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Stoichiometric Production of Protein-Complex Components in Eukaryotes Without Widespread Feedback Regulation

ABSTRACT: Constituents of multi-protein complexes are required at well-defined levels relative to each other. However, it remains unknown whether eukaryotic cells typically produce precise amounts of subunits, or instead rely on degradation to mitigate imprecise production. Here we quantified the production rates of multi-protein complexes in single- and multi-cellular eukaryotes using ribosome profiling. By resolving read-mapping ambiguities present in a large fraction of ribosome footprints which often distorts quantitation accuracy in eukaryotes, we found that obligate components of multi-protein complexes are produced in proportion to their stoichiometry, indicating that their abundances are already precisely tuned at the synthesis level. By systematically interrogating the impact of gene dosage variations in budding yeast, we found a general lack of negative feedback regulation protecting the normally precise rates of subunit synthesis. These results reveal a core principle of proteome homeostasis and highlight the evolution towards quantitative control at every stage of the central dogma.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE118676 | GEO | 2018/12/03

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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Stoichiometric Production of Protein-Complex Components in Eukaryotes Without Widespread Feedback Regulation

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