Split-Chloramphenicol Acetyl Transferase Assay to Study Protein-Protein Interactions and Ubiquitylation in Escherichia coli

Florentin, Amir; Kordonsky, Alina; Yariv, Elon; Avishid, Reut; Efron, Noa; Akogwu, Edache; Prag, Gali

doi:10.21769/bioprotoc.4497

Search citation statements

Order By: Relevance

Paper Sections

Select...

Citation Types

Supporting

Mentioning

Contrasting

Year Published

2023

2024

Publication Types

Select...

Book1

Article1

Relationship

Self Cite0

Independent2

Authors

Journals

Cited by 2 publications

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

Protein–Protein Interactions: Bimolecular Fluorescence Complementation and Cytology Two Hybrid

Purkait,

Ilyas,

Atmakuri

2023

Methods in Molecular Biology

View full text Add to dashboard Cite

Protein–Protein Interactions: Bimolecular Fluorescence Complementation and Cytology Two Hybrid

Purkait,

Ilyas,

Atmakuri

2023

Methods in Molecular Biology

View full text Add to dashboard Cite

Proximal Co-Translation Facilitates Detection of Weak Protein-Protein Interactions

Kordonsky,

Gabay,

Rosinoff

et al. 2024

IJMS

View full text Add to dashboard Cite

Ubiquitin (Ub) signals are recognized and decoded into cellular responses by Ub-receptors, proteins that tether the Ub-binding domain(s) (UBDs) with response elements. Typically, UBDs bind mono-Ub in highly dynamic and weak affinity manners, presenting challenges in identifying and characterizing their binding interfaces. Here, we report the development of a new approach to facilitate the detection of these weak interactions using split-reporter systems where two interacting proteins are proximally co-translated from a single mRNA. This proximity significantly enhances the readout signals of weak protein–protein interactions (PPIs). We harnessed this system to characterize the ultra-weak UBD and ENTH (Epsin N-terminal Homology) and discovered that the yeast Ent1-ENTH domain contains two Ub-binding patches. One is similar to a previously characterized patch on STAM1(signal-transducing adaptor molecule)-VHS (Vps27, Hrs, and STAM), and the other was predicted by AlphaFold. Using a split-CAT selection system that co-translates Ub and ENTH in combination with mutagenesis, we assessed and confirmed the existence of a novel binding patch around residue F53 on ENTH. Co-translation in the split-CAT system provides an effective tool for studying weak PPIs and offers new insights into Ub-receptor interactions.

show abstract

Split-Chloramphenicol Acetyl Transferase Assay to Study Protein-Protein Interactions and Ubiquitylation in Escherichia coli

Cited by 2 publications

References 23 publications

Protein–Protein Interactions: Bimolecular Fluorescence Complementation and Cytology Two Hybrid

Protein–Protein Interactions: Bimolecular Fluorescence Complementation and Cytology Two Hybrid

Proximal Co-Translation Facilitates Detection of Weak Protein-Protein Interactions

Contact Info

Product

Resources

About