Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling

Abstract

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

The role of 3’UTR-protein complexes in the regulation of protein multifunctionality and subcellular localization

“…Indeed, a large number of intermediate proteins are kinases that can modify nascent proteins, illustrating the fact that moonlighting functions may be partly commanded by short linear motifs 17,48 . Recently, it has been shown that 4 out of 9 S. cerevisiae protein complexes that form co-translationally 49 , possibly according to a 3'UTR-complex formation model 50 , involve 6 known multifunctional and moonlighting yeast proteins 49 . Interestingly, 2 of those, MetRS and GluRS, possess nuclear and mitochondrial localization signals, which are only revealed when they proteins are not in complex with each other 51 , indicating that their co-translational complex assembly -and possibly 3'UTR-protein complex formation -may regulate their moonlighting function.…”

Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes

“…The fact that we did not find evidence for cotranslational assembly of eIF3c with eIF3a in the eIF3a::80S data ( Figure S13B) suggests a uni-directional co-translational binding of the nascent PCI domain in eIF3a to fully synthesized eIF3c, but not vice versa. This type of uni-directional cotranslational assembly was suggested to prevent increased propensity for aggregation/misfolding of proteins prone to these expression pathologies (Shiber et al, 2018).…”

“…This trimer then co-assembles with eIF3i but only once it is fully synthetized, contrary to eIF3g that can be picked up co-translationally. Targeting the remaining eIF3 subunits is required to examine whether there are alternative orders of co-assembly events; nonetheless, it is clear that eIF3 belongs to the growing class of multiprotein complexes that have been shown to undergo co-translational assembly by a ribosome profiling-based technique called SeRP (Shiber et al, 2018;Shieh et al, 2015).…”

N-terminal protein acetylation by NatB modulates the levels of Nmnats, the NAD+biosynthetic enzymes inSaccharomyces cerevisiae

“…It was shown that unassembled proteins are prone to degradation through exposure of degrons that are normally shielded when in complex [64]. Alternatively, studies in bacteria, yeast and mammalian cells suggest many multi-subunit complexes are built during translation, where proteins undergoing translation already begin interacting with their partners [65][66][67]. Preventing cotranslational binding and folding often results in degradation of the translating partners, suggesting a competition between protein folding and complex formation with degradation.…”