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Split chloramphenicol acetyl-transferase assay reveals self-ubiquitylation-dependent regulation of UBE3B

Persaud, Avinash, Prag, Gali, Shusterman, Anna, Biswas, Sagnik, Elias, Sivan, Florentin, Amir, Levin-Kravets, Olga, Borisova, Ekaterina, Simpson-Lavy, Kobi, Kupiec, Martin, Kordonsky, Alina, Yariv, Elon, Avishid, Reut, Sror, Mor, Almog, Ofir, Marshanski, Tal, Kadosh, Shira, David, Nichole, Manori, Bar, Fischer, Zohar, Lilly, Jeremiah, Ambrozkiewicz, Mateusz, Tarabykin, Victor, Rotin, Daniela and Thaker, Maulik (2021) Split chloramphenicol acetyl-transferase assay reveals self-ubiquitylation-dependent regulation of UBE3B. Journal of molecular biology, 433 (23). ISSN 00222836


Split reporter protein-based genetic section systems are widely used to identify and
characterize protein-protein interactions (PPI). Assembly of split markers that
antagonize toxins, rather than required for synthesis of missing essential metabolites,
facilitate the seeding of high density of cells and selective growth. Here we present adeveloped 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 of a postulated protein 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 growth on
selective media. The fact that proteins do not have to be purified from bacteria and the
high sensitivity of the split-CAT reporter, enable the detection of challenging protein
cascades and post-translation modifications. 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 demonstrate that the spit-CAT
system provides a readout for the known self-ubiquitylation-dependent inactivation of
NEDD4. Subsequently, we harnessed the system to explore if UBE3B, a HECT ligase
not belonging to the Nedd4 subfamily, is also regulated by self-ubiquitylation. We found
that self-ubiquitylation of UBE3B at residue K665 inactivates the enzyme in the E. coli
system and in mammalian cells due to its oligomerization.

Item Type: Article
Date Deposited: 01 Mar 2022 00:45
Last Modified: 01 Mar 2022 00:45


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