Regulation of the endocytosis and prion-chaperoning machineries by yeast E3 ubiquitin ligase Rsp5 as revealed by orthogonal ubiquitin transfer

Yiyang Wang, Shuai Fang, Geng Chen, Rakhee Ganti, Tatiana A. Chernova, Li Zhou, Duc Duong, Hiroaki Kiyokawa, Ming Li, Bo Zhao*, Natalia Shcherbik, Yury O. Chernoff, Jun Yin

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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 languageEnglish (US)
Pages (from-to)1283-1297.e8
JournalCell Chemical Biology
Volume28
Issue number9
DOIs
StatePublished - Sep 16 2021

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

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