Mechanistische Untersuchungen zur Funktion der ribosomen-assoziierten Chaperone RAC und Ssb während der Expression von nonstop- und Polylysin-Proteinen

• Antragstellerin: Professorin Dr. Sabine Karola Rospert
• Fachliche Zuordnung: Biochemie ; Zellbiologie
• Förderung: Förderung von 2013 bis 2021
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 244586127

• Erstellungsjahr: 2022

Zusammenfassung der Projektergebnisse

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.

Projektbezogene Publikationen (Auswahl)

• (2014). Release factor eRF3 mediates premature translation termination on polylysine-stalled ribosomes in Saccharomyces cerevisiae. Mol Cell Biol 34, 4062-4076
  Chiabudini, M., Tais, A., Zhang, Y., Hayashi, S., Wölfle, T., Fitzke, E., and Rospert, S.
  
• (2016). Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and rRNA depends on its lid domain. Nat Commun 7, 1-12
  Gumiero, A., Conz, C., Gesé, G.V., Zhang, Y., Weyer, F.A., Lapouge, K., Kappes, J., von Plehwe, U., Schermann, G., Fitzke, E., Wölfle, T., Fischer, T., Rospert, S., and Sinning, I.
  
• (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, V., Mudholkar, K., Chiabudini, M., Fitzke, E., Wölfle, T., Pfeifer, D., Drepper, F., Warscheid, B., and Rospert, S.
  
• (2017). The Hsp70 homolog Ssb affects ribosome biogenesis via the TORC1-Sch9 signaling pathway. Nat Commun 8, 1-14
  Mudholkar, K., Fitzke, E., Prinz, C., Mayer, M.P., and Rospert, S.
  
• (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, V., Mudholkar, K., and Rospert, S.
  
• (2017). Two chaperones locked in an embrace: Structure and function of the ribosome-associated complex RAC. Nat Struct Mol Biol 24, 611-619
  Zhang, Y., Sinning, I., and Rospert, S.
  
• (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, A.S., Chiabudini, M., Zhang, Y., Qiu, Z., Scazzari, M., Wölfle, T., Wohlwend, D., and Rospert, S.
  
• (2019). Polyamines and eIF5A Hypusination Modulate Mitochondrial Respiration and Macrophage Activation. Cell Metab 30, 352-363 e358
  Puleston, D.J., Buck, M.D., Klein Geltink, R.I., Kyle, R.L., Caputa, G., O'Sullivan, D., Cameron, A.M., Castoldi, A., Musa, Y., Kabat, A.M., Zhang, Y., Flachsmann, L.J., Field, C.S., Patterson, A.E., Scherer, S., Alfei, F., Baixauli, F., Austin, S.K., Kelly, B., Matsushita, M., Curtis, J.D., Grzes, K.M., Villa, M., Corrado, M., Sanin, D.E., Qiu, J., Pallman, N., Paz, K., Maccari, M.E., Blazar, B.R., Mittler, G., Buescher, J.M., Zehn, D., Rospert, S., Pearce, E.J., Balabanov, S., and Pearce, E.L.
  
• (2020). The ribosome-associated complex RAC serves in a relay that directs nascent chains to Ssb. Nat Commun 11, 1504
  Zhang, Y., Valentin Gese, G., Conz, C., Lapouge, K., Kopp, J., Wölfle, T., Rospert, S., and Sinning, I.
  
• (2021). Ribosome-bound Get4/5 facilitates the capture of tail-anchored proteins by Sgt2 in yeast. Nat Commun 12, 782
  Zhang, Y., De Laurentiis, E., Bohnsack, K.E., Wahlig, M., Ranjan, N., Gruseck, S., Hackert, P., Wölfle, T., Rodnina, M.V., Schwappach, B., and Rospert, S.