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Principles of cotranslational mitochondrial protein import revealed by selective ribosome profiling

ABSTRACT: The biogenesis of nearly all mitochondrial proteins begins with translation on cytosolic ribosomes. How these proteins are subsequently delivered to mitochondria remains poorly understood. Here, we systematically investigated the coupling of mitochondrial protein translation and import using selective ribosome profiling in human cells. Cotranslational targeting requires an N-terminal presequence on the nascent protein and contributes to mRNA localization at the mitochondrial surface. This pathway is predominantly used by large, multidomain and topologically complex proteins, whose import efficiency is enhanced when targeted cotranslationally. In contrast to protein targeting to the endoplasmic reticulum (ER), cotranslational mitochondrial import does not favor membrane proteins and initiates late during translation, specifically upon the exposure of a complex globular fold in the nascent protein. Our findings reveal a multi-layered protein sorting system that recognizes both the targeting signal and protein folding status during translation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE255657 | GEO | 2025/08/01

REPOSITORIES: GEO

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Project description:Folding of newly synthesized proteins to the native state is a major challenge within the crowded cellular environment, since non-productive interactions can lead to misfolding, aggregation and degradation1. Cells cope with this challenge by coupling synthesis with polypeptide folding and by employing molecular chaperones to safeguard folding already cotranslationally2. However, little is known about the final step of folding, the assembly of polypeptides into complexes, although most of the cellular proteome forms oligomeric assemblies3. In prokaryotes, a proof-of-concept study showed that assembly of heterodimeric luciferase is an organized cotranslational process, facilitated by spatially confined translation of the subunits encoded on a polycistronic mRNA4. In eukaryotes, however, fundamental differences such as rarity of polycistronic mRNAs and different chaperone constellations raise the question whether assembly is also coordinated with translation. Here we provide a systematic and mechanistic analysis of protein complex assembly in eukaryotes using ribosome profiling. We determined the in vivo nascent subunits interactions of 12 hetero-oligomeric protein complexes of Saccharomyces cerevisiae at near-residue resolution. We find 9 complexes assemble cotranslationally; the 3 complexes that do not show cotranslational interactions are regulated by dedicated assembly chaperones5-7. Cotranslational assembly often occurs uni-directionally, with one fully synthesized subunit engaging its nascent partner subunit(s), thereby counteracting its aggregation propensity. The onset of cotranslational subunit association coincides sharply with full exposure of the nascent interaction domain at the ribosomal tunnel exit. The action of the ribosome-associated Hsp70 chaperone Ssb8 is coordinated with assembly. Ssb transiently engages partially synthesized interaction domains, then dissociates before the onset of partner subunit association, presumably to prevent premature assembly interactions. Our study shows that cotranslational subunit association is a prevalent mechanism for hetero-oligomers assembly in yeast and indicates that translation, folding and assembly of protein complexes are integrated processes in eukaryotes.

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Principles of cotranslational mitochondrial protein import revealed by selective ribosome profiling

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