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.