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

Methods Mol Biol. 2019:2004:79-90. doi: 10.1007/978-1-4939-9520-2_7.

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.

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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / metabolism
  • Chromosomes, Fungal / physiology
  • Multiprotein Complexes / metabolism*
  • Protein Interaction Maps / physiology*
  • Protein Subunits / metabolism
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / metabolism
  • Two-Hybrid System Techniques

Substances

  • Cell Cycle Proteins
  • Multiprotein Complexes
  • Protein Subunits
  • Saccharomyces cerevisiae Proteins