bims-tricox 2025-02-09 papers

Issue of 2025–02–09
three papers selected by
Yash Verma, University of Zurich

STAR Protoc. 2025 Jan 31. pii: S2666-1667(25)00023-1. [Epub ahead of print]6(1): 103617

Protocol for assessing the clogging of the mitochondrial translocase of the outer membrane by precursor proteins in human cells.

John Kim, Hilla Weidberg.

Protein import into the mitochondria is required for organellar function. Inefficient import can result in the stalling of mitochondrial precursors inside the translocase of the outer membrane (TOM) and blockage of the mitochondrial entry gate. Here, we present a protocol to assess the clogging of TOM by mitochondrial precursors in human cell lines. We describe how the localization of mitochondrial precursors can be determined by cellular fractionation. We then show how co-immunoprecipitation can be used to test the stalling of precursors inside TOM. For complete details on the use and execution of this protocol, please refer to Kim et al.1.

Keywords: cell biology; cell culture; cell separation/fractionation; molecular biology; protein biochemistry; protein expression and purification

DOI: https://doi.org/10.1016/j.xpro.2025.103617

bioRxiv. 2025 Jan 26. pii: 2025.01.26.634913. [Epub ahead of print]

Translational activators align mRNAs at the small mitoribosomal subunit for translation initiation.

Joseph B Bridgers, Andreas Carlström, Dawafuti Sherpa, Mary T Couvillion, Urška Rovšnik, Jingjing Gao, Bowen Wan, Sichen Shao, Martin Ott, L Stirling Churchman.

Mitochondrial gene expression is essential for oxidative phosphorylation. Mitochondrial-encoded mRNAs are translated by dedicated mitochondrial ribosomes (mitoribosomes), whose regulation remains elusive. In the baker's yeast Saccharomyces cerevisiae , nuclear-encoded mitochondrial translational activators (TAs) facilitate transcript-specific translation by a yet unknown mechanism. Here, we investigated the function of TAs containing RNA-binding pentatricopeptide repeats (PPRs) using selective mitoribosome profiling and cryo-EM structural analysis. These analyses revealed that TAs exhibit strong selectivity for mitoribosomes initiating on their target transcripts. Moreover, TA-mitoribosome footprints indicated that TAs recruit mitoribosomes proximal to the start codon. Two cryo-EM structures of mRNA-TA complexes bound to post-initiation/pre-elongation-stalled mitoribosomes revealed the general mechanism of TA action. Specifically, the TAs bind to structural elements in the 5' UTR of the client mRNA as well as to the mRNA channel exit to align the mRNA in the small subunit during initiation. Our findings provide a mechanistic basis for understanding how mitochondria achieve transcript-specific translation initiation without relying on general sequence elements to position mitoribosomes at start codons.

DOI: https://doi.org/10.1101/2025.01.26.634913

Nat Cell Biol. 2025 Jan 31.

An avoidance segment resolves a lethal nuclear-mitochondrial targeting conflict during ribosome assembly.

Michaela Oborská-Oplová, Alexander Gregor Geiger, Erich Michel, Purnima Klingauf-Nerurkar, Sven Dennerlein, Yury S Bykov, Simona Amodeo, André Schneider, Maya Schuldiner, Peter Rehling, Vikram Govind Panse.

The correct sorting of nascent ribosomal proteins from the cytoplasm to the nucleus or to mitochondria for ribosome production poses a logistical challenge for cellular targeting pathways. Here we report the discovery of a conserved mitochondrial avoidance segment (MAS) within the cytosolic ribosomal protein uS5 that resolves an evolutionary lethal conflict between the nuclear and mitochondrial targeting machinery. MAS removal mistargets uS5 to the mitochondrial matrix and disrupts the assembly of the cytosolic ribosome. The resulting lethality can be rescued by impairing mitochondrial import. We show that MAS triages nuclear targeting by disabling a cryptic mitochondrial targeting activity within uS5 and thereby prevents fatal capture by mitochondria. Our findings identify MAS as an essential acquisition by the primordial eukaryote that reinforced organelle targeting fidelity while developing an endosymbiotic relationship with its mitochondrial progenitor.

DOI: https://doi.org/10.1038/s41556-024-01588-4