BPS2026 – In situ characterization of mitochondrial Hsp60-Hsp10 chaperone complex under folding stress

Mitochondrial proteostasis is critical for maintaining mitochondrial function, and its disruption triggers mitochondrial unfolded protein response, which alleviates protein-folding stress by upregulating chaperones. However, the mechanisms by which these chaperones mitigate protein-folding stress remain unclear. Here, utilizing cryo-electron tomography, we show that folding stress induces marked morphological alterations in mitochondria, including the accumulation of amorphous protein aggregates and is associated with an increase in the abundance and spatial clustering of the mitochondrial Hsp60-Hsp10 (mtHsp60-Hsp10) complex. Subtomogram analysis revealed the in situ architecture and conformational heterogeneity of the mtHsp60-Hsp10 complex under stress conditions. While retaining its canonical double-ring structure, the complex adopted multiple conformational states, including football, half-football, and bullet-like forms. Notably, there were significant shifts of conformational ensembles in response to folding stress, resulting in predominance of football conformation. Furthermore, mtHsp60-Hsp10 complex encapsulated unstructured substrates utilizing conserved hydrophobic residues. We further demonstrated that knockdown of the mtHsp60-Hsp10 complex exacerbates folding stress, as evidenced by elevated cellular stress responses and activation of mitophagy. In conclusion, our study defines the in situ structural properties of the mtHsp60-Hsp10 complex and provides mechanistic insight into how it mitigates mitochondrial protein-folding stress.