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Related Experiment Video

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Deciphering Protein Interaction Dynamics Using the Yeast Three-Hybrid System.

Qianwei Liu1, Jathish Ponnu2

  • 1Joseph Gottlieb Kölreuter Institute for Plant Sciences (JKIP), Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|April 1, 2026
PubMed
Summary

The yeast three-hybrid (Y3H) system was used to study ternary protein interactions. Researchers found that SUPPRESSOR OF PHYA-105 1 (SPA1) enhances the interaction between CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and CRYPTOCHROME 1 (CRY1).

Keywords:
COP1CRY1LiAcPEGPNPGQuantitative assaySPA1TernaryY2HY3HYeast three-hybridpACTpBridgeα-galα-galactosidase

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Area of Science:

  • Molecular Biology
  • Plant Biology
  • Biochemistry

Background:

  • Proteins function in complexes, and understanding ternary interactions is crucial for biological processes.
  • The yeast three-hybrid (Y3H) system allows for the study of three-protein interactions ex planta.
  • CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and CRYPTOCHROME 1 (CRY1) are key regulators in plant photomorphogenesis, with SUPPRESSOR OF PHYA-105 1 (SPA1) mediating their interaction.

Purpose of the Study:

  • To present a practical yeast three-hybrid (Y3H) protocol for dissecting ternary protein interactions.
  • To investigate the role of SPA1 in mediating the interaction between COP1 and CRY1 in Arabidopsis thaliana.

Main Methods:

  • Utilized the yeast three-hybrid (Y3H) system with pBridge and pACT2 AD vectors in Saccharomyces cerevisiae Y2HGold strain.
  • Co-expressed COP1, CRY1, and SPA1 to assess ternary interactions.
  • Quantified interactions via MEL1 reporter gene activation using p-nitrophenyl alpha-D-galactopyranoside (PNPG) assay.

Main Results:

  • The Y3H system successfully detected ternary interactions.
  • Co-expression of SPA1 significantly enhanced the interaction between COP1 and CRY1.
  • MEL1 reporter gene activation confirmed the enhanced COP1-CRY1 interaction in the presence of SPA1.

Conclusions:

  • The developed Y3H protocol provides a robust framework for studying ternary protein interactions.
  • SPA1 plays a significant role in mediating and enhancing the interaction between COP1 and CRY1.
  • This finding contributes to understanding the molecular mechanisms of plant photomorphogenesis.