Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B

Olga Levin-Kravets, Alina Kordonsky, Anna Shusterman, Sagnik Biswas, Avinash Persaud, Sivan Elias, Yael Langut, Amir Florentin, Kobi J. Simpson-Lavy, Elon Yariv, Reut Avishid, Mor Sror, Ofir Almog, Tal Marshanski, Shira kadosh, Nicole Ben David, Bar Manori, Zohar Fischer, Jeremiah Lilly, Ekaterina BorisovaMateusz C. Ambrozkiewicz, Victor Tarabykin, Martin Kupiec, Maulik Thaker, Daniela Rotin, Gali Prag

Research output: Contribution to journalArticlepeer-review

Abstract

Split reporter protein-based genetic section systems are widely used to identify and characterize protein–protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.

Original languageEnglish
Article number167276
JournalJournal of Molecular Biology
Volume433
Issue number23
DOIs
StatePublished - 19 Nov 2021

Keywords

  • Kaufman oculocerebrofacial syndrome
  • protein-protein interaction assay
  • ubiquitylation

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