Physical map and dynamics of the chaperone network inEscherichia colimmi_8054 736..747
Mohit Kumar and Victor Sourjik*Zentrum für Molekulare Biologie der UniversitätHeidelberg, DKFZ-ZMBH Alliance, Im NeuenheimerFeld 282, D-69120 Heidelberg, Germany.
Summary
Diverse families of molecular chaperones cooperateto effect protein homeostasis, but the extent anddynamics of direct interactions among chaperonesystems within cells remain little studied. Here weused fluorescence resonance energy transfer to sys-tematically map the network of pairwise interactionsamong the major Escherichia coli chaperones. Wedemonstrate that in most cases functional coopera-tion between chaperones within and across familiesinvolves physical complex formation, which pre-exists even in the absence of folding substrates. Theobserved connectivity of the overall chaperonenetwork confirms its partitioning into sub-networksthat are responsible for de novo protein folding andmaturation and for refolding/disaggregation of mis-folded proteins, respectively, and are linked by theHsp70 system. We further followed heat-inducedchanges in the cellular chaperone network, revealingtwo distinct pathways that process heat-denaturedsubstrates. Our data suggest that protein foldingwithin cells relies on highly ordered and direct chan-nelling of substrates between chaperone systemsand provide a comprehensive view of the underlyinginteractions and of their dynamics.
Introduction
Molecular chaperones are proteins that bind to, stabilizeand/or fold, mature and remodel proteins in healthy andstressed cells (Hartl et al., 2011). The fundamental impor-tance of molecular chaperones can be appreciated bytheir conservation through the boundaries of bacteria,archaea and eukarya. Chaperones are classified intodistinct groups based on their sequence similarityand their distinct functions. In Escherichia coli, de novo
protein folding is mainly brought about by three systems:the ribosome-associated trigger factor (TF), the Hsp70system DnaK/DnaJ/GrpE and the chaperonin systemGroES/GroEL (Genevaux et al., 2007; Hartl et al., 2011),although the folding is also assisted by the ribosome itself(Kramer et al., 2009; Wilson and Beckmann, 2011) andin some cases by specific factors such as SecB (Ullerset al., 2007).
By virtue of its location near the peptide exit tunnel, TFis the first chaperone that interacts with emergent nascentpolypeptide chains, protecting them and promoting theircotranslational folding. TF may also sequester polypep-tides as they leave the ribosome or even bind andstabilize natively folded proteins in the cytoplasm, but ithas a very low affinity for client proteins and is generallydispensable for cell survival (Hoffmann et al., 2010). Incontrast, the deletion of DnaK (Hsp70) or GrpE stronglyaffects growth, particularly at high temperatures(Genevaux et al., 2007). DnaK/DnaJ/GrpE is consideredto be the most versatile chaperone system in E. coli(Mogk et al., 1999; Tomoyasu et al., 2001), which pre-vents aggregation of proteins and refolds them to theirnative state both during normal growth and under foldingstress. This is achieved by repetitive ATP-driven cycles ofbinding and subsequent release of the substrate polypep-tide by DnaK. Binding is promoted by the co-chaperoneDnaJ that delivers the substrates and increases the sub-strate affinity of DnaK by stimulating ATP hydrolysis,whereas release is facilitated by GrpE that lowers DnaKaffinity for substrates by mediating the exchange of ADPto ATP (Genevaux et al., 2007). DnaJ has two homo-logues in E. coli, CbpA and DjlA, which can also transferclient substrates to DnaK. Finally, the GroE (GroEL/GroES) chaperonin system is responsible for maturationof polypeptides with complex structure and high-energybarrier of folding, by providing a cage-like environmentwithin the oligomeric GroEL ring. GroEL and its cofactorGroES are believed to act downstream of and/or parallelto the Hsp70 system and are essential for cell viabilityunder all growth conditions (Hartl et al., 2011).
Yet another highly conserved chaperone system isHsp90 (HtpG in E. coli ). In eukaryotic cells, the Hsp90dimer is involved in maturation and conformational remod-elling of a variety of multi-domain signalling proteins, nor-mally receiving its substrates directly from the Hsp70
system. In bacteria, however, the function of Hsp90remains enigmatic, despite its high conservation amongbacteria and high abundance in the cell. The deletion ofHtpG has only a minor effect on growth at elevated tem-peratures (Bardwell and Craig, 1988), but shows no othersignificant phenotypic change.
The other broadly conserved families of molecularchaperones, the Hsp100 (ClpB in E. coli ) and the smallheat shock proteins (sHsps; IbpA and IbpB in E. coli ),are primarily involved in refolding of the unfolded oraggregated proteins. While the oligomers of IbpA andIbpB passively bind and stabilize denatured proteinsin a folding-competent state (Haslbeck et al., 2005),ClpB hexamer can actively solubilize and reactivateaggregated proteins using the energy of ATP hydrolysis(Barends et al., 2010; Mayer, 2010). As a second line ofdefence against protein-folding stress, cells also possessmultiple protease systems, which degrade misfolded pro-teins that can no longer be efficiently refolded by thechaperone systems. One of the most prominent proteasecomplexes in E. coli is ClpX/ClpP (or ClpXP), where ClpXis a ClpB-homologous ATPase and ClpP is the catalyti-cally active protease. Normally, ClpX is responsible forrecognition, unfolding and translocation of client sub-strates to ClpP (Sauer and Baker, 2011), but it might alsofunction as an Hsp100-type chaperone independently ofClpP (Baker and Sauer, 2012).
In E. coli, as in other organisms, individual chaperonesystems are likely to cooperate in de novo folding orrefolding of proteins (Richter et al., 2010), and functionalcooperation has indeed been shown between DnaK andGroEL (Buchberger et al., 1996), DnaK and ClpB (Hasl-berger et al., 2007), DnaK and HtpG (Genest et al., 2011),and between IbpA/B and DnaK/ClpB (Mogk et al., 2003).It was further demonstrated that most chaperone sys-tems colocalize to the heat-induced protein aggregates inE. coli (Winkler et al., 2010). However, it remains unclearwhether cooperation in intracellular substrate processingamong chaperones is merely functional, i.e. a result ofindependent but synergistic action of different chaperoneson the same substrates, or whether it relies on directphysical interactions between different chaperonesystems, as has been recently suggested for buddingyeast (Gong et al., 2009). Here we used fluorescenceresonance energy transfer (FRET) to obtain a physicalmap of interactions among different molecular chaper-ones in E. coli and to follow the changes in these inter-actions upon a heat shock. Our analysis suggests thatmost – but not all – cases of functional cooperationbetween different chaperone systems in E. coli involvedirect physical interactions among components of thesesystems. Moreover, we also describe the dynamics andorder of processing of the heat-denatured substrates atthe early stages of a heat shock.
Results
Localization of chaperone fusions
To monitor physical interactions among chaperones andto investigate their dynamics, we created a library of chap-erone fusions to yellow and cyan fluorescent proteins(YFP and CFP respectively). With the exception of GrpE,fluorescent proteins were fused to the C-terminus ofchaperones (Table S1). GroEL could not be included inour screen, because both its termini are unexposed andcould not be tagged with a fluorophore. The functionalityof fusions to DnaK, DnaJ, GrpE, IbpA and IbpB wasverified by complementation assays (see Experimentalprocedures and Fig. S1); the functionality of fusions toClpB, ClpX and ClpP was demonstrated in a previousstudy (Winkler et al., 2010). The resulting library was thenused to investigate localization of the fusion proteins inthe wild-type background (Fig. 1). At 30°C, most fusionsshowed uniform cytoplasmic localization, consistent withthe expected distribution of molecular chaperones duringnormal growth. In response to a heat shock, most chap-erones, except GrpE, TF and GroES, rapidly relocalizedto the poles, apparently following localization of emergingprotein aggregates, such as those formed by a thermallyunstable protein, firefly luciferase (Winkler et al., 2010).This is consistent with the physiological functions of thesechaperones in refolding protein aggregates and with arecent observation that DnaK, DnaJ, ClpB, ClpX and ClpPas well as a protease HslU – but not GroEL or a proteaseLon – localize to the heat-induced aggregates (Winkleret al., 2010). IbpA and to a lesser extent IbpB and ClpXshowed propensity for polar localization even before theheat shock, which was already reported for IbpA and wasproposed to indicate existence of subcellular sites of weakprotein aggregation even under native, non-stressed con-ditions (Lindner et al., 2008).
Fluorescence resonance energy transfer screen ofchaperone interactions at permissive temperatures
To investigate the extent of binary chaperone interactionsat permissive temperatures, we performed acceptor-photobleaching FRET experiments on wild-type strainsthat expressed pairwise combinations of CFP and YFPfusions to all chaperones in question (Fig. 2). Here cellcultures were grown at 30°C, and the experimentsthemselves were performed at 20°C (see Experimentalprocedures). These FRET measurements determinethe extent of energy transfer from CFP to YFP by pho-tobleaching YFP and assessing the consequent increasein the CFP emission. Energy transfer occurs only whenthe CFP and YFP are brought into the immediate proxim-ity in a molecular complex, with the extent of energytransfer being dependent on the distance between fluoro-
Physical map of the bacterial chaperone network 737
phores and on the fraction of the CFP-tagged protein thatis in the complex with the YFP-tagged protein. Therefore,even for a pair of interacting proteins, a false-negativeFRET result might arise due to an unfavourable orienta-tion of fluorophores in the complex, the competition forbinding with untagged proteins that are also expressedin the wild-type strain used here, or to a small fraction ofthe CFP fusion in the complex (Sourjik et al., 2007). Thetwo latter factors are particularly sensitive to the relativeexpression levels of donor and acceptor fusions andexplain a partial asymmetry of the interaction map(Fig. 2A). False negatives can be in principle eliminatedby tuning the relative expression levels of fusion proteinsto achieve an excess of the acceptor over the donor(Fig. 2A), but a systematic titration of this kind was notpracticable on the scale of a larger screen performedhere. Notably, due to the substantial contribution ofthe background to the overall fluorescence in the CFPchannel, even for positive pairs thus determined invivo FRET values may be substantially lower than the
theoretical efficiency of energy transfer within the samepair (see Experimental procedures).
Most chaperone interactions identified in this screen areconsistent with our current understanding of intracellularchaperone activity (Fig. 2B). Although DnaK was seen tointeract with DnaJ and GrpE, no interaction was observedbetween the latter two proteins, in accordance with theirsequential binding to DnaK. A relatively strong FRET wasobserved for DnaJ with itself, consistent with it being adimer (Shi et al., 2005). More surprisingly, no FRET wasobserved between GrpE fusions, despite this protein alsobeing a dimer (Moro et al., 2007). In addition, DnaK andDnaJ showed interactions with most other chaperones –TF, IbpA/B, ClpB, and HtpG – in line with the central roleof the Hsp70 system in the protein-folding homeostasis inE. coli. A very similar pattern of interactions was observedfor the two analogues of DnaJ (CbpA and DjlA), therebyaffirming the specificity of the screen (Fig. S2). Consistentwith its role in promoting the substrate release from DnaK,GrpE was observed to interact with HtpG and ClpB, the
Fig. 1. Intracellular localization of chaperonefusions before and after heat shock. MG1655(wild-type) cells expressing the indicatedchaperones fused to YFP were washed andresuspended in tethering buffer, before a heatshock was applied for 20 min at 42°C. Seetext, Table S1 and Fig. S1 for a descriptionand functionality of the fusions.
two potential acceptors of the DnaK-processed sub-strates, but not with sHsps that are expected to donatesubstrates to DnaK (Haslbeck et al., 2005).
In addition to their interaction with DnaK, IbpA and IbpBshowed a number of interactions, both among themselvesand with ClpB, ClpX and the protease ClpP. These inter-actions are consistent with the role of sHsps in theprocessing of misfolded and aggregated proteins andsuggest that they can directly transfer client substratesto several different chaperone systems as well as to theproteolytic system. The functional grouping of sHsps,DnaK and ClpB into the refolding chaperone network wasconfirmed by their interactions with the easily unfoldedprotein luciferase. Interestingly, the fluorescent protein
fusion to luciferase also showed a weak interaction withTF, which is unlikely to be purely cotranslational sincefluorescent protein maturation in E. coli takes severalminutes (M. Kumar and V. Sourjik, unpublished). This isconsistent, however, with a recently proposed function ofTF in protein refolding (Martinez-Hackert and Hendrick-son, 2009). In contrast, no interaction was observedbetween luciferase and DnaJ, likely due to the transientnature of DnaJ binding to the substrates that are rapidlypassed to DnaK (Gamer et al., 1996).
Other than an interaction with itself, no interactions weredetected for GroES. This is not surprising, because GroESis recruited at a later stage of the chaperonin cycle and maynot participate in the substrate transfer to GroEL. Alterna-
Fig. 2. FRET map of the chaperoneinteraction network.A. The results of the acceptor-photobleachingFRET screen at 20°C. Positive interactionsare highlighted in green. Negative controlswere performed for individual pairs bycoexpressing both respective chaperone–CFPfusions with free YFP (expressed from a pTrcpromoter induced with 100 mM IPTG) andrespective chaperone–YFP fusions with freeCFP (expressed from a pBAD promoterinduced with 0.01% arabinose), with theobtained significance threshold valuesindicated in light blue and light yellow axesrespectively. The standard error of themeasurement is indicated for each pair.Additionally, the interactions that showedreduction after substrate depletion(chloramphenicol treatment, see Experimentalprocedures) are indicated with red boxes.Asterisk indicates an example of afalse-negative interaction, which testedpositive after the expression of DnaK–CFPwas sufficiently reduced. Double asteriskindicates a pair that showed strong positiveFRET at higher expression levels.B. A graphical summary of the FRET screen.Individual interactions are marked by arrows,with dashed lines indicating interactions thatwere significantly reduced uponchloramphenicol treatment. Sub-networksinvolved in de novo folding and in proteinrefolding/disaggregation are indicated byyellow and blue ovals, respectively, with theHsp70 system being shared by the twosub-networks (green).GroE
TF
HtpG
DnaK
DnaJ
GrpE
IbpA
IbpB
ClpB
ClpX
ClpP
de novo folding Re-foldingB
A x CF P DnaK DnaJ GrpE IbpA IbpB ClpB HtpG TF GroES ClpX ClpP Luci
x YF P 0.57 0.70 0.82 0.58 0.87 0.40 1.13 0.51 0.71 0.19 0.63 0.55
DnaK 0.30 -1.2 ± 0.0
1.5 ± 0.4
1.0 ± 0.06
0.9 ± 0.2
0.8 ± 0.07
1.3 ± 0.1
- -0.7 ± 0.05
-0.8 ± 0.08
DnaJ 0.64 -1.1 ± 0.1
-1.3 ± 0.04
-0.6 ± 0.03
- - -0.9 ± 0.06
- -
GrpE 0.350.6 ± 0.01
- - - -0.7 ± 0.04
- - - - - -
IbpA 0.440.6 ± 0.07
1.0 ± 0.08
-11.8 ±
2.39.0 ± 1.7
0.7 ± 0.2
1.3 ± 0.4
- - -0.9 ± 0.2
0.9 ± 0.3
IbpB 0.00 - - -7.6 ± 1.8
6.4 ± 1.0
- - - - - - -
ClpB 0.67 -0.7 ± 0.08
-1.3 ± 0.08
1.13 ± 0.3
5.5 ± 0.4
- - - - - -
HtpG 0.91 -* -1.0 ± 0.2
- - -7.7 ± 2.00
- - - - -
TF 0.54 -0.8 ± 0.01
- - - - - - - - - -
GroES 0.1 0 - - - - - - - -2.7 ± 0.4
- - -
ClpX 0.51 - - -0.9 ± 0.1
- - - - -7.7 ± 2.1 -** -
ClpP 0.00 - - - - - - - - - -** 6.0 ± 1.1
-
Luci 0.54 - - -1.7 ± 0.02
-0.7 ± 0.06
-0.6 ± 0.003
- - -0.9 ± 0.3
Physical map of the bacterial chaperone network 739
tively, a relatively uninteractive GroES could also suggestthat GroE functions largely independently of other chaper-ones in the later stages of protein maturation, and/or onlywith a specialized set of substrates (Kerner et al., 2005).
Substrate dependence of chaperone interactions
To determine whether interactions between individualchaperones depend on their binding to substrates, wetreated cells with a bacteriostatic translation inhibitorchloramphenicol for the last 30 min of growth. This treat-ment apparently reduced not only the load of de novoprotein folding but also the amount of misfolded proteins,as judged by the reduced self-aggregation of luciferase(Fig. 2A). Consequently, we observed a substantial(> 30%) weakening or abolishment of the interactions ofluciferase with IbpA and DnaK. Moreover, FRET wasreduced for several pairs of interactions that appear to playa role in protein refolding (Fig. 2), whereby the interactionbetween DnaK and ClpB was completely abolished. Otherinteractions, including all interactions that are involvedin de novo folding, appear to be substrate-independentand are thus likely to reflect direct interactions betweenchaperones.
Heat shock induction of chaperone–substrateinteractions
We next investigated how the observed chaperone inter-action network reacts to the heat shock, which drasticallyincreases the load of unfolded and aggregated proteins. Toachieve that, cells expressing individual positive FRETpairs were subject to a rapid (within 80 s) increase oftemperature from 20°C to 42°C under the microscope, andthen kept at 42°C for 5 min. We first observed that fluores-cence of both YFP and CFP decreased with temperature,whereby a stronger relative decrease in the CFP channelled to the change in the YFP/CFP ratio even for free CFPand YFP (Fig. S3). This temperature dependence of fluo-rescence signals made it impossible to follow changes inFRET during the heating stage, particularly because thevalue of the observed change in the YFP/CFP ratiodepended on expression levels of YFP and CFP and thuscould not be easily corrected for. However, the YFP/CFPratio of the control sample stabilized as soon as the tem-perature became constant, enabling measurements ofchanges in FRET from that point onwards. Moreover, thecontrol ratio returned to the initial level when the tempera-ture was lowered back to 20°C, confirming that it was notcaused by the denaturation or co-aggregation of YFP andCFP themselves. Similar effect of temperature on fluores-cence was observed for a mixed population of CFP- andYFP-expressing cells (data not shown). A very differentpicture was observed for a pair of luciferase fusions to YFPand CFP, where the heat shock resulted in a rapid and
steady increase in the YFP/CFP ratio even after tempera-ture stabilization at 42°C (Figs 3A and S3). This increasewas a consequence of the enhanced energy transferbetween CFP and YFP fusions to luciferase, as evidencedby a decrease in the CFP signal and an increase in the YFPsignal consistent with the thermally induced gradual self-aggregation of this thermolabile protein.
Aggregation of luciferase induced its interactions with anumber of chaperones (Fig. 3). The most pronouncedincrease was observed in the interaction with IbpA andIbpB, which started at the onset of the heat shock andcontinued to rise concomitant with increasing luciferaseaggregation (Fig. 3A). This is consistent with the roleof the small heat shock proteins as holdases, whichstoichiometrically bind denatured proteins and sequesterthe exposed hydrophobic surfaces to prevent proteinaggregation, forming large oligomeric assemblies in thesubstrate-bound state (Haslbeck et al., 1999). Both thekinetics and the amplitude of the observed changes inprotein interactions were well reproducible among experi-ments (Fig. S4).
A steep initial increase was also observed in the interac-tion of luciferase with DnaJ, which was followed by a rapidsaturation (Fig. 3B). In contrast, the interaction with DnaKshowed a much more gradual increase. This suggests thatin the short period succeeding a heat shock, DnaJ rapidlysequesters the substrates and transfers them to DnaK,which binds substrates more stably (Gamer et al., 1996).Even slower kinetics and longer delays were observed inthe interactions of luciferase with Clp proteins (Fig. 3C),whereby binding of ClpB was slightly faster than that ofClpX. This may indicate a sequential transfer of the sub-strates from the Hsp70 system to ClpB and/or ClpX (seeDiscussion). The slowest binding kinetics in these experi-ments was observed for ClpP, in an apparent accord withits function at the later stage of the stress response.
Little, if any, increase at the time scale of our experimentwas observed in the interactions with GroES and HtpG,and no increase was observed for TF (Fig. 3D). Thus,strong inducibility of interactions of the thermally unfoldedluciferase was primarily limited to the chaperones thatfunction in protein disaggregation and refolding, which isapparently consistent with the functional partitioning ofthe chaperone network in E. coli (Fig. 2). Nevertheless,we cannot exclude that the chaperones assigned to thede novo folding pathway might interact with the substratesother than luciferase or become involved at the laterstages of protein refolding.
Heat shock induction of chaperone–chaperoneinteractions
In addition to their enhanced interactions with the sub-strates, many positive interactions between chaperones
themselves also increased upon the heat shock (Fig. 4).Both the extent of this increase and its kinetics werechaperone pair-specific, but in general the increase wascomparable to the extent of complex formation betweenchaperones prior to heat shock (Fig. 2A). The inductionkinetics of the DnaJ/DnaK interaction (Fig. 4A) was similarto the kinetics of the DnaK/luciferase interaction, confirm-ing that the latter is limited by the substrate transferfrom DnaJ. Interestingly, we also observed a slowergradual increase in FRET between DnaJ fusions, possiblybecause multiple DnaJ molecules are brought into imme-diate proximity through binding to aggregating substrates(Han and Christen, 2003; Acebron et al., 2008). Theinduction of interactions within the Hsp70 system wasfollowed by the increased interaction between DnaK andClpB (Fig. 4B), consistent with the proposed order of sub-strate transfer from DnaK to ClpB. Notably, DnaJ does notseem to be involved in this process, as its interaction withClpB increases only weakly and with a large delay. Suchincrease might result from the simultaneous binding ofDnaJ and ClpB to the same aggregates substrates, butmight also indicate that DnaJ can accept the partly foldedsubstrates from ClpB. Little increase was observed in theinteraction of the Hsp70 chaperones with ClpX, suggest-ing that no extensive substrate transfer occurs between
these two systems in the first few minutes after a heatshock.
Among the other molecular chaperones interactingwith the Hsp70 system, only a very weak increase wasobserved in the interactions of DnaK or DnaJ with TF,confirming that it plays little or no role in the heat shockresponse. In contrast, we observed a substantial steadyincrease in FRET between HtpG and DnaK (Fig. 4C). Thiswas unexpected, because the interaction of HtpG with themodel substrate luciferase showed little increase upon theheat shock. Nevertheless, the recruitment to the Hsp70machinery might at least partly mediate HtpG localizationto the protein aggregates (Fig. 1). We thus tested local-ization of the HtpG–YFP fusion in a dnaK mutant, whichdisplays extensive protein aggregation even at moderatetemperature due to severe defects in protein folding.Consistent with our expectations, all of IbpB, DnaJ andClpP were abundantly localized at polar foci in this strainalready at 30°C, whereas no localization of HtpG could beobserved (Fig. 5). Furthermore, the defect in the localiza-tion of HtpG–YFP in this strain could be corrected by thetransient expression of DnaK–CFP, thereby confirmingthe DnaK-dependent targeting of HtpG to potentialsubstrates under these conditions. Nevertheless, HtpGseems to also possess a second, direct, binding mode to
Fig. 3. Heat shock-induced interactions ofchaperones with the substrate. Cellsexpressing indicated protein pairs weresubjected to a rapid temperature increaseunder the microscope from 20°C to 42°C,starting at time point zero, and changes in theratio of YFP to CFP fluorescence wasfollowed over time. The change in the ratioduring heating period, up to 100 s, wasprimarily due to the direct effect oftemperature on the fluorescence of CFP andYFP (as seen in control cells expressingunfused CFP and YFP). Subsequent changesafter temperature stabilization at 42°Creflected increase in FRET (see Fig. S3) ofluciferase–CFP with YFP fusions to sHspsand luciferase (A), to the Hsp70 system (B),to chaperones/protease of the AAA+superfamily (C), and to TF, GroES and HtpG(D). Each curve is an average of at leastthree independent measurements, which weregenerally highly reproducible (see Fig. S4). Tofacilitate comparisons between individualcurves, the values of the YFP/CFP signal ratiowere normalized to the ratio before the onsetof the heat shock and unity was subtracted(see Experimental procedures).
0.1
0.15
0.2
0.25
100 200 300
ControlLuci-Luci
Luci-IbpALuci-IbpB
A
Time (s)
0.06
0.07
0.08
0.09
100 200 300
ControlLuci-DnaJ
Luci-DnaKB
Time (s)
0.06
0.07
0.08
0.09
0.1
0.11
0.12
100 200 300
ControlLuci-ClpB
Luci-ClpXLuci-ClpP
C
Time (s)
Ra
tio (
YF
P/C
FP
)-1
Ra
tio (
YF
P/C
FP
)-1
0.06
0.07
0.08
0.09
100 200 300
ControlLuci-GroES
Luci-HtpGLuci-TF
D
Time (s)
Physical map of the bacterial chaperone network 741
Fig. 4. Heat shock-induced interaction between chaperones. Time-course of changes in pairwise interactions between chaperones upon heatshock, performed as described in Fig. 3; only FRET pairs that were positive at permissive temperature (Fig. 2) were studied.A. Interactions between members of the Hsp70 system.B–D. Interactions of the Hsp70 system with AAA+ chaperones (B), with TF and HtpG (C), and with small heat shock proteins (D). Changes inFRET between fusions to HtpG are also shown in (C).E. Interactions between small heat shock proteins.F. Interactions of small heat shock proteins with the AAA+ chaperones.G. Interactions between ClpX and ClpP fusions. For measurements of interactions between different chaperones, both combinations of CFPand YFP fusions were used and the resulting curves were averaged. Each curve is an average of at least two or three concordantmeasurements. The negative control is as in Fig. 3.
protein aggregates, since exposure of DdnaK::kan cells to42°C did result in HtpG localization resembling that in thewild-type cells (data not shown). Apart from the increasedinteraction between HtpG and DnaK, we further observedfast changes in FRET between HtpG fusions, which mightreflect heat shock-induced conformational changes withinthe HtpG dimer.
The increase in the interactions of sHsps with DnaK orDnaJ showed a clear initial delay (Fig. 4D), whereby theinteraction of both IbpA and IbpB with DnaK was moredelayed than their interaction with DnaJ. The interactionsbetween sHsps themselves increased steadily, from theonset of the heat shock, with similar kinetics as theirinteraction with the substrate luciferase (Figs 4E and 3A),suggesting that this increase is due to the binding ofmultiple sHsp molecules to the same substrate. Com-pared with the rapid stimulation of the Hsp70 system itself(Figs 3B and 4A), its delayed interaction with sHsps sug-gests that DnaJ initially interacts with the free unfoldedsubstrates and only later with the sHsp-bound aggregat-ing proteins. In contrast, the interactions of sHspswith ClpB and ClpX showed no clear delay, but ratherincreased slowly but steadily after the heat shock(Fig. 4F). Notably, the specificities of IbpA and IbpB toHsp100 proteins were different: IbpA showed a strongincrease in its interaction with ClpB but not with ClpX,whereas IbpB showed the inverse pattern of interactions.The observed differences in the kinetics of sHsp interac-tions indicate that ClpB and ClpX might specifically binddifferent types of the sHsp-associated substrates in anHsp70-independent manner. These differences may bealso related to the differential heat induction of interac-tions among the sHsps themselves, whereby IbpB/IbpBinteraction increased slower than IbpA/IbpA. Neverthe-less, further processing of the unfolded or aggregatedsubstrates is likely to require the assistance of the Hsp70system (see Discussion).
We further observed a heat shock-induced decreaseof FRET within the ClpX/ClpX and ClpX/ClpP pairs(Fig. 4G). Such a decrease may be due to structural rear-rangements within oligomers upon the engagementof chaperones with their substrates. The reduction inthe FRET ratio between ClpX and ClpP might also beexplained by a disengagement of the ClpX rings from theClpP barrel when the cells are profoundly challenged witha thermal insult, consistent with the slower interaction ofClpP with the substrates as compared to ClpX (Fig. 3C).
Discussion
The intracellular network of chaperones in E. coli
Our FRET-based mapping has demonstrated a complexnetwork of physical interactions between the chaperonesin E. coli. Illustrating the advantage of FRET in detectingtransient intracellular interactions, most of these physi-cal complexes were not previously revealed by othermethods, either in vitro or in vivo, with the notable excep-tion of recently published interactions of DnaK with ClpB(Miot et al., 2011) and HtpG (Genest et al., 2011). Ourresults suggest that the functional cooperation betweenmost chaperone systems in the cell involves direct physi-cal transfer of client substrates, which may largelyenhance the efficiency of protein folding in the extremelycrowded environment of the bacterial cytoplasm. Our datamay even indicate existence of higher-order chaperoneassemblies, as proposed for yeast cells (Gong et al.,2009), where the client substrate may be passed througha series of molecular chaperones held together in a loosecomplex (akin to a ‘protein-folding factory’) until produc-tive folding has been accomplished. Although somechaperone interactions (e.g. between the Hsp70 andHsp100 systems) are promoted by the client substrates,most complexes appear to exist even under conditions ofsubstrate depletion.
Fig. 5. DnaK-mediated localization of HtpGto polar aggregates at 30°C.A. Fluorescence images showing thelocalization of ClpP–YFP, DnaJ–YFP,IbpB–YFP and HtpG–YFP in DdnaK::kan cellscultivated and maintained at/below 30°C.B. Expression of DnaK–CFP relocalizesHtpG–YFP to sites of protein aggregation.
ClpP-YFP DnaJ-YFP IbpB-YFP HtpG-YFP
HtpG-YFP DnaK-CFP Merge
A
B
Physical map of the bacterial chaperone network 743
The observed connectivity within the chaperonenetwork is well consistent with our understanding ofcellular proteostasis (Hartl et al., 2011). It separateschaperones into two groups (Fig. 2B): those primarilyinvolved in de novo protein folding and maturation (TF,Hsp70, Hsp90 and chaperonin systems) and those func-tioning in protein disaggregation and refolding (sHsps,Hsp70 and Hsp100 systems). Notably, only the lattergroup showed substantial dependence of interactions onthe availability of client substrates, possibly becausethese chaperone systems engage simultaneously with thesame molecules of unfolded substrates, while transferringthem between systems. The Hsp70 system plays the roleof the central hub connecting the two groups, apparentlyboth accepting client substrates from other chaperonesystems and transferring them further (Morano, 2007).
Sequence of substrate processing
The observed interactions and the kinetics of their induc-tion upon a heat shock allow us to draw some importantconclusions about the sequence of substrate processingby different chaperone systems. In de novo protein foldingand maturation, TF shows a clear interaction with DnaJ,suggesting that it can directly pass substrates to theHsp70 system while cooperating with it in protein folding(Deuerling et al., 1999; Teter et al., 1999). Notably, thisinteraction was apparently independent of protein synthe-sis and showed little induction upon a heat shock. Evenmore interesting are the observed interactions betweenDnaK and GrpE (but not DnaJ) with HtpG, another chap-erone responsible for protein maturation. In agreementwith a recent study (Genest et al., 2011), our observationssuggest that the two systems also directly cooperatein bacteria, with HtpG operating downstream from DnaK
and receiving client substrates for further maturation viadirect interaction between the two systems. Such sub-strate transfer might be aided by the nucleotide exchangefactor GrpE, which induces substrate release from DnaK.Although HtpG is expected to function in protein matura-tion and showed no interaction with the thermally unfoldedluciferase, it was recruited to protein aggregates, and thephysical interaction with DnaK was at least partly respon-sible for this recruitment. Moreover, the heat shockinduced a steady increase in the DnaK–HtpG interaction,indicating that certain substrates may be directly passedfrom DnaK to HtpG during protein refolding.
We further investigated the sequence of substratepassage during the initial stages of refolding and disag-gregation of heat-denatured proteins. Our analysis sug-gests existence of two distinct refolding pathways (Fig. 6),with the denatured proteins being either directly pro-cessed by the Hsp70 system or initially sequesteredwithin the sHsp-associated aggregates. The increase inthe interactions of DnaJ with the substrate was only tran-sient, apparently followed by the rapid substrate transferto DnaK. This agrees well with the DnaJ/DnaK interactionkinetics and with a previous biochemical analysis (Gameret al., 1996). Interestingly, we also observed a muchslower and gradual increase in the DnaJ–DnaJ interac-tion, possibly due to the recruitment of multiple DnaJmolecules to the same aggregating proteins. DnaKappears to subsequently channel the substrates to theHsp100 machinery, as was already suggested by a pre-vious biochemical study (Weibezahn et al., 2004), first toClpB and later to ClpX. The much delayed weak increasein the interaction of DnaJ with ClpB and ClpX towardsthe end of our experiment (5 min) might indicate that thesubstrates may be passed again from the Hsp100 to theHsp70 machinery through DnaJ, or reflect the delayed
Severe denaturation Aggregation
Recruitment of
sHsp
Refolded
protein
Degradation by ClpXP
Unfolding by
ClpX
Unfolding by
ClpB
Moderate denaturation
Recruitment of
Hsp70
Folding by HtpG
Fig. 6. Refolding pathways of heat-denatured proteins. Proposed sequence of chaperone interactions with substrates and among each otherafter a heat shock, based on the interaction dynamics shown in Fig. 4. Dashed lines indicate more hypothetical interactions; see text fordetails.
binding of DnaJ to aggregating proteins that are alreadyassociated with ClpB and ClpX. The increase in the inter-action of luciferase with the protease ClpP was muchslower, and ClpX even apparently became disengagedfrom ClpP at the early stages of the heat shock response,indicating that substrate proteolysis is delayed comparedto refolding.
Within the second pathway, interactions between sHspsand luciferase, as well as among sHsps, increasedsteadily throughout the course of the heat shock. This isconsistent with formation of large oligomeric assembliesbetween sHsps molecules and aggregated protein sub-strates (Ehrnsperger et al., 1997; Lee et al., 1997; Hasl-beck et al., 1999). The observed difference between therates of self-association of individual sHsps after a heatshock agrees with a recently proposed model, where theholdase function is primarily played by IbpA whereas IbpBsubsequently associates with the aggregates via its inter-action with IbpA and makes them competent for disaggre-gation (Ratajczak et al., 2009).
Subsequent interactions of sHsps with the Hsp70system show a delay of several minutes, suggestingthat the substrates bound by sHsps are not immediatelypassed to DnaJ or DnaK. We speculate that it may beexplained by the initial engagement (and saturation) of theHsp70 system with the substrates that are not yet sHsp-bound, which prevents it from binding to already aggre-gating proteins. In contrast, the sHsp interactions withClpB and ClpX did not show such a delay but ratherincreased steadily – albeit slowly – after the heat shock.This increase suggests that ClpB and ClpX can associatewith the sHsp-held substrates independently of DnaKalready at the early stages of protein aggregation, bindingeither to sHsps or to the substrates themselves (Schliekeret al., 2004; Barnett et al., 2005). We observed that ClpBpreferentially interacts with IbpA and ClpX with IbpB,which might be related to different functions of the twosHsps during formation of aggregates and their subse-quent disaggregation (Ratajczak et al., 2009). Neverthe-less, the subsequent interaction of ClpB with DnaK islikely to be necessary for efficient protein disaggregation(Weibezahn et al., 2004), illustrating the complexity ofprotein interactions at the later stages of the stressresponse.
Experimental procedures
Construction of fluorescent protein fusions
All fusion constructs were created as previously described(Kentner et al., 2006). Target genes were amplified andligated into pTrc99A (Pharmacia; pBR ori, pTrc promoter,AmpR) derivatives pDK112 or pDK4 carrying the eyfpA206K
sequence, or pDK113 or pDK2 carrying the ecfpA206K
sequences. The CFP fusions were further re-cloned into
pBAD18K derivative pDK5 (pACYC ori, pBAD promoter,KanR). Immunoblotting with a GFP-specific antibody (JL8; BDBiosciences) was performed to confirm that the full-lengthfusion was being expressed. All fusion constructs are listed inTable S1.
Complementation assays
Functionality of the fusion proteins was tested whereverdirect complementation assays have been described. Over-night cultures of respective knockout strains, harbouring theplasmid expressing the fusion proteins, were serially dilutedand spotted in duplicate on Luria–Bertani agar plates withvarying amounts of the inducers. Plates were incubated for16 h at permissive or at elevated temperatures, and function-ality was assessed by the rescue of the wild-type growthphenotype at high growth temperature. Representativeimages of the plates are shown in Fig. S1. All of the testedfusions were at least partly functional. Functionality of otherfusion proteins has been tested in a recent study (Winkleret al., 2010).
Strains and cultivation
All experiments were done with wild-type MG1655 cells, cul-tivated at 30°C in tryptone broth (TB; 1% tryptone and 0.5%sodium chloride) with added antibiotics. Ampicillin and kana-mycin were used at final concentrations of 100 and 50 mg ml-1
respectively. Overnight cultures grown at 30°C were diluted1:100 and grown for 4 h at 30°C, in the presence of theinducers isopropyl b-D-thiogalactoside (IPTG) or L-arabinoseat concentrations indicated in Table S1. When specified,35 mg ml-1 final concentration of chloramphenicol was addedto the culture for the last 30 min of growth. The cells wereharvested by centrifugation (at 3200¥ g for 10 min), washedonce and suspended in tethering buffer (10 mM potassiumphosphate, 0.1 mM EDTA, 1 mM L-methionine, 67 mM sodiumchloride, 10 mM sodium lactate, pH 7.0) for microscopy.
Fluorescence imaging
A small volume of the cell suspension was overlaid onan agarose pad and imaged with a Zeiss Axio Imager.Z1microscope equipped with an ORCA AG CCD camera(Hamamatsu), a 100¥ NA 1.45 objective, and HE YFP (Exci-tation BP 500/25; Emission BP 535/30) and HE CFP (Exci-tation BP 436/25; Emission BP 480/40) filter sets. All imageswere acquired under identical conditions, and subsequentlyanalysed and quantified using the ImageJ software (WRasband; http://rsb.info.nih.gov/ij).
Quantification of fluorescent proteins expression
Quantification of fluorescent protein levels was performedessentially as previously described (Kentner and Sourjik,2009), whereby the expression of CFP and YFP fusions in acell population was compared against reference strains withpreviously determined expression levels of YFP and CFPusing flow cytometry and/or fluorescence microscopy. Cul-
Physical map of the bacterial chaperone network 745
tures expressing the YFP fusions were harvested, washedwith tethering buffer and examined by flow cytometry on aFACScan (BD Biosciences) equipped with a 488 nm Argonlaser. Fluorescence micrographs of the CFP fusions wereanalysed with ImageJ to ascertain the mean fluorescence ofthe cells.
Acceptor-photobleaching FRET measurements
The cells were concentrated 10-fold and overlaid on anagarose pad. Measurements were performed at room tem-perature (20°C) on a custom-modified Zeiss Axiovert 200microscope, as described previously (Kentner and Sourjik,2009), whereby CFP and YFP signals from a field of cells wasdetected using H7421-40 photon counters (Hamamatsu). Foreach measurement point, photons were counted over 0.5 susing a counter function of the PCI-6034E board, controlledby a custom-written LabView 7.1 program (both from NationalInstruments). Bleaching of YFP was accomplished by a 20 sillumination with a 532 nm diode laser (Rapp OptoElectronic),reflected by the 495DCSP dichroic mirror into the light path.CFP emission was recorded before and after bleaching ofYFP, and FRET was calculated as the CFP signal increasedivided by the total signal after bleaching (Sourjik et al.,2007). Notably, thus determined apparent FRET efficienciesare much lower than the theoretical FRET efficienciesexpected for the same pairs based on the distance betweenCFP and YFP in the complex. This is due to the contributionof the background fluorescence to the CFP channel, whichdiminishes the calculated FRET signal by a factor ofCCFP/(CCFP + CB), where CCFP is the specific CFP fluorescenceand CB is fluorescence background. At moderate levels offluorescent protein expression used here, the contribution ofCB is comparable with CCFP or even higher.
Measurements of heat shock-induced changes in FRET
The same setup as described above but implementedon a Zeiss Axio Imager.Z1 microscope equipped with thetemperature-controlled stage was used to measure heatshock-induced changes in FRET for positive pairs. Tempera-ture of the stage was rapidly (within approximately 80 s)raised from 20°C to 42°C using a custom-made program-mable Peltier controller and subsequently maintained at 42°Cfor 5 min. Changes in FRET curves were deduced from theYFP/CFP ratio as described in Results. To facilitate compari-sons between individual curves in Figs 3 and 4, the YFP/CFPratio was normalized to the ratio before the onset of the heatshock (YFP/CFP)0 and unity was subtracted from all values,giving the normalized ratio (YFP/CFP)/(YFP/CFP)0 - 1. Asdiscussed above for the steady-state FRET signals, the mag-nitude of the relative change in the ratio of YFP to CFPfluorescence may be substantially smaller than the change inthe number of interacting FRET pairs.
Acknowledgements
We thank Bernd Bukau, Matthias Mayer, Axel Mogk, JulianeWinkler and Jennifer Reed for their helpful advice during thecourse of this work and for critical reading of the manuscript.
We further thank Gabriele Malengo and Hui Li for technicalhelp, and Willim Ryu for providing the temperature controller.This work was partly funded by the Bioquant GraduateProgram of Land Baden-Württemberg ‘Molecular machines:mechanisms and functional interconnections’.
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Physical map of the bacterial chaperone network 747
