Periodic Reporting for period 1 - srpabsotcpfaaieps (Selective ribosome profiling and biochemistry studies on the co-translational protein folding and assembly in eukaryotic protein synthesis)
Berichtszeitraum: 2018-05-01 bis 2020-04-30
The objective of this project was to dissect the chaperone network which assists in the folding of newly synthesized proteins during ongoing translation in cells of the model organism S. cerevisiae, and to correlate the action of this network with co-translational protein assembly. In particular, this project used a high throughput technology, termed ribosome profiling, to determine the interaction profiles of the major chaperones of yeast with the entire nascent proteome at all stages of translation. This knowledge was then used to correlate chaperone action with the process of subunit association driving co-translational assembly. The focus was on the chaperones of the yeast cytosol, Ssa1 and Ssb1, two homologs of the Hsp70 chaperone family, with their co-chaperones RAC, Ydj1 and Sis1, as well as on the yeast Hsp90, Hsc82. The results allowed us to reveal principles of functional networking of the chaperone machineries that promote the formation of correctly folded proteins. Intriguingly, the association of chaperones is tuned to the onset of co-translational assembly, suggesting a high degree of functional coordination.
This fundamental research broadens our view on cellular organization and protein homeostasis. It establishes the basis for a rational understanding of protein biogenesis, thereby providing new opportunities for wider applications of the results for research, such as cell biology and synthetic biology, for understanding of protein folding diseases and for industry.
In a second step, we compared the binding profiles of Ssa1, its cochaperones, and Hsc82 with the nascent chain binding profiles of Ssb1 and RAC (additional data from our lab). This allowed us to get a uniquely comprehensive dataset on the co-translational action of the chaperone network acting in yeast. Furthermore, this dataset allows to correlate the chaperone binding profiles with the onsets of co-translational protein assembly.
The data show that upon emergence at the tunnel exit, nascent chains typically interact with Ssb (and RAC) first, before Ssa1 and Hsc82 associate at later stages of translation. The Ydj1 and Sis1 profiles closely resemble those of Ssa1, indicating that they cooperate closely with Ssa1. The binding patterns of Ssa1 and Hsc82 chaperones are similar, consistent with functional cooperation. For all chaperones we detected binding/release cycles during translation that typically encompass 10-30 codons, suggesting residence times of a few seconds per event. However, for some nascent chain species, Hsc82 stays bound until the end of translation, perhaps for continued chaperoning of post-translational folding steps. An intriguing finding was that chaperone action and co-translational subunit joining are timely coordinated such that at the onset of subunit association, chaperones are not bound to the nascent chains.
The data furthermore allowed to correlate nascent chain folding states with chaperone association. Binding occurs at nascent chain lengths at which tertiary structures are already formed co-translationally. Further investigation of selected proteins revealed that Hsc82 and Ssa1 binding typically occurs while folding domains are formed in the nascent chains, and they dissociate upon ribosome exposure of critical structural elements that stabilize these domains.
The results are prepared for publication and were disseminated at international conferences as follows:
◦ Invited talk at St Jude Children´s Research Hospital, Memphis USA, March 10, 2023.
◦ Invited talk at National Institute of Health (NIH), Bethesda, USA, March 20, 2023.
◦ Invited talk at National Cancer Institute (NIC), Frederick, USA, March 21, 2023.
◦ Invited Talk at John Hopkins University, Baltimore, USA, March 23, 2023.
◦ Invited talk at the Annual Meeting of the American Chemical Society, March 26-30, 2023. Invited talk at the ERC Synergy Grant “CoTransFold”Annual Retreat at the ETH Zurich, April 18-20, 2023.
◦ Keynote talk at the Symposium “Stress and Resilience of Biological Systems” in Ulm, Germany, May 3, 2023.
◦ Keynote talk at the EMBO workshop “Protein quality control” in Dubrovnik, Croatia, May 21-26, 2023.
◦ Invited talk at the EMBO/FEBS “Protein Folding, Aggregation and Compartmentalization”, Spetses, Greece, Sept. 1-8, 2023.
◦ Invited talk at the FEBS Advanced Course “Susan Lindquist School of Proteostasis”, Ingelheim, Germany, Oct. 3-6, 2023.
◦ Invited talk at the IMB-Symposium at the Institute for Molecular Biology, Academia Sinica, Taiwan, Nov 1, 2023.
◦ The work will be presented at future conferences and job interviews.