Abstract
Attachment of the ubiquitin (UB) peptide to proteins via the E1-E2-E3 enzymatic machinery regulates diverse biological pathways, yet identification of the substrates of E3 UB ligases remains a challenge. We overcame this challenge by constructing an “orthogonal UB transfer” (OUT) cascade with yeast E3 Rsp5 to enable the exclusive delivery of an engineered UB (xUB) to Rsp5 and its substrate proteins. The OUT screen uncovered new Rsp5 substrates in yeast, such as Pal1 and Pal2, which are partners of endocytic protein Ede1, and chaperones Hsp70-Ssb, Hsp82, and Hsp104 that counteract protein misfolding and control self-perpetuating amyloid aggregates (prions), resembling those involved in human amyloid diseases. We showed that prion formation and effect of Hsp104 on prion propagation are modulated by Rsp5. Overall, our work demonstrates the capacity of OUT to deconvolute the complex E3-substrate relationships in crucial biological processes such as endocytosis and protein assembly disorders through protein ubiquitination.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1283-1297.e8 |
| Journal | Cell Chemical Biology |
| Volume | 28 |
| Issue number | 9 |
| DOIs | |
| State | Published - Sep 16 2021 |
Funding
This work was supported by grants from the National Institutes of Health (NIH) of the United States (R01GM104498 to J.Y. and H.K. R01GM114308 to N.S. and R01GM133873 to M.L.) and the National Science Foundation of the United States (NSF) (1817976 to Y. O. C. and 1710460 to J.Y.). B.Z. was supported by grants from the National Natural Science Foundation of China, China (31770921 and 31971187 to B.Z.). We thank Lu Zhu and Scott D. Emr (Cornell University), David J. Katzmann (Mayo Clinic College of Medicine), Rosine Haguenauer-Tsapis (Institut Jacques Monod, France) and Pierre Morsomme (Louvain Institute of Biomolecular Science and Technology, Belgium) for kindly sharing materials and helpful discussions. We also thank Milo Fasken and Anita Corbett (Emory University) for technical advice. B.Z. N.S. Y.O.C. and J.Y. conceived the overall idea and designed the experiments. Y.W. S.F. G.C. R.G. L.Z. D.D. and N.S. performed experiments. T.A.C. M.L. and H.K. provided technical guidance and/or intellectual input. Y.W. S.F. G.C. R.G. B.Z. N.S. Y.O.C. and J.Y. analyzed data, interpreted results, drafted and edited the manuscript. B.Z. N.S. Y.O.C. and J.Y. acquired funding. All authors reviewed and approved the final version of the manuscript. The authors declare no competing interests. This work was supported by grants from the National Institutes of Health (NIH) of the United States ( R01GM104498 to J.Y. and H.K., R01GM114308 to N.S., and R01GM133873 to M.L.) and the National Science Foundation of the United States (NSF) ( 1817976 to Y. O. C. and 1710460 to J.Y.). B.Z. was supported by grants from the National Natural Science Foundation of China , China ( 31770921 and 31971187 to B.Z.). We thank Lu Zhu and Scott D. Emr (Cornell University), David J. Katzmann (Mayo Clinic College of Medicine), Rosine Haguenauer-Tsapis (Institut Jacques Monod, France) and Pierre Morsomme (Louvain Institute of Biomolecular Science and Technology, Belgium) for kindly sharing materials and helpful discussions. We also thank Milo Fasken and Anita Corbett (Emory University) for technical advice.
Keywords
- Hsp104
- Ssb
- Sup35
- amyloid
- chaperone
- endocytosis
- protein engineering
- ubiquitin ligase
- yeast cell surface display
- yeast prion
ASJC Scopus subject areas
- Biochemistry
- Molecular Medicine
- Molecular Biology
- Pharmacology
- Drug Discovery
- Clinical Biochemistry
