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Multicomponent Yeast Two-Hybrid System: Applications to Study Protein–Protein Interactions in SMC Complexes

  • Jan Josef PalečekEmail author
  • Lucie Vondrová
  • Kateřina Zábrady
  • Jakub Otočka
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2004)

Abstract

Analysis of protein–protein interactions (PPI) is key for the understanding of most protein assemblies including structural maintenance of chromosomes (SMC) complexes. SMC complexes are composed of SMC proteins, kleisin, and kleisin-interacting subunits. These subunits interact in specific ways to constitute and regulate the closed structure of the complexes. Specifically, kleisin molecules bridge the SMC dimers and the kleisin-interacting subunits modulate stability of the bridge. Here we describe a multicomponent version of a yeast two-hybrid (Y2H) method and its application for analysis of the bridging role of the Nse4 kleisin in the SMC5/6 complex. Using this technique, we also show a stabilizing effect of KITE (kleisin-interacting tandem winged-helix element) proteins on SMC5/6.

Key words

Protein–protein interactions Multicomponent yeast two-hybrid (Y2H) system Structural maintenance of chromosomes (SMC) complexes SMC5/6 Non-SMC Element (Nse) subunits Kleisin bridge KITE proteins 

Notes

Acknowledgments

Czech Science Foundation grant GA18-02067S and the Ministry of Education, Youth and Sports of the Czech Republic project CEITEC 2020 (LQ1601) are acknowledged for their financial support. This chapter reflects only the author’s view, and the Research Executive Agency is not responsible for any use that may be made of the information it contains.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Jan Josef Paleček
    • 1
    • 2
    Email author
  • Lucie Vondrová
    • 1
  • Kateřina Zábrady
    • 3
  • Jakub Otočka
    • 1
  1. 1.National Centre for Biomolecular Research, Faculty of ScienceMasaryk UniversityBrnoCzech Republic
  2. 2.Mendel Centre for Plant Genomics and Proteomics, Central European Institute of TechnologyMasaryk UniversityBrnoCzech Republic
  3. 3.Genome Damage and Stability CentreUniversity of SussexBrightonUK

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