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Dynamic map of protein interactions in the Escherichia coli chemotaxis pathway

2009; Springer Nature; Volume: 5; Issue: 1 Linguagem: Inglês

10.1038/msb.2008.77

1744-4292

David Kentner, Victor Sourjik,

Gene Regulatory Network Analysis

Article20 January 2009Open Access Dynamic map of protein interactions in the Escherichia coli chemotaxis pathway David Kentner David Kentner Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Victor Sourjik Corresponding Author Victor Sourjik Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author David Kentner David Kentner Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Victor Sourjik Corresponding Author Victor Sourjik Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Author Information David Kentner1 and Victor Sourjik 1 1Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Heidelberg, Germany *Corresponding author. Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany. Tel.: +49 6221 54 6858; Fax: +49 6221 54 5894; E-mail: [email protected] Molecular Systems Biology (2009)5:238https://doi.org/10.1038/msb.2008.77 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Protein–protein interactions play key roles in virtually all cellular processes, often forming complex regulatory networks. A powerful tool to study interactions in vivo is fluorescence resonance energy transfer (FRET), which is based on the distance-dependent energy transfer from an excited donor to an acceptor fluorophore. Here, we used FRET to systematically map all protein interactions in the chemotaxis signaling pathway in Escherichia coli, one of the most studied models of signal transduction, and to determine stimulation-induced changes in the pathway. Our FRET analysis identified 19 positive FRET pairs out of the 28 possible protein combinations, with 9 pairs being responsive to chemotactic stimulation. Six stimulation-dependent and five stimulation-independent interactions were direct, whereas other interactions were apparently mediated by scaffolding proteins. Characterization of stimulation-induced responses revealed an additional regulation through activity dependence of interactions involving the adaptation enzyme CheB, and showed complex rearrangement of chemosensory receptors. Our study illustrates how FRET can be efficiently employed to study dynamic protein networks in vivo. Synopsis The chemotaxis system controlling the swimming behavior of the bacterium Escherichia coli is one of best-studied model signaling pathways and has been used to illustrate many fundamental principles of biological signal processing (Sourjik, 2004; Wadhams and Armitage, 2004). The pathway regulates flagellar rotation dependent on chemotactic stimuli and consists of five ligand-specific membrane-associated receptors and six cytoplasmic proteins. Excitation signaling is mediated by histidine kinase CheA, coupling protein CheW and response regulator CheY, whereas CheY phosphatase CheZ plays the role in signal termination, and receptor methyltransferase CheR and methylesterase CheB constitute a simple adaptation system. Despite its simplicity, the pathway shows large signal amplification (Li and Weis, 2000; Gestwicki and Kiessling, 2002; Sourjik and Berg, 2002b, 2004; Lai et al, 2005) and impressive robustness against such perturbations as gene expression noise (Alon et al, 1999; Kollmann et al, 2005). Fluorescence resonance energy transfer (FRET), which is based on the distance-dependent energy transfer from an excited donor to an acceptor fluorophore, allows the detection of intracellular interactions of fluorescently labeled proteins non-invasively and with high selectivity (Wouters and Bastiaens, 2001). It was recently used to study stimulation dependence of several interactions in the chemotaxis pathway, and to investigate signal processing by receptor–kinase complexes (Sourjik and Berg, 2002a, 2002b; Vaknin and Berg, 2006, 2007; Sourjik et al, 2007). In the current study, we extend FRET-based interaction mapping to the entire chemotaxis pathway, using the library of fusions to cyan and yellow fluorescent proteins (CFP and YFP, respectively) as donor–acceptor pairs. To reduce the chances of false negatives due to the large distance or unfavorable orientation of proteins in the complex and to find pairs with strongest FRET efficiency for subsequent investigation of stimulation dependence, the library contains both N- and C-terminal fusions of CFP and YFP to all chemotaxis proteins, the aspartate receptor Tar and the serine receptor Tsr. Most fusions are functional and show the expected localization in the cell. Initial FRET mapping of protein interactions in our assay is made by acceptor photobleaching (Figure 2), whereby acceptor (YFP) is bleached by short high-intensity laser illumination. After the identification of positive FRET pairs, the dependence of these interactions on chemotactic stimulation is examined using a flow assay (Figure 2B). This analysis enables us to picture the entire network of protein interaction in the chemotaxis pathway, with 19 positive FRET pairs being identified out of the 28 possible protein combinations (Figure 2C). Most positive pairs interact even in the absence of all other chemotaxis proteins (Figure 2C, solid lines), whereas interactions of other pairs depend on a common binding partner (Figure 2C, dotted and dashed lines). Nine FRET pairs are responsive to chemotactic stimulation, five of which clearly correspond to direct protein interactions (Figure 2C, black circles). Although stimulation dependence of most pairs could be expected based on existing biochemical data (Li et al, 1995; McEvoy et al, 1999) or FRET experiments (Sourjik and Berg, 2002a, 2002b; Vaknin and Berg, 2006), the observed attractant-induced decrease in affinity between the adaptation enzyme CheB and both of its interaction partners, receptors and CheA, was novel. Such dependence implies an additional enhancement of a negative feedback from the level of kinase activity to that of receptor activity, which is provided by CheB phosphorylation. This feedback is essential to maintain robust output of the chemotaxis system under such perturbations as gene expression noise (Kollmann et al, 2005), and a recent computational analysis suggested that strong binding of phosphorylated CheB to receptors and weak binding of phosphorylated CheY to CheA are important for robust adaptation in chemotaxis (Matsuzaki et al, 2007). Equally important for the overall picture of the chemotaxis pathway is the observation that many pathway's interactions are not stimulation dependent. This allows us to rule out many potential regulation mechanisms, such as activity-dependent CheZ oligomerization (Blat and Eisenbach, 1996) or large conformational changes in the sensory complexes. We further demonstrate differences in concentration dependence and kinetics of response to stimulation at different levels of signal processing. Upon a step-like attractant stimulation, changes in kinase activity—reflected by the level of phosphorylation-dependent FRET between CheY and CheZ—show fastest kinetics, followed by rearrangement of receptors in the sensory complexes, and then by changes in CheB binding to receptors and CheA. Receptor rearrangement shows different dependence on the stimulus strength than the downstream kinase regulation, confirming previous observations (Vaknin and Berg, 2007) and suggesting that amplification of chemotactic signals takes place among signaling domains of interacting receptors. Moreover, both dose–response and kinetics measurements reveal two distinct stimulation-induced movements or rearrangements of receptors. Our study provides a holistic picture of chemotactic signaling in E. coli and demonstrates that FRET can be successfully used to systematically map and quantify all intracellular protein interactions in a signaling network, including transient interactions, with no false-positives and nearly no false negatives. A strong advantage of FRET is that it enables systematic mapping of activity dependence of interactions and measuring response kinetics, and therefore yields a dynamic picture of the network. In addition to direct interactions, protein proximities—mediated by association with a common interaction partner—can be identified, facilitating the characterization of multiprotein complexes and their dynamics in vivo. FRET-based interaction mapping thus stands as a simple and reliable means for the investigation of other protein networks in bacteria or eukaryotes. Introduction The chemotaxis pathway in Escherichia coli senses gradients of attractants and repellents to direct cellular movement toward favorable environments. Being a comparatively simple system with few components, chemotaxis in E. coli is an excellent model for the detailed quantitative study of signaling networks (Sourjik, 2004; Wadhams and Armitage, 2004). The sensory complex is formed by ligand-specific membrane-associated receptors (Tsr, Tar, Trg, Tap and Aer), histidine kinase CheA and coupling protein CheW. Ligand binding to receptor homodimers alters the autophosphorylation activity of the receptor-associated CheA. The kinase subsequently donates the phosphoryl group to response regulator CheY, which diffuses to flagellar motors and modulates their rotation. Dephosphorylation of CheY is catalyzed by the phosphatase CheZ. Adaptation is exerted by methyltransferase CheR and its antagonist, methylesterase CheB. CheB consists of a C-terminal catalytic domain and an N-terminal CheY-like regulatory domain, which is subject to activatory phosphorylation by CheA. Adaptation enzymes tune the ability of receptors to activate CheA by adjusting the level of receptor methylation on four specific glutamate residues in an activity-dependent manner, thereby returning kinase activity to a constant steady-state level under conditions of continuous stimulation. Two of the four glutamates at the methylation sites are originally translated as glutamines, which are functionally equivalent to methylated glutamates, and are converted to glutamates by the deamidase activity of CheB. Immunoelectron (Maddock and Shapiro, 1993), fluorescence (Sourjik and Berg, 2000) and cryo-electron (Zhang et al, 2007) microscopy have shown that thousands of chemoreceptors are organized in polar and lateral clusters, to which all other chemotaxis proteins localize, forming a large sensory machinery (Sourjik, 2004; Wadhams and Armitage, 2004). Allosteric interactions between receptors in clusters appear to play a central role in the amplification and integration of chemotactic signals (Li and Weis, 2000; Gestwicki and Kiessling, 2002; Sourjik and Berg, 2002b, 2004; Lai et al, 2005). To obtain a comprehensive view of the network dynamics in living cells, we tested all intracellular protein interactions in the pathway and their dependence on chemotactic stimulation by fluorescence resonance energy transfer (FRET). We further quantified concentration dependence and kinetics of stimulation-induced changes in protein interactions. Our results provide a holistic picture of the pathway and of its intracellular dynamics, and demonstrate the efficiency of the FRET-based interaction mapping approach. Results FRET mapping of protein interactions FRET allows the detection of intracellular interactions of fluorescently labeled proteins non-invasively by the energy transfer from an excited donor to an acceptor fluorophore (Wouters and Bastiaens, 2001). The transfer efficiency depends on the distance between the fluorophores as R06/(R6+R06), with the Förster radius R0—at which the energy is transferred with 50% efficiency—being around the size of a typical protein. Such steep dependence on spacing and short characteristic distance make the energy transfer highly specific for proteins that are part of the same complex, either interacting directly or binding to a common scaffolding protein. High distance selectivity, however, can also impair FRET in a protein complex where the spacing between fluorescent labels is above the critical distance, approximately 2 × R0. In our interaction screen, we used cyan and yellow fluorescent proteins (CFP and YFP, respectively) as a donor–acceptor pair with R0∼49 Å, approximately the same size as a fluorescent protein monomer (Tsien and Miyawaki, 1998; Sourjik and Berg, 2002a). To reduce the chances of false negatives due to the large distance or unfavorable orientation of proteins in the complex and to find pairs with strongest FRET efficiency for subsequent investigation of stimulation dependence, we constructed a library of both N- and C-terminal fusions of CFP and YFP to all chemotaxis proteins, the aspartate receptor Tar and the serine receptor Tsr (Supplementary Table SI). E. coli CheA is endogenously expressed from two alternative start codons, yielding a long and a short variant, CheAL and CheAS, respectively (Smith and Parkinson, 1980). To independently analyze both versions of CheA, we made separate fusions to CheA98–655 (CheAS) and to CheAM98I, a mutant of CheAL that does not express CheAS (Sanatinia et al, 1995). Expression of full-length fusions was verified by immunoblot analysis (data not shown). Fusions with the fluorophore at the N terminus of receptors or CheB were omitted from further analysis because of failed membrane insertion or very low expression levels. Fusions to CheY, CheZ, CheR and CheB were fully functional, as tested by their ability to complement respective null mutants for chemotaxis-driven swarming in soft agar (Figure 1A), whereas fusions to the core components of the signaling complex—Tar, CheA and CheW—did not promote swarming. Nevertheless, all fusions localized to the chemosensory clusters (Figure 1B) as expected (Sourjik and Berg, 2000; Shiomi et al, 2002; Banno et al, 2004; Kentner et al, 2006). Figure 1.Functionality and localization of YFP fusions. (A) Complementation assay for chemotactic swarming on soft agar plates. Plates were inoculated with wild-type cells (top), the respective mutant containing the fusion (bottom right) and the mutant without the fusion (bottom left). The Tar fusion was tested in the strain UU1250, which lacks all receptor genes, and CheAL and CheAS fusions in the strain VS166, which lacks the entire cheA gene. Respective mutants expressing Tar, CheA and CheW fusions did not swarm at any induction level tested (plate shown contained 50 μM IPTG), other fusions were expressed at 50 μM (CheR–YFP and YFP–CheY), 40 μM (CheY–YFP), 20 μM (CheZ fusions, YFP–CheR) or without (CheB–YFP) IPTG. Although the CheB–YFP construct only partly complemented the ΔcheB mutant even without induction when expressed from the pTrc promoter, complementation was nearly 100% for the same construct expressed under tighter control of the arabinose promoter (not shown). (B) Localization to clusters in the wild-type strain RP437. IPTG inducer levels were 5 μM (CheZ–YFP and CheB–YFP), 10 μM (CheAL–YFP, CheAS–YFP), 20 μM (Tar–YFP, YFP–CheAL and YFP–CheAS) or 50 μM (CheW, CheY and CheR fusions; YFP–CheZ). Download figure Download PowerPoint FRET mapping of protein interactions was made by acceptor photobleaching (Figure 2A; Supplementary Figure S1A). For simplicity, all protein pairs were tested in the same wild-type E. coli strain RP437 (Parkinson and Houts, 1982). To avoid false negatives that could arise from competitive binding of native proteins, negative results were confirmed in respective knockout mutants. For pairs including YFP–CheR or CheB–YFP, the respective catalytic mutants, YFP–CheRD154A and CheBS164C–YFP (Barnakov et al, 2002; Shiomi et al, 2002), were also tested, as expression of the enzymatically active fusions influences the receptors' methylation level and activity. After the identification of positive FRET pairs, the dependence of these interactions on chemotactic stimulation was examined using a flow assay (Sourjik et al, 2007), whereby cells were attached to a coverslip in a flow chamber and changes in the YFP/CFP ratio were recorded in response to a stepwise addition and subsequent removal of 1 mM α-methyl-DL-aspartate (MeAsp), a non-metabolizable analog of the Tar-specific attractant aspartate (Figure 2B; Supplementary Figure S1B). This analysis enabled us to picture the entire network of protein interaction in the chemotaxis pathway, with 19 positive FRET pairs being identified out of the 28 possible protein combinations, not counting the identical interactions of CheAL and CheAS separately (Figure 2C; Supplementary Table SII). Most pairs showed FRET even in the absence of all other chemotaxis proteins, arguing for direct interactions (Figure 2C, solid lines), whereas other combinations showed FRET only in the presence of either CheA or receptors (Figure 2C, dotted and dashed lines, respectively), indicating their dependence on a common binding partner. Additionally, several interactions are direct but depend on the kinase activity of CheA (see below). Figure 2.FRET analysis of the chemotaxis pathway. (A) FRET measurement by acceptor bleaching. FRET is seen as an increase in CFP emission upon bleaching of YFP for 20 s using a 532-nm laser. Bleaching eliminates energy transfer to the YFP acceptor, causing an unquenching of CFP emission. Example shows CheW–CFP/CheW–YFP pair expressed in Δ[cheA-cheZ] cells. See Materials and methods and Supplementary Figure S1A for details. FRET efficiency for a given pair (Supplementary Tables SII and SIII) was derived from the data as a fractional change in CFP fluorescence. (B) FRET responses to chemostimulation, seen as changes in the YFP/CFP ratio. Examples show VS153 (Δ[cheR-cheZ] Δtsr) cells expressing CFP–CheAS/CheY–YFP (orange line; left Y axis) and CFP–CheAS/CheBS164C–YFP (green line; right Y axis) pairs. Cells were stimulated with 100 μM MeAsp, added at 200 s and removed at 400 s. See Materials and methods and Supplementary Figure S1B for details. (C) FRET interaction map of the chemotaxis pathway. Positive FRET pairs (lines) correspond to direct (solid lines) or presumably indirect interactions that depend either on receptors (dashed lines) or on CheA (dotted lines). Receptors and CheZ are present as homodimers; CheAS and CheAL can form homo- or heterodimers and are depicted as a heterodimer. FRET signal amplitudes are summarized in Supplementary Tables SII and SIII. FRET between Tar and CheW was detected using a truncated Tar1–425 fusion. Receptor–receptor FRET occurs between receptor dimers as it could be measured with both Tar–Tar and Tar–Tsr. Interactions were further classified into stimulation-independent (open circles), direct stimulation-dependent (black circles) and those with indirect or unclear stimulation-dependent (grey circles; see text for details). The stimulus dependence of CheA–CheB and Tar–CheB FRET was measured with a catalytic CheBS164C mutant. The CheY–FliM FRET pair was not included in our initial interaction mapping analysis, but was identified previously (Sourjik and Berg, 2002a). Download figure Download PowerPoint In most cases where four possible combinations of N- and C-terminal fusions could be tested, interactions were observed with all of these combinations (Supplementary Table SII). A notable exception was CheR, for which only the N-terminal fusion showed interactions with other proteins, although both fusions localized to receptor clusters (Figure 1B). The only previously reported interactions that were not detected in the initial screen were those between Tar and CheW or CheA, which was expected because of the large distance between the C terminus of Tar and the binding site for both CheA and CheW at the signaling domain. A truncated Tar1–425–CFP construct, with CFP being positioned closer to the signaling domain, indeed showed FRET in combination with CheW–YFP, though not with YFP–CheW or with N-terminal fusions to CheAL and CheAS or to a CheA509–655 fragment, which corresponds to the CheW homologous receptor-binding P5 domain of CheA. The experimentally observed FRET efficiency for individual combinations could be indicative of the relative binding affinity of the respective fusions but also depends on the distance between protein termini in the complex and relative orientation of the fluorophores. It is always lower than the theoretical FRET efficiency for the respective donor–acceptor pair and depends on the expression levels of the donor and acceptor, due to the contribution of the autofluorescent cell background to the overall cyan signal, and also because in most cases the acceptor is not in sufficient excess to saturate all donor molecules. Stimulation-induced changes in protein interactions Nine FRET pairs were responsive to chemotactic stimulation, five of which clearly correspond to direct protein interactions (black circles in Figure 2C; Supplementary Table SIII). Among them, CheY–CheZ (Sourjik and Berg, 2002b) and receptor–receptor (Vaknin and Berg, 2006) interactions were previously detected using in vivo FRET, along with an additional direct stimulation-dependent interaction between CheY and FliM (Sourjik and Berg, 2002a). The stimulation dependence of the other interactions involving CheY, CheB and CheZ (grey circles in Figure 2C) could be indirect (see Discussion). Stimulus-induced changes in interactions involving CheY or CheB were phosphorylation dependent, and were not observed for non-phosphorylatable mutants CheYD57A or CheBD56E/S164C, whereas receptor–receptor FRET responses are believed to reflect a change in the spacing between receptor dimers upon ligand binding (Vaknin and Berg, 2006, 2007, 2008). Consistent with that, stimulation-induced fractional changes in FRET were strongest for interactions that involved CheY and weakest for interactions that involved receptors (Supplementary Table SIII). To determine the sensitivity of interactions to stimulation, we recorded response amplitudes across a range of MeAsp concentrations (Figure 3). Although the absolute values of FRET efficiency depend on several parameters, relative changes in FRET allow direct quantification of stimulation effects on the concentration of the FRET complex or on the conformational changes in this complex. Measurements were made with a minimal set of pathway components in the background. Strain VS116, which does not express any chemotaxis genes, was used to measure receptor–receptor responses; strain VS153, which contains only a core sensory complex of Tar, CheA and CheW, along with minor receptors Trg and Aer, was used to investigate the phosphorylation-dependent responses. All pairs except CheA–CheY showed a decrease in FRET with kinase inhibition (addition of attractant) in strains VS116 and VS153, respectively (Figure 3). Notably, the CheA–CheY response also showed a decrease in FRET with kinase inhibition when measured in the wild-type RP437 (CheZ+) cells. Figure 3.Dose–response curves for direct stimulation-dependent interactions. Responses to steps of MeAsp for CheZ–CheY (black circles), CheAS–CheY (blue triangles), CheAS–CheBS164C (red squares), Tar–CheBS164C (green diamonds), and FliM–CheY (yellow triangles) fusion pairs in the strain VS153, and Tar–Tar (magenta open circles) and Tar–Tsr (pink open squares) in the strain VS116 (flhC). Cells were equilibrated in the buffer before each stimulation. Response amplitudes were calculated as described in Materials and methods and normalized to the maximal response upon stimulation with 1 mM MeAsp. Smooth curves are fits to the data using a multisite Hill model. Absolute values of stimulation-dependent and total FRET and protein expression levels are summarized in Supplementary Tables SIII and SIV, respectively. Source data isavailable for this figure at www.nature.com/msb. Download figure Download PowerPoint FRET between CheBS164C and Tar or CheA responded to stimulation, and we tested whether the responses represent independent changes in the interaction of CheB with each target. The Tar–CheBS164C response was seen in the strain VS177, where CheA lacks the response regulator-binding domain, whereas no FRET was observed in the strain DK1, a derivative of VS153 in which Tar lacks the CheB-binding C-terminal pentapeptide sequence. This confirms that stimulation-dependent Tar–CheBS164C FRET is due to CheBS164C–YFP binding to the C terminus of receptors and not to CheA. On the other hand, the CheAS–CheBS164C FRET response was still present, albeit much weaker, in the strain DK1. FRET was also observed between a CheA98–257 fusion, which cannot bind receptor clusters, and fusions to CheBS164C or to the N-terminal CheB domain, CheB1–134. We further determined kinetics of responses to a saturating stimulus of attractant for most direct stimulation-dependent interactions. All kinetics could be well fitted by an exponential decay function, yielding characteristic response times, τ (Figure 4). For all interactions involving CheY, the response kinetics was apparently determined by dephosphorylation of phospho-CheY. CheY–CheZ FRET decayed with τ∼0.33 s (Figure 4A), consistent with the in vivo rate of CheZ-stimulated CheY dephosphorylation estimated before (Sourjik and Berg, 2002a). CheY–CheA and CheY–FliM FRET showed slower increase and decay kinetics, respectively (Figure 4B and C), which corresponded to the autodephosphorylation rate of phospho-CheY in the absence of CheZ. Similarly, decay in the interaction of CheB with Tar (Figure 4D) presumably reflected kinetics of CheB dephosphorylation. The characteristic time, τ=2.7 s, and the derived first-order dephosphorylation rate constant, 1/τ∼0.37 s−1, were close to the previously measured in vitro rate constant for CheB dephosphorylation, 0.35 s−1, at the room temperature (Stewart, 1993). Small response amplitude of the CheB–CheA FRET pair precluded reliable measurement of its kinetics, but it is likely to be similar to that of the CheB–Tar pair. Response kinetics of Tar–Tar and Tar–Tsr pairs had characteristic response times of 1.1 and 1.7 s, respectively (Figure 4E and F). However, monoexponential function did not perfectly fit the data, indicating biphasic response kinetics, which was consistent with the biphasic response observed in dose–response measurements (Figure 3). Figure 4.Response kinetics of direct stimulation-dependent interactions. Responses to a saturating step of 10 mM aspartate is shown for CheZ–CheY (A), FliM–CheY (B), CheAS–CheY (C), Tar–CheBS164C (D), Tar–Tar (E) and Tar–Tsr (F). As in Figure 3, receptor pairs were measured in the strain VS116 (flhC), and all other pairs in the strain VS153. FRET signals were normalized to the pre-stimulus value in the buffer. Cells were stimulated at time points indicated by the vertical dashed line. Smooth red curves are fits to the data using an exponential decay model (see Materials and methods). Characteristic response time, τ, is shown for each response kinetics. Source data is available for this figure at www.nature.com/msb. Download figure Download PowerPoint Discussion Altogether, our FRET analysis of protein interactions in the chemotaxis pathway of E. coli identified 19 positive FRET pairs out of the 28 possible protein combinations, with 9 pairs being responsive to chemotactic stimulation. The obtained map of protein interactions is in good agreement with previous biochemical data and localization analyses (Sourjik, 2004; Wadhams and Armitage, 2004), and offers a better insight into the pathway regulation. Core of the sensory complex The core of the chemosensory complex is formed primarily by the receptor–receptor interactions, and is further stabilized by the binding of CheA and CheW (Maddock and Shapiro, 1993; Kim et al, 1999; Ames et al, 2002; Kentner et al, 2006). This ternary complex is stable on the time scale of signaling and adaptation, with characteristic equilibration time being 12 min for CheA and CheW, and more than 30 min for receptors (Schulmeister et al, 2008). We observed energy transfer among most fusions to the core proteins. Receptor–receptor FRET results primarily from the interactions between homodimers, rather than from an intradimeric energy transfer, as FRET signals were similar for the Tar–Tsr and Tar–Tar pairs (Supplementary Table SII). This is consistent with previous reports (Vaknin and Berg, 2006, 2007, 2008) and suggests that receptors are tightly packed in the cluster, with distances between the C termini of different dimers being shorter that the respective intradimeric distances. We further observed FRET for the CheA–CheA, CheA–CheW and CheW–CheW pairs. CheA–CheA FRET was largely intradimeric, as the strength of the signal was not affected by the lack of all other chemotaxis proteins (Supplementary Table SII). This could mean that CheA dimers are less densely packed in the sensory complexes than receptor dimers, consistent with a high ratio of receptors to CheA in the active complexes (Levit et al, 2002). Energy transfer efficiency was similar for all combinations of CheA fusions, indicating similar distances between all termini in the dimer. FRET between CheA and CheW was also independent of the other proteins and thus reflects the expected direct interaction between the two. FRET between two CheW fusions required receptors, suggesting that CheW fusion proteins come into proximity by binding to receptor oligomers and possibly also to CheA dimers. Interactions of receptors with CheW and CheA were the only two established protein interactions in the chemotaxis pathway that were initial