bims-mitpro 2025-10-26 papers

Issue of 2025–10–26
four papers selected by
Andreas Kohler, Umeå University

Mol Cell. 2025 Oct 20. pii: S1097-2765(25)00815-9. [Epub ahead of print]

Delayed protein translocation protects mitochondria against toxic CAT-tailed proteins.

Nils Bertram, Toshiaki Izawa, Felix Thoma, Serena Schwenkert, Stéphane Duvezin-Caubet, Sae-Hun Park, Nikola Wagener, Anne Devin, Christof Osman, Walter Neupert, Dejana Mokranjac.

Ribosome-associated protein quality control (RQC) protects cells against the toxic effects of faulty polypeptides produced by stalled ribosomes. However, mitochondria are vulnerable to C-terminal alanyl and threonyl (CAT)-tailed proteins that are generated in this process, and faulty nuclear-encoded mitochondrial proteins are handled by the recently discovered mitoRQC. Here, we performed a genome-wide screen in yeast to identify additional proteins involved in mitoRQC. We found that peptidyl-tRNA hydrolase 2 (Pth2), present in the mitochondrial outer membrane, influences aggregation of CAT-tailed proteins without majorly affecting the CAT-tailing process itself. Peptidyl-tRNA hydrolase activity is essential during this process, yet the activity of Pth2 can be substituted by another peptidyl-tRNA hydrolase upon proper localization. Our data suggest that Pth2 acts by modulating protein translocation and that the mitochondrial proteostasis network is relieved through increased access of CAT-tailed proteins to cytosolic chaperones. Other hits obtained in the screen show that, in general, delayed protein translocation protects mitochondria against toxic CAT-tailed proteins.

Keywords: RQC; TOM complex; cellular homeostasis; mitoRQC; mitochondria; peptidyl-RNA hydrolase; protein translocation

DOI: https://doi.org/10.1016/j.molcel.2025.09.030

FEBS Lett. 2025 Oct 25.

The IQ-compete assay for measuring mitochondrial protein import efficiencies in living yeast cells.

Yasmin Hoffman, Annika Egeler, Saskia Rödl, Johannes M Herrmann.

Most mitochondrial proteins are synthesized in the cytosol and imported into the organelle. Here, we describe a novel Import and de-Quenching Competition (IQ-compete) assay which monitors the import efficiency of model proteins by fluorescence in living cells. For this method, the sequence of the tobacco etch virus (TEV) protease is fused to a mitochondrial precursor and coexpressed with a cytosolic reporter which becomes fluorescent upon TEV cleavage. Thus, inefficient import of the fusion protein leads to a fluorescent signal. With the IQ-compete assay, the import efficiency of proteins can be reliably analyzed in fluorescence readers, by flow cytometry, by microscopy, and by western blotting. We are convinced that the IQ-compete assay will be a powerful strategy for many different applications. Impact statement This article describes a novel method to monitor the mitochondrial import efficiency for a given protein in living yeast cells. With this IQ-compete assay, protein import efficiencies can be quantified by fluorescent microscopy, flow cytometry, fluorescence spectrometry or western blotting.

Keywords: fluorescence quenching; genetically encoded sensors; mitochondria; presequences; protein targeting

DOI: https://doi.org/10.1002/1873-3468.70206

FEBS J. 2025 Oct 23.

TbTim20 facilitates protein import at a low membrane potential in trypanosomes lacking the mitochondrial genome.

Corinne von Känel, Salome Aeschlimann, Michaela Husová, Silke Oeljeklaus, Philip Stettler, Achim Schnaufer, Bettina Warscheid, Alena Zíková, André Schneider.

Protein import across the mitochondrial inner membrane typically depends on two protein translocases of the inner membrane (TIM) complexes and the membrane potential. The protozoan parasite Trypanosoma brucei, however, has a single, divergent TIM complex. Unlike other trypanosomal TIM subunits, TbTim20 is not essential for the normal growth of the insect or bloodstream forms of T. brucei, leaving its role uncertain. Specific mutations in the γ-subunit of the F1FO-ATPase, such as γL262P, permit bloodstream form trypanosomes to grow without mitochondrial DNA (kinetoplast or kDNA). Here, we show that RNAi-mediated depletion of TbTim20 inhibits growth of this cell line, but only if it lacks the kDNA. Titration of mitochondrial uncouplers and direct membrane potential measurements reveal that TbTim20 becomes more critical as the membrane potential decreases across all tested cell lines. Proteomic analysis of the uninduced and induced γL262P TbTim20-RNAi cell line, which lacks kDNA and exhibits the lowest membrane potential, shows depletion of a subset of imported proteins. This subset includes ATPase subunits, suggesting a mechanism by which TbTim20-silenced cell lines become more sensitive to uncouplers. Thus, we propose that TbTim20 supports the import of a subset of proteins whose import is hypersensitive to a low membrane potential.

Keywords: TIM complex; Trypanosoma brucei; kinetoplast DNA; mitochondrial membrane potential; mitochondrial protein import

DOI: https://doi.org/10.1111/febs.70297

Sci Adv. 2025 Oct 24. 11(43): eadw6064

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

Mingyu Jung, Minjung Kim, Su Jin Ham, Jongkyeong Chung, Soung-Hun Roh.

Mitochondrial proteostasis is critical for maintaining mitochondrial function, and its disruption induces mitochondrial unfolded protein response, which up-regulates chaperones to alleviate protein-folding stress. However, how these chaperones mitigate protein-folding stress remains unclear. Here, using correlated cryo-electron tomography, we show that folding stress triggers marked mitochondrial morphological changes, including the accumulation of amorphous protein aggregates and increased abundance and spatial clustering of the mitochondrial heat shock protein 60-heat shock protein 10 (mtHsp60-Hsp10) complex. Subtomogram analysis revealed the in situ architecture and conformational heterogeneity of mtHsp60-Hsp10 under stress, which retains its canonical double-ring structure while adopting distinct football, half-football, and bullet-like states. Notably, the mtHsp60-Hsp10 complex encapsulates unstructured substrates through conserved hydrophobic interactions. We further demonstrate that knockdown of the mtHsp60-Hsp10 complex exacerbates folding stress, as evidenced by elevated cellular stress responses and activation of mitophagy. Our study defines the in situ structural properties of the mtHsp60-Hsp10 complex and provides mechanistic insight into how it safeguards mitochondrial proteostasis under folding stress.

DOI: https://doi.org/10.1126/sciadv.adw6064