Mechanistic investigations on the role of the ribosome-bound chaperones RAC and Ssb during nonstop- and polylysine protein expression

• Applicant: Professorin Dr. Sabine Karola Rospert
• Subject Area: Biochemistry ; Cell Biology
• Term: from 2013 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 244586127

• Final Report Year: 2022

Final Report Abstract

Yeast contains a specialized chaperone system composed of the Hsp70 homolog Ssb and the ribosome associated complex (RAC), which is composed of the non-canonical Hsp70 homolog Ssz1 and the Hsp40 homolog Zuo1. One of the most important functions of the RAC/Ssb system is to assist the folding of emerging polypeptide chains. However, a general characteristic of chaperones is their distinctive multifunctionality. This applies also to the RAC/Ssb system, which is involved in a multitude of processes besides co-translational protein folding. In this work we have investigated the crucial role of RAC/Ssb in maintaining the fidelity of translation termination. Yeast cells lacking the RAC/Ssb system suffer from two distinct defects in translation termination. On the one hand, nascent chains are prematurely released from ribosomes pausing on stalling-prone poly-AAA/G sequences. This translational error produces C-terminally truncated polypeptide chains. On the other hand, stop codon readthrough is significantly enhanced leading to the production of C-terminally extended translation products. In this project we found that stalling on poly-AAA/G was directly affected by RAC/Ssb, indicating that the interaction of RAC/Ssb with the ribosome facilitated translation of a stalling-prone transcript. In contrast, enhanced stop codon readthrough was not solely connected to the absence of RAC/Ssb during translation. Rather, readthrough was due to structurally altered ribosomal particles, pointing at the role of RAC/Ssb during ribosome biogenesis. Our analysis revealed that structural alterations of ribosomes, purified from cells lacking RAC/Ssb, occur in the decoding and peptidyltransferase center and lead to strongly enhanced affinity for the aminoglycoside paromomycin. The combined data support a model in that RAC/Ssb is required for the proper discrimination between stop codons and sense codons and for the biogenesis of fully functional ribosomal particles.

Publications

• (2014). Release factor eRF3 mediates premature translation termination on polylysine-stalled ribosomes in Saccharomyces cerevisiae. Mol Cell Biol 34, 4062-4076
  Chiabudini, Marco; Tais, Arlette; Zhang, Ying; Hayashi, Sachiko; Wölfle, Tina; Fitzke, Edith & Rospert, Sabine
  
• (2016). Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and rRNA depends on its lid domain. Nat Commun 7, 1-12
  Gumiero, Andrea; Conz, Charlotte; Gesé, Genís Valentín; Zhang, Ying; Weyer, Felix Alexander; Lapouge, Karine; Kappes, Julia; von Plehwe, Ulrike; Schermann, Géza; Fitzke, Edith; Wölfle, Tina; Fischer, Tamás; Rospert, Sabine & Sinning, Irmgard
  
• (2016). The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae. Nucleic Acids Res 44, 5629-5645
  Hübscher, Volker; Mudholkar, Kaivalya; Chiabudini, Marco; Fitzke, Edith; Wölfle, Tina; Pfeifer, Dietmar; Drepper, Friedel; Warscheid, Bettina & Rospert, Sabine
  
• (2017). The yeast Hsp70 homolog Ssb: a chaperone for general de novo protein folding and a nanny for specific intrinsically disordered protein domains. Curr Genet 63, 9-13
  Hübscher, Volker; Mudholkar, Kaivalya & Rospert, Sabine
  
• (2017). The Hsp70 homolog Ssb affects ribosome biogenesis via the TORC1-Sch9 signaling pathway. Nat Commun 8, 1-14
  Mudholkar, Kaivalya; Fitzke, Edith; Prinz, Claudia; Mayer, Matthias P. & Rospert, Sabine
  
• (2017). Two chaperones locked in an embrace: Structure and function of the ribosome-associated complex RAC. Nat Struct Mol Biol 24, 611-619
  Zhang, Ying; Sinning, Irmgard & Rospert, Sabine
  
• (2019). A dual role of the ribosome-bound chaperones RAC/Ssb in maintaining the fidelity of translation termination. Nucleic Acids Res 7018-7034
  Gribling-Burrer, Anne-Sophie; Chiabudini, Marco; Zhang, Ying; Qiu, Zonghao; Scazzari, Mario; Wölfle, Tina; Wohlwend, Daniel & Rospert, Sabine
  
• (2019). Polyamines and eIF5A Hypusination Modulate Mitochondrial Respiration and Macrophage Activation. Cell Metab 30, 352-363 e358
  Puleston, Daniel J.; Buck, Michael D.; Klein Geltink, Ramon I.; Kyle, Ryan L.; Caputa, George; O’Sullivan, David; Cameron, Alanna M.; Castoldi, Angela; Musa, Yaarub; Kabat, Agnieszka M.; Zhang, Ying; Flachsmann, Lea J.; Field, Cameron S.; Patterson, Annette E.; Scherer, Stefanie; Alfei, Francesca; Baixauli, Francesc; Austin, S. Kyle; Kelly, Beth; ... & Pearce, Erika L.
  
• (2020). The ribosome-associated complex RAC serves in a relay that directs nascent chains to Ssb. Nat Commun 11, 1504
  Zhang, Ying; Valentín Gesé, Genís; Conz, Charlotte; Lapouge, Karine; Kopp, Jürgen; Wölfle, Tina; Rospert, Sabine & Sinning, Irmgard
  
• (2021). Ribosome-bound Get4/5 facilitates the capture of tail-anchored proteins by Sgt2 in yeast. Nat Commun 12, 782
  Zhang, Ying; De Laurentiis, Evelina; Bohnsack, Katherine E.; Wahlig, Mascha; Ranjan, Namit; Gruseck, Simon; Hackert, Philipp; Wölfle, Tina; Rodnina, Marina V.; Schwappach, Blanche & Rospert, Sabine