A bacterial genetic selection system for ubiquitylation cascade discovery

Olga Levin-Kravets, Neta Tanner, Noa Shohat, Ilan Attali, Tal Keren-Kaplan, Anna Shusterman, Shay Artzi, Alexander Varvak, Yael Reshef, Xiaojing Shi, Ori Zucker, Tamir Baram, Corine Katina, Inbar Pilzer, Shay Ben-Aroya, Gali Prag*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

About one-third of the eukaryotic proteome undergoes ubiquitylation, but the enzymatic cascades leading to substrate modification are largely unknown. We present a genetic selection tool that utilizes Escherichia coli, which lack deubiquitylases, to identify interactions along ubiquitylation cascades. Coexpression of split antibiotic resistance protein tethered to ubiquitin and ubiquitylation target together with a functional ubiquitylation apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media. We applied the selection system to uncover an E3 ligase from the pathogenic bacteria EHEC and to identify the epsin ENTH domain as an ultraweak ubiquitin-binding domain. The latter was complemented with a structure-function analysis of the ENTH-ubiquitin interface. We also constructed and screened a yeast fusion library, discovering Sem1 as a novel ubiquitylation substrate of Rsp5 E3 ligase. Collectively, our selection system provides a robust high-throughput approach for genetic studies of ubiquitylation cascades and for small-molecule modulator screening.

Original languageEnglish
Pages (from-to)945-952
Number of pages8
JournalNature Methods
Volume13
Issue number11
DOIs
StatePublished - 1 Nov 2016

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