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Related Concept Videos

Protein Complexes with Interchangeable Parts01:57

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Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order...
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Related Experiment Video

Updated: Mar 18, 2026

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions
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A Split-Ubiquitin Based Strategy Selecting for Protein Complex-Interfering Mutations.

Thomas Gronemeyer1, Julian Chollet1, Stefan Werner1

  • 1Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, D89081, Germany.

G3 (Bethesda, Md.)
|July 13, 2016
PubMed
Summary
This summary is machine-generated.

Researchers developed a new method to identify mutations disrupting protein interactions. This technique, using a split-ubiquitin assay, successfully pinpointed a specific mutation affecting the Bem1-Cdc24 interaction in yeast.

Keywords:
mutagenesisprotein structureprotein-protein interactionsplit-ubiquitin

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Yeast Genetics

Background:

  • Understanding protein interaction networks is crucial for deciphering cellular functions.
  • Selective disruption of specific protein-protein interactions is essential for studying network topology and function.
  • Existing methods may lack the precision needed to isolate single-interaction disrupting mutations.

Purpose of the Study:

  • To introduce and validate a novel selection strategy for identifying mutations that impair specific protein-protein binding.
  • To apply this strategy to the interaction between yeast proteins Bem1 and Cdc24.
  • To compare the efficacy of Sanger sequencing and next-generation sequencing (NGS) for analyzing mutation pools.

Main Methods:

  • Development of a selection strategy utilizing a split-ubiquitin based protein interaction assay.
  • Enrichment of mutant alleles that disrupt a specific protein interaction within a yeast system.
  • Application of the assay to the PB domains of Bem1 and Cdc24.
  • Analysis of selected mutations using both Sanger sequencing and next-generation sequencing (NGS).

Main Results:

  • The selection strategy successfully enriched for mutations that specifically impair the binding of Cdc24 to Bem1.
  • A single mutation at position 833 of Cdc24 was consistently identified by both sequencing methods.
  • Biochemical analysis confirmed this mutation disrupts the Bem1-Cdc24 interaction without affecting protein folding.
  • NGS provided a more comprehensive dataset, revealing a greater portion of the interaction interface.

Conclusions:

  • The developed selection strategy is effective for isolating mutations that disrupt specific protein-protein interactions.
  • This method, particularly with NGS, offers a powerful tool for mapping protein interaction interfaces.
  • The findings provide insights into the structural basis of the Bem1-Cdc24 interaction.