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To whom correspondence may be addressed: Munich Center for Integrated Protein Science, Dept. of Biology I, Microbiology, Ludwig-Maximilians-Universität München, Grosshaderner Strasse 2–4, D-82152 Planegg-Martinsried, Germany. Tel.: 49-89-2180-74506; Fax: 49-89-2180-74520;
To whom correspondence may be addressed: Munich Center for Integrated Protein Science, Dept. of Biology I, Botany, Ludwig-Maximilians-Universität München, Grosshaderner Strasse 2–4, D-82152 Planegg-Martinsried, Germany. Tel.: 49-89-2180-74760; Fax: 49-89-2180-74752;
* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 1035, Project A04 (to J. S. and S. S.), Fonds der Chemischen Industrie Grant Do 187/22 (to R. S.), and the Exc114/1. This article contains supplemental Fig. 1.
The three tetratricopeptide repeat domain-containing docking proteins Toc64, OM64, and AtTPR7 reside in the chloroplast, mitochondrion, and endoplasmic reticulum of Arabidopsis thaliana, respectively. They are suggested to act during post-translational protein import by association with chaperone-bound preprotein complexes. Here, we performed a detailed biochemical, biophysical, and computational analysis of the interaction between Toc64, OM64, and AtTPR7 and the five cytosolic chaperones HSP70.1, HSP90.1, HSP90.2, HSP90.3, and HSP90.4. We used surface plasmon resonance spectroscopy in combination with Interaction Map® analysis to distinguish between chaperone oligomerization and docking protein-chaperone interactions and to calculate binding affinities for all tested interactions. Complementary to this, we applied pulldown assays as well as microscale thermophoresis as surface immobilization independent techniques. The data revealed that OM64 prefers HSP70 over HSP90, whereas Toc64 binds all chaperones with comparable affinities. We could further show that AtTPR7 is able to bind HSP90 in addition to HSP70. Moreover, differences between the HSP90 isoforms were detected and revealed a weaker binding for HSP90.1 to AtTPR7 and OM64, showing that slight differences in the amino acid composition or structure of the chaperones influence binding to the tetratricopeptide repeat domain. The combinatory approach of several methods provided a powerful toolkit to determine binding affinities of similar interaction partners in a highly quantitative manner.
Background: Tetratricopeptide repeat proteins at organellar surfaces serve as docking proteins for chaperone-bound preproteins.
Results: Binding affinities of docking proteins and chaperones were determined using surface plasmon resonance spectroscopy, Interaction Map® analysis, and microscale thermophoresis.
Conclusion: Docking proteins of the chloroplast, mitochondrion, and endoplasmic reticulum bind differentially to various cytosolic chaperones.
Significance: Tetratricopeptide repeat docking proteins possibly discriminate between chaperones in the cytosol.
as well as almost the entire proteome of chloroplasts and mitochondria rely on being synthesized in the cytosol and transported to and across the correct membranes. All chloroplast and mitochondrial proteins are imported post-translationally, whereas for the endoplasmic reticulum both co-translational and post-translational import has been described (
All preprotein-translocon complexes are equipped with a central channel protein embedded into the lipid bilayer thus allowing preproteins to travel from the cytosol into the respective organelles. The translocation process through the pore is assisted by associated docking or receptor proteins, which often harbor large cytosolic domains to mediate interaction with preproteins and cytosolic factors. Docking proteins containing tetratricopeptide repeat (TPR) domains are found in almost all organellar membranes and organisms as parts of the translocon complexes (
). Although they do not represent an essential feature for cell viability, causing only mild defects upon deletion, they act as regulators under stress conditions and in concert with other receptor proteins (
). The contact between the TPR domains of membrane docking proteins and cytosolic preproteins can either occur directly or indirectly utilizing chaperones bound to the preproteins as scaffold proteins. The C-terminal EEVD motif conserved in cytosolic chaperones such as heat shock proteins HSP70s and HSP90s can be coordinated by the clamp-type TPR domain, which consists of three repetitive motifs of 34 degenerate amino acids together forming a helix-turn-helix structure (
In mammals and yeast mitochondria, Tom70 is the most prominent TPR domain-containing receptor, which has 11 TPR motifs organized in three distinct domains. The three N-terminal TPR motifs form a clamp-type domain by which it associates with HSP70 in yeast, as well as with HSP90 in mammals. In mammals, Tom70 is assisted by the membrane-associated Tom34, which harbors two TPR domains that interact with HSP70 as well as HSP90, suggesting a possible role as co-chaperone in the cytosol (
). Recognized chaperone-preprotein complexes are subsequently released to the Tom translocon, and preproteins are translocated across the outer mitochondrial membrane. Post-translational import into the ER in yeast is also facilitated with the aid of a TPR domain-containing protein, Sec72, that is soluble by itself but anchored to the membrane via Sec71, a membrane-spanning component of the Sec translocon (
). However, in plants no preproteins of the post-translational translocation pathway into the ER are known to date, which might utilize chaperone guidance. Therefore, it will be interesting to identify candidate proteins in plants in the future and to analyze the role of HSP70 or HSP90 in their delivery to the ER membrane in vivo.
In plants, complexity is added to post-translational targeting by the chloroplast as an additional organelle. Likewise, cytosolic components have been described to associate with chloroplast preproteins, such as 14-3-3 proteins as well as HSP70 and HSP90. HSP90-binding candidates are recognized indirectly by the TPR domain-containing protein Toc64 (
), a loosely associated component of the chloroplast Toc translocation machinery in the outer envelope membrane. The composition of the Tom complex in plant mitochondria differs distinctively from the complex in yeast and mammals, especially with respect to the receptor proteins. Tom70 is not found in plant genomes; however, a close homologue of Toc64, OM64, has been identified in the outer mitochondrial membrane. Mutants lacking OM64 show reduced import of some mitochondrial proteins, corroborating the idea of a catalytic function of OM64 in protein import dependent on chaperone-assisted translocation (
). Although plants, yeast, and mammals share the central components of the ER Sec translocon Sec61, Sec62, and Sec63, the TPR domain containing Sec72 is only found in yeast. However, we have recently identified AtTPR7 as an interaction partner of Arabidopsis Sec63 and Sec62 (
). As we could also show that AtTPR7 can complement the function of Sec72 in yeast and interacts with both HSP70 and HSP90 in pulldown experiments, AtTPR7 is most likely involved in post-translational translocation into the ER in plants. In the plant cytosol, four HSP90 and five HSP70 isoforms exist. Some of these are constitutively produced at high levels, i.e. show a minor response to stress exposure (HSP90.2, HSP90.3, HSP90.4, and HSP70.1), whereas other isoforms are heat shock-induced and produced at higher levels under stress conditions (HSP90.1, HSP70.2, HSP70.3, HSP70.4, and HSP70.5) (
In this study, we investigated whether Toc64, OM64, and AtTPR7 exhibit preferences for either HSP70 or any of the HSP90 isoforms to investigate a potential supporting function of chaperones in discrimination between organelles during protein sorting. Individual TPR domains of HSP90 co-chaperones have previously been shown to distinguish between HSP70 and HSP90, for example in the HSP70/90-organizing protein, which contains three TPR domains all showing different binding affinities for HSP70 and HSP90 (
). Therefore, we utilized a combination of several biochemical, biophysical, and computational methods to quantify these interactions, including surface plasmon resonance spectroscopy (SPR) with Interaction Map® (IM) evaluation, microscale thermophoresis (MST), as well as in vitro pulldown experiments. Interestingly, significant differences were observed with respect to the individual binding affinities of Toc64 and OM64 to HSP70.1 and the HSP90 isoforms. Although the TRP domains are highly similar, OM64 binds preferentially to HSP70.1 and Toc64 binds to both HSP70.1 and the HSP90 isoforms. AtTPR7 binds to HSP70.1 and the HSP90 isoforms in the same manner except for HSP90.1, the heat-induced isoform, for which it shows a reduced binding affinity. Using a combination of SPR and IM analyses, we were able to determine binding kinetics and to quantify these interactions. MST was used as a novel and surface immobilization-independent method to additionally analyze the AtTPR7-chaperone binding affinities.
The aim of our study was to compare binding affinities of three TPR domain-containing docking proteins, Toc64 at the outer envelope of chloroplasts, OM64 at the outer membrane of mitochondria, and AtTPR7 at the ER membrane, with the cytosolic chaperones HSP70.1 and the four isoforms of HSP90. Members of the HSP70 and HSP90 family have a suggested function in post-translational protein import into the respective compartments. In this context, we aimed to investigate a potential role of HSP70 or HSP90 in the sorting process of preproteins to distinct organelles, because TPR domains of various proteins (e.g. HSP70/90-organizing protein) have previously been shown to selectively discriminate between the two chaperones (
). As a first step, we performed in vitro pulldown experiments that showed the binding potential of the three TPR domain-containing docking proteins to all tested chaperones, albeit with different intensities. However, because pulldown assays cannot supply quantitative data, we chose SPR combined with IM evaluation as the central method to determine binding affinities; the obtained results are summarized in Fig. 8.
Previous data on Toc64, the chloroplast-docking protein, has shown that the pea Toc64 isoform preferentially binds the C-terminal HSP90 peptide over the HSP70 peptide as determined by semi-quantitative pulldown experiments (
). We could show in our initial pulldown with the Arabidopsis Toc64 along with the constitutively produced full-length HSP70.1 and HSP90 isoforms that it has the potential to interact with all chaperones. SPR analyses revealed that binding affinities for all chaperones tested were in the micromolar range and showed no significant differences. However, by determining the on- and off-rates, it became evident that association constants are higher toward HSP70.1 and HSP90.4 compared with the other HSP90 isoforms, although dissociation showed comparable values for all tested chaperones. Considering that the chaperones are suggested to play a role in preprotein recognition at organellar surfaces (
), we suggest that chloroplast preproteins can be delivered with the aid of both, HSP70 and HSP90, in vivo. HSP90 isoforms are not discriminated and therefore are possibly functionally redundant in this context.
OM64 is phylogenetically very closely related to Toc64, showing an overall sequence identity of 51% (68% within the TPR domain) (
). Therefore, it is likely that OM64 is functionally similar to the yeast and mammalian Tom70. The clamp-type TPR domain of Tom70 functions as docking site for HSP70 to receive mitochondrial preproteins, whereas mammalian Tom70 additionally binds to HSP90 (
). Surprisingly, our data revealed that OM64 binds to HSP70.1 with a much higher affinity in direct comparison with the HSP90 isoforms. This tendency is evident already from the pulldown experiments, and KD values calculated for OM64-HSP70.1 were 100 times lower, in the nanomolar range, compared with the HSP90 isoforms. In an in vivo situation, with HSP70 and HSP90 present in the cytosol, preferential binding of HSP70 to OM64 can be expected. Although these data are surprising, especially considering the high sequence identity between OM64 and Toc64, it favors a model in which chloroplast preproteins are assisted by HSP90, whereas mitochondrial preproteins are preferentially bound to HSP70 in the cytosol. Moreover, initial results support this hypothesis, as we have so far been unable to demonstrate HSP90 binding to plant mitochondrial preproteins, not even to hydrophobic carrier proteins,
) (AtTPR7 is designated as OEP61 in this study). All binding affinities calculated for AtTPR7 and chaperones were found to be in the micromolar range. Although AtTPR7-HSP70.1 showed the strongest interaction, binding of AtTPR7 to HSP90 is also likely to occur in vivo. Interestingly, the binding affinity of HSP90.1 to AtTPR7 and OM64 was the weakest. This might indicate that the constitutively produced chaperone isoforms (HSP70.1, HSP90.2–4) play a predominant role in preprotein targeting in contrast to the mainly heat shock-induced isoform HSP90.1. Moreover, the HSP90.2, HSP90.3, and HSP90.4 isoforms are highly homologous (97–99% identity), suggesting a redundant function, whereas HSP90.1 shows only 85–87% identity to the other isoforms. The fact that we observed differences in the binding affinities of HSP90.1 to AtTPR7 and OM64 in comparison with the other HSP90 isoforms indicates that binding characteristics to the TPR domain are not only influenced by the presence of the C-terminal MEEVD motif, which is present in all HSP90 isoforms. Binding affinities seem also to be influenced by the entire protein, its amino acid composition, as well as its higher order structure. In this respect, the C-terminal amino acids adjacent to the MEEVD motif may play a role, because they could come into close proximity to the TPR clamp of the receptor. Sequence identity in the C-terminal 45 amino acids is reduced to 73–75% when comparing HSP90.1 with the other isoforms (Fig. 9). Moreover, future structural analyses and amino acid replacements within the individual TPR domains will reveal which residues interplay with the different chaperones and participate in conferring specificity.
In addition to the SPR data set for AtTPR7, we used MST as a novel interaction analysis approach, which is surface immobilization independent. The obtained binding affinities showed the same tendency as the corresponding SPR results. Slight differences in the determined KD values between the two methods could result from the different principles of the two techniques. Whereas in MST measurements the two proteins are allowed to interact for several minutes until the interaction has reached a steady state equilibrium, only a transient interaction is monitored by determining on- and off-rates in SPR due to the quick change between buffer and binding partner. However, clearly the same tendencies are observed, and both methods are ideally suited to act as complementary approaches.
In this study, we have used a combinatory approach of biochemical, biophysical, and computational methods to investigate protein-protein interactions and to quantify binding affinities and kinetics of three TPR receptor proteins as well as five different full-length chaperones. SPR in combination with IM and MST has proven to be a powerful approach to distinguish individual binding constants.
We thank Nano Temper (Munich, Germany), Dr. Karl Andersson (Ridgeview Instruments, Uppsala, Sweden), and Dr. Anja Drescher (GE Healthcare, Munich, Germany) for support and helpful discussions. We are grateful to Dr. Susanne Gebhard (LMU München) for critical reading of the manuscript. Katharina Schöngruber and Stefanie Rapp are acknowledged for excellent technical assistance.