 50∞C. Viability of DclpB mutant not observed for the single mutant strains (Fig. 4). Levels cells was also largely unaffected by the 42∞C pre-shock of aggregated proteins in DibpAB DclpB mutant cells were in contrast to wild-type and DibpAB mutants. Survival of still low compared with those found in DdnaK52 mutants. DibpAB DclpB double mutant cells at 50∞C was slightly To test for the ability to develop thermotolerance, we deter- reduced compared with DclpB mutants if cells were directly mined the survival rates of E. coli wild-type, DibpAB, DclpB heat-shocked to 50∞C. Together these ﬁndings indicate an and DibpAB DclpB mutant cells either directly upon heat activity of sHsps in preventing and/or reversing protein shock from 30∞C to 50∞C, or after applying a 15 min pre- aggregation. The dominating effect in thermotolerance shock to 42∞C for cell adaptation (Fig. 5A–B). In both development, however, is provided by ClpB.

© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 585–595 sHsps, ClpB and DnaK form a functional triade 589 cant delay in the shut-off phase of the heat shock response (Fig. 6B and C). Downregulation of Hsp synthesis is mediated by the DnaK chaperone system (Straus et al., 1990). A delayed downregulation suggests that especially the lack of ClpB function leads to a requirement for increased DnaK levels to cope with heat stress at 42∞C, which in turn might compensate for lacking ClpB activity.

IbpA/IbpB and ClpB become essential for viability in cells with reduced DnaK levels Based on the above ﬁndings, we tested whether increased levels of KJE can compensate for IbpA/IbpB and ClpB. We took advantage of an E. coli strain in which the dnaKJ operon is placed under lacI control (KJ- Fig. 4. Escherichia coli cells lacking sHsp and ClpB function exhibit protein aggregation at 42∞C. E. coli wild-type, DibpAB, DclpB, DibpAB DclpB and DdnaK mutant cells were grown in LB medium at 30∞C to mid-exponential phase and shifted to 42∞C for 30 min. Aggregated proteins were isolated and analysed by SDS–PAGE followed by staining with Coomassie brilliant blue. The amount of aggregated proteins was quantiﬁed by Bradford assay and calculated in relation to the total protein content (set at 100%). The position of IbpA/B is indicated.

Lack of ClpB and sHsp function inﬂuences the heat shock response

The mildness of phenotypes of DibpAB, DclpB and DibpAB DclpB mutant cells might result from a regulatory adjustment in the mutant cells involving overproduction of other chaperones to compensate for the lack of IbpA/IbpB and ClpB activity. In particular, increased levels of the KJE system would reduce the levels of misfolded proteins and the formation of protein aggregates and thereby decrease the necessity for chaperones acting at the level of protein aggregates. To test this possibility, we analysed the levels of DnaK at 37∞C in E. coli wild-type and chaperone mutant cells (Fig. 6A). Immunoblot analysis revealed that DnaK levels were indeed 1.5- to twofold elevated at 37∞C in DclpB and DibpAB DclpB mutant cells compared with wild- type cells, whereas the levels of Trigger factor, which is not regulated as part of the heat shock response, remained constant (Fig. 6A). These increased levels of DnaK, and conceivably also of other heat shock proteins, are expected to mask potential phenotypes of DibpAB and DclpB mutant cells. It is also possible that the kinetics of the heat shock Fig. 5. Escherichia coli cells missing sHsp or ClpB function exhibit reduced thermotolerance. E. coli wild-type, DibpAB, DclpB and Dib- response is affected in cells lacking sHsp and/or ClpB pAB DclpB mutant strains were grown in LB medium at 30∞C to mid- function. To test this possibility E. coli wild-type, DibpAB, exponential phase. 10-2 dilutions of each strain were heat-shocked to DclpB and DibpAB DclpB mutant cells were grown at 50∞C and incubated for the indicated time (A). Alternatively aliquots of all strains were ﬁrst shifted to 42∞C for 15 min to allow induction of 30∞C in M9 minimal medium and heat-shocked to 42∞C the heat shock response and transferred to 50∞C afterwards (B). (Fig. 6B–C). The heat shock response was transient for Various dilutions of the stressed cells were spotted on LB plates and E. coli wild-type and DibpAB mutant cells with a peak in incubated at 30∞C. After 24 h, colony numbers were counted, and survival rates were calculated in relation to unstressed cells. Mean GroEL synthesis at 10 min after upshift. In contrast, values of three independent experiments are shown. The average DclpB and DclpB DibpAB mutant cells exhibited a signiﬁ- variation between the experiments was less than 20%.

Fig. 6. Loss of ClpB and IbpA/IbpB function alters the heat shock response. A. Levels of DnaK and Trigger factor were determined by immunoblotting of total lysates (5 mg) prepared from E. coli wild-type (WT), DibpAB, DclpB and DibpAB DclpB mutant cells grown in LB medium at 37∞C. B. E. coli wild-type (WT), DibpAB, DclpB and DibpAB DclpB mutant cells were grown at 30∞C in M9 minimal medium without L-methionine to mid- exponential growth phase and shifted to 42∞C. At the indicated time points, aliquots were pulse-labelled with [35S]-L-methionine (7.5 mCi) for 1 min and subsequently precipitated by TCA (10% (v/v)). Samples were subjected to SDS–PAGE (12%) followed by development with a phosphoimager and synthesis rates of GroEL were determined (C). Values were normalized to the maximal value of E. coli WT (set as 100%). The positions of GroEL and DnaK are indicated.

regulatable strain) and thus is no longer subject to heat production to levels found in wild-type cells at 30∞C. shock regulation (Tomoyasu et al., 1998). This strain Growth of DclpB mutants required 70 mM IPTG, allowing allows adjustment of the cellular levels of DnaK and for production of DnaK and DnaJ to increased levels and DnaJ according to the IPTG concentration in the growth even higher IPTG concentrations (80 mM) were neces- medium. We introduced the DibpAB, DclpB or DibpAB sary for growth of DibpAB DclpB double knockout cells. DclpB mutations into this strain background and deter- At 42∞C, the KJ-regulatable wild-type strain also required mined whether the levels of DnaK and DnaJ affect cell IPTG for growth, as expected from the known tempera- viability on LB agar plates at various growth temperature sensitive growth phenotype of dnaK mutant cells. tures (Fig. 7). At 30∞C, all three KJ-regulatable mutant The different mutant cells showed a similar relative strains (DibpAB, DclpB, DibpAB DclpB) remained viable requirement for KJ as compared with 37∞C, except that even in the absence of IPTG (i.e. with greatly reduced KJ the IPTG threshold concentration was generally higher. levels). At 37∞C, none of the mutants was able to form Taking these data together, the DibpAB, DclpB or DibpAB colonies in the absence of IPTG, while the KJ-regulat- DclpB mutant cells require KJ function for growth at able wild-type control strain showed no growth defects. ≥37∞C. This requirement follows a hierarchy, with Dib- The lack of IbpA/IbpB and/or ClpB function thus causes pAB, DclpB and DibpAB DclpB mutant cells showing synthetic lethality in cells with limiting KJ levels (Fig. 7). increasing requirements in that order, indicating that the Our experiment further revealed that different KJ levels IbpA/IbpB, ClpB and KJE chaperone systems form a co- were required to compensate for missing IbpA/IbpB or operative functional network that is essential for viability ClpB function. Growth of DibpAB mutants was restored at regular growth temperature and under heat shock at low IPTG concentrations (40 mM), which allows for KJ conditions.

Fig. 7. sHsps and ClpB become essential at elevated temperatures in E. coli cells with reduced DnaK/DnaJ levels. BB7352 (PA1/lacO-1 dnaK, dnaJ, q q -1 q q lacI ), BB6406 (PA1/lacO-1 dnaK, dnaJ, lacI , DibpAB), BB6410 (PA1lacO -1 dnaK, dnaJ, lacI , DclpB) and BB6411 (PA1/lacO-1 dnaK, dnaJ, lacI , DibpAB, DclpB) cells were grown at 30∞C in LB medium supplemented with 1 mM IPTG until late log phase. Various dilutions (10-3-10-6) were spotted on LB plates containing the indicated IPTG concentrations and incubated at 30∞C, 37∞C or 42∞C for 24 h.

IbpA/IbpB, ClpB and the DnaK system act co-operatively operative action of IbpA/IbpB and ClpB in the KJE- to reverse protein aggregation mediated removal of protein aggregates in vivo. To elucidate the functional interplay between IbpA/IbpB, KJE and ClpB in the protein quality control network more Discussion precisely, we determined the degree of protein aggregation in DibpAB, DclpB and DibpAB DclpB mutants that have In this study, we analysed the functions of sHsps in the KJ adjusted to various levels (Fig. 8). Because ClpB and protein quality control system of E. coli. We provide in vitro IbpA/IbpB do not prevent protein aggregation in vivo and in vivo evidence that sHsps co-operate with the ClpB/ (Mogk et al., 1999), increased amounts of aggregated KJE bi-chaperone system in reversing protein aggrega- proteins in the respective mutants would indicate a less tion. This functional triade of chaperones is operative at efficient protein disaggregation. At 30∞C no protein aggre- regular growth temperatures and during heat stress and gation was detectable for all tested mutant strains, even becomes essential if KJE levels are limiting. in cells with greatly reduced KJ levels. After a 30 min Using MDH and luciferase as thermolabile model sub- incubation at 42∞C, 5% of cellular proteins aggregated in strates, we could demonstrate that in vitro refolding of all mutant cells, provided that IPTG was omitted from the substrates bound to IbpB is exclusively mediated by KJE growth medium (Fig. 8). Increasing KJ levels (by addition or, more efficiently, by ClpB/KJE. The dependency of sub- of IPTG) reduced the amount of aggregated proteins in strate refolding on KJE and ClpB/KJE can likely be each strain, but to different degrees dependent on the explained by the high stability of sHsp/substrate com- mutant background. While 50 mM IPTG in the growth plexes (Ehrnsperger et al., 1999). Indeed, substrates medium was sufficient to eliminate aggregates in DibpAB bound to sHsps are not released spontaneously to signif- cells, 2% and 5% of total proteins still aggregated in DclpB icant amounts (Mogk et al., 2003). We therefore suggest and DibpAB DclpB mutant cells respectively. Even in pres- that sHsp/substrate complexes represent protein aggre- ence of DnaK/DnaJ levels corresponding to heat shock gates and, consequently, substrate refolding relies on the conditions (100 mM IPTG), 2% of cellular proteins active extraction of substrates by KJE or ClpB/KJE. remained aggregated in DibpAB DclpB mutant cells. While efficient in forming soluble complexes with These findings are in complete agreement with the hier- unfolded proteins in vitro, sHsps extensively co-localize archal complementation of growth defects of these mutant with insoluble protein aggregates in heat treated E. coli cells at high temperatures and demonstrate the co- cells. In view of our findings, this suggests that their pri-

Fig. 8. Escherichia coli cells missing sHsp and ClpB function accumulate aggregated proteins at high temperatures. BB7352 (PA1/lacO-1 dnaK, q q q q dnaJ, lacI ), BB6406 (PA1/lacO-1 dnaK, dnaJ, lacI , DibpAB), BB6410 (PA1/lacO-1 dnaK, dnaJ, lacI , DclpB) and BB6411 (PA1/lacO-1 dnaK, dnaJ, lacI , DibpAB, DclpB) cells were grown at 30∞C overnight in LB medium supplemented with 1 mM IPTG. Pre-cultures were washed twice with LB medium and inoculated into LB medium containing the indicated IPTG concentrations. Cells were cultured further to mid-exponential growth phase and shifted to 42∞C for 30 min or left at 30∞C. Aggregated proteins were isolated and analysed by SDS–PAGE followed by staining with Coomassie brilliant blue. The amount of aggregated proteins was quantified by Bradford assay and calculated in relation to the total protein content (set at 100%). Two prominent bands visible at 30∞C were identified as membrane proteins OmpA and OmpF and do not represent protein aggregates. mary function is to facilitate the solubilization process of tion of sHsp/substrate complexes by DnaK alone is pos- aggregated proteins. Although it is difficult to determine sible in vivo. However, solubilization of such complexes the nature of the in vivo formed sHsp/substrate com- was much faster in the presence of ClpB and represented plexes, a large heterogenity of species can be assumed. only a subfraction of stress-induced protein aggregates. In E. coli wild-type cells, approximately 50% of IbpA/IbpB Thus ClpB and sHsps make different contributions to the was found associated with aggregated proteins after heat KJE-mediated protein disaggregation. While ClpB plays a stress, while the remaining sHsps stayed soluble, poten- direct and dominant role in this process, sHsps exert their tially representing smaller sHsp/substrate complexes. function indirectly by facilitating the extraction of unfolded Based on our in vitro findings it is likely that the size of proteins from aggregates. This hierarchal function of IbpA/ such aggregates determines the chaperone requirement IbpB and ClpB in the chaperone triade is also indicated for solubilization of the aggregated proteins. In vitro KJE by increased requirement for KJE if one member of the alone can dissociate small, soluble sHsp/substrate com- network is missing. Importantly, by influencing different plexes, whereas ClpB is additionally needed for efficient aspects of the disaggregation reaction sHsps and ClpB solubilization of insoluble sHsp/substrate complexes. Fur- can work co-operatively and, consistent with this sugges- thermore, the presence of sHsps in protein aggregates tion, DibpAB DclpB double mutants needed the highest aggregates facilitates the solubilization process by ClpB/ KJE levels for growth at 37∞C or 42∞C and exhibited KJE (Mogk et al., 2003). This latter finding parallels the strongly increased protein aggregation compared with the delayed protein disaggregation in DibpAB mutant cells single knockout strains (Figs 7 and 8). Recent findings (Fig. 2). indicate that the functional interplay between sHsps, ClpB The analysis of dissociation of IbpA/IbpB from aggre- and KJE is not restricted to E. coli, but may represent a gated proteins in DclpB mutant cells revealed a slow dis- conserved principle in bacteria to guarantee efficient pro- aggregation reaction, which was no longer detectable in tein disaggregation. Co-operation of sHsps and ClpB has DdnaK mutant cells (Fig. 3). This indicates that dissocia- been suggested in the development of thermotolerance in

© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 585–595 sHsps, ClpB and DnaK form a functional triade 593 Synechocystis sp. PCC6803 (Giese and Vierling, 2002). activity of sHsps. Different mechanisms guarantee low The existence of the sHsp/ClpB/KJE triade in Mycobacte- levels of active sHsps under non-stress conditions to pro- rium tuberculosis is also suggested by their specific tect cells from their extraordinary binding capacity. Induc- HspR-dependent co-regulation (Stewart et al., 2002). tion of sHsps at stress conditions allows association with The functions of sHsps and ClpB in KJE-mediated pro- unfolded proteins destined for aggregation, if other chap- tein disaggregation are most important during severe erone systems are overburdened. This co-aggregation stress; however, this network is already operative at 37∞C. represents a second defence line and has important con- The absence of phenotypes in DibpAB and DclpB mutant tributions to survival of cells during severe stress by accel- cells can be explained by a redundancy of the cellular erating the ClpB/KJE-dependent protein disaggregation. protection machinery. The high abundance of DnaK in the E. coli cytosol guarantees a high potential to prevent pro- Experimental procedures tein aggregation. However, we could show that sHsps or ClpB become essential for viability of E. coli cells at reg- Strains and culture conditions ular and heat shock temperatures under conditions of low E. coli strains used were derivatives of MC4100 [araD139 DnaK levels. Small changes in the DnaK/J levels had D(argF-lac)U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 dramatic effects on the viability of the chaperone mutant rbsR]. Mutant alleles of chaperone genes were introduced cells as compared with wild-type control cells. Depletion by P1 transduction into MC4100 background to generate of DnaK increases protein aggregation and at the same strains BB4561 (DclpB::kan), BB4563 (DibpAB::kan), time reduces the disaggregation potential of the cells. BB4566 (DibpAB::kan, zic4901::Tn10, DclpB::kan), BB2395 Such conditions may exist in cells subjected to prolonged (lon146::Tn10), BB7999 clpP::kan, BB7352 (PA1/lacO-1 dnaK, q q dnaJ, lacI ), BB6406 (PA1/lacO-1 dnaK, dnaJ, lacI , DibpAB), or repeated stress treatment. Importantly, increased levels q BB6410 (PA1/lacO-1 dnaK, dnaJ, lacI , DclpB) and BB6411 of other chaperone systems or proteases, caused by heat q (PA1/lacO-1 dnaK, dnaJ, lacI , DibpAB, DclpB). Bacterial strains shock induction in presence of reduced DnaK concentra- were cultured at 30∞C in Luria broth (LB) or M9 minimal tions, cannot replace the missing sHsp or ClpB function. medium supplemented with glucose (0.2%) and all L-amino The co-aggregation of IbpA/IbpB with misfolded pro- acids (30 mg ml-1) except L-methionine. Chloramphenicol, teins during heat stress is likely explained by low sHsp kanamycin and tetracycline were used at final concentrations -1 levels in unstressed E. coli cells. sHsp levels become of 10, 20, and 10 mg ml respectively. Temperature-shift abundant only after induction of the heat shock response experiments were performed in orbital shaking water baths. For pulse-labelling with [35S]-L-methionine, cells were grown (Mogk et al., 1999; Richmond et al., 1999). We speculate, in M9 minimal medium (see above). Labelling was done by that most bacteria tend to keep the sHsp concentration adding [35S]-L-methionine to heat shocked cells (Amersham low, in order to direct the flux of unfolded proteins to SJ1515; 15 mCi ml-1, 1000 Ci mmol-1) to 30 mCi ml-1 cell cul- chaperones with folding capacity such as ELS and KJE. ture for 5 min, followed by addition of unlabeled L-methionine Interestingly, high sHsp levels can also interfere with pro- to 200 mg ml-1. tein disaggregation, as overproduction of IbpA/B retarded the resolubilization of protein aggregates in vivo (data not Proteins shown; Kuczynska-Wisnik et al., 2002). Furthermore, sHsps assemble into oligomeric structures that may con- Purifications of DnaK, DnaJ, GrpE, ClpB, GroEL, GroES and stitute inactive resting states. Activation appears to involve IbpB were performed as described previously (Goloubinoff dissociation of the oligomers into dimers; for some sHsps et al., 1999; Mogk et al., 1999). Pyruvate kinase was pur- chased from Sigma, pig heart muscle MDH and firefly heat shock temperatures shifts the oligomer-dimer equi- Luciferase from Roche. Protein concentrations were deter- librium to the active dimer form (Haslbeck et al., 1999; van mined with the Bio-Rad Bradford assay using BSA as stan- Montfort et al., 2001). Therefore increased levels and acti- dard. Protein concentrations refer to the protomer. vation of sHsps seem to be restricted to stress conditions. We also noticed a third strategy to ensure low sHsp levels even after strong heat shock induction: only a fraction of Protein denaturation and chaperone activity assays IbpA/IbpB, after the dissociation from protein aggregates, MDH (2 mM) was denatured at 47∞C for 30 min in buffer A was regained in the soluble cell fraction, indicating degra- (50 mM Tris, pH 7.5, 150 mM KCl, 20 mM MgCl2, 2 mM DTT) dation of sHsps after dissociation from the aggregates in the presence of IbpB (16 mM). Luciferase (0.2 mM) was (Fig. 3B and C). Proteolysis of IbpA/IbpB would guarantee denatured at 43∞C for 15 min in buffer A in the presence of that high sHsp levels are restricted to heat stress condi- 1.2 mM IbpB. Protein refolding was started by diluting IbpB/ MDH or IbpB/luciferase complexes 1:1 into the indicated final tions, but are actively reduced during the recovery phase concentrations of chaperones in buffer A at 30∞C. All assays of cells. were performed in presence of an ATP-regenerating system Together these findings demonstrate how the quality (3 mM phosphoenol pyruvate; 20 mg ml-1 pyruvate kinase; control system of cells regulates and uses the chaperone 2 mM ATP). Determination of enzymatic activities followed

© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 50, 585–595 594 A. Mogk et al. published protocols (Schröder et al., 1993; Goloubinoff et al., the DFG (Bu617/14-1) and the Fond der Chemischen Indus- 1999). Refolding rates were calculated from the linear trie to B.B. and A.M. increase of substrate activities.

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