Journal of Biological Chemistry
Volume 291, Issue 36, 2 September 2016, Pages 18620-18631
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Protein Structure and Folding
Heat Shock Protein 90 kDa (Hsp90) Has a Second Functional Interaction Site with the Mitochondrial Import Receptor Tom70*

https://doi.org/10.1074/jbc.M115.710137Get rights and content
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To accomplish its crucial role, mitochondria require proteins that are produced in the cytosol, delivered by cytosolic Hsp90, and translocated to its interior by the translocase outer membrane (TOM) complex. Hsp90 is a dimeric molecular chaperone and its function is modulated by its interaction with a large variety of co-chaperones expressed within the cell. An important family of co-chaperones is characterized by the presence of one TPR (tetratricopeptide repeat) domain, which binds to the C-terminal MEEVD motif of Hsp90. These include Tom70, an important component of the TOM complex. Despite a wealth of studies conducted on the relevance of Tom70·Hsp90 complex formation, there is a dearth of information regarding the exact molecular mode of interaction. To help fill this void, we have employed a combined experimental strategy consisting of cross-linking/mass spectrometry to investigate binding of the C-terminal Hsp90 domain to the cytosolic domain of Tom70. This approach has identified a novel region of contact between C-Hsp90 and Tom70, a finding that is confirmed by probing the corresponding peptides derived from cross-linking experiments via isothermal titration calorimetry and mitochondrial import assays. The data generated in this study are combined to input constraints for a molecular model of the Hsp90/Tom70 interaction, which has been validated by small angle x-ray scattering, hydrogen/deuterium exchange, and mass spectrometry. The resultant model suggests that only one of the MEEVD motifs within dimeric Hsp90 contacts Tom70. Collectively, our findings provide significant insight on the mechanisms by which preproteins interact with Hsp90 and are translocated via Tom70 to the mitochondria.

heat shock protein 90 (Hsp90)
mitochondrial transport
molecular chaperone
protein assembly
protein folding

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*

This work was supported in part by Fundação de Amparo à Pesquisa do Estado de São Paulo Grant FAPESP 2012/50161-8, Ministério da Ciência e Tecnologia/Conselho Nacional de Desenvolvimento Científico e Tecnológico (MCT/CNPq), and fellowships from FAPESP and the Canadian Institutes of Health Research (MOP-130332). The authors declare they have no conflict of interest with the contents of this article.

This article contains supplemental Figs. S1 and S2 and Videos S1–S5.

1

Both authors contributed equally to the results of this work.

2

Holds a Canada Research Chair in Molecular Chaperones.