Periodic Reporting for period 4 - TransFold (Molecular Biology of Nascent Chains: Co-translational folding and assembly of proteins in eukaryotes)
Okres sprawozdawczy: 2022-04-01 do 2023-03-31
The faithful production of functional proteins is critical for the integrity of all cells. Translation of genetic information on mRNAs to synthesize linear polypeptides is executed by ribosomes. In addition to their synthesis activity, ribosomes also act as integration hubs of different protein maturation processes, including protein folding and assembly supported by chaperones, amino-terminal processing by enzymes and protein translocation across or insertion into membranes supported by targeting factors.
Overall objectives of the project was to explore basic features and prevalence of co-translational protein assembly, how chaperones guide co-translational protein folding to affect assembly, whether translation of subunit-encoding mRNAs is spatially organized, and if so, how this occurs, and to what extent translation speed variations affect assembly. Studying the physical coupling of translation, folding and maturation will significantly advance our understanding of functional protein synthesis and provide a deeper understanding of the highly intricate organization of eukaryotic cells.
Overall objectives of the project was to explore basic features and prevalence of co-translational protein assembly, how chaperones guide co-translational protein folding to affect assembly, whether translation of subunit-encoding mRNAs is spatially organized, and if so, how this occurs, and to what extent translation speed variations affect assembly. Studying the physical coupling of translation, folding and maturation will significantly advance our understanding of functional protein synthesis and provide a deeper understanding of the highly intricate organization of eukaryotic cells.
We have completed a study on the mechanism of the ribosome-associated yeast chaperone Ssb, which was published during the early phase of the grant period (Döring et al., 2017, Cell). A second study verified the key hypothesis of the ERC proposal, that the co-translational assembly of hetero- oligomeric protein complexes is a widespread phenomenon in yeast cells (Shiber et al., 2018, Nature). The assembly pathway studied in this project is now termed co-post assembly, as it involves a subunit that is still being synthesized (co-translational) and a second one that is already completed (post-translational). A comprehensive review on co-translational folding and assembly of proteins was published (Kramer et al., 2019, Annual Review of Biochemistry). We have then extended our studies towards the analysis of a second translation-coupled assembly pathway that involves two nascent proteins, termed co-co assembly (Bertolini et al., Science, 2021). Using a newly developed method termed disome-selective profiling (DiSP), we identified hundreds of human co-co assembling protein complexes, among them many coiled-coil proteins (e.g. nuclear lamins), as well as proteins that interact via BTB, BAR, SCAN or RHD domains. Co-co assembly is coordinated with nascent chain engagements by molecular chaperones and maturation factors and in many cases additionally coincides with transient changes of translation speed.
Studying the role of molecular chaperones, we have discovered a so far unappreciated mechanism of chaperone-substrate interaction at the ribosome, that is driven by emerging cysteine residues, suggesting a role of chaperones in controlling the redox state of nascent proteins. Furthermore, we identified a group of nascent yeast proteins that are co-translationally engaged by Hsp70 chaperones (Ssa) and Hsp90 chaperones (Hsp82, Hsc82). Hsp70s and Hsp90s act synergistically at specific periods of translation and engage a specific subset of nascent proteins, among them many protein kinases implicated in signal recognition processes and -propeller proteins. In addition, we have determined the nascent chain interactome of yeast NAC and established that NAC function critically depends on the co-translational action of the chaperone triad consisting of Zuo1, Ssz1 and Ssb. Finally, we discovered that NAC binding is coordinated with the onset of co-co assembly, implying a functional integration of both processes. These studies are close to completion and results will be published in the near future. Some of the unpublished data were already presented at international conferences and received major attention in the scientific community.