Cells have evolved organelle-specific responses to maintain protein homeostasis (proteostasis). During proteostatic stress, mitochondria downregulate translation and enhance protein folding, yet the underlying mechanisms remain poorly defined. Here, we employed cryo-electron tomography to observe the structural consequences of mitochondrial proteostatic stress within human cells. We detected protein aggregates within the mitochondrial matrix, accompanied by a marked remodeling of cristae architecture. Concomitantly, the number of mitochondrial ribosome complexes was significantly reduced. Mitochondrial Hsp60 (mHsp60), a key protein folding machine, underwent major conformational changes to favor complexes with its co-chaperone mHsp10. We visualized the interactions of mHsp60 with native substrate proteins, and determined in vitro mHsp60 cryo- EM structures enabling nucleotide state assignment of the in situ structures. These data converge on a model of the mHsp60 functional cycle and its essential role in mitochondrial proteostasis. More broadly, our findings reveal structural mechanisms governing mitochondrial protein biosynthesis and their remodeling under proteostatic stress.

Fluorescence imaging was performed on a DMi8 microscope (Leica) equipped with an HC PL APO 63×/1.40 oil immersion objective and a Leica K5-14401415 detector. Image acquisition was carried out as 2×2 binned Z-stacks comprising 20 steps and three channels corresponding to the respective signals (MitoSpy Red CMXRos in the TXT channel, PINK1- GFP in the GFP channel, and DAPI in the DAPI channel). Further processing, including histogram adjustment and denoising with THUNDER, was performed using the LAS X software platform (Leica).