Multicomponent Yeast Two-Hybrid System: Applications to Study Protein–Protein Interactions in SMC Complexes
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 wordsProtein–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
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.
- 10.Hazbun TR, Malmstrom L, Anderson S, Graczyk BJ, Fox B, Riffle M, Sundin BA, Aranda JD, McDonald WH, Chiu CH, Snydsman BE, Bradley P, Muller EG, Fields S, Baker D, Yates JR III, Davis TN (2003) Assigning function to yeast proteins by integration of technologies. Mol Cell 12:1353–1365CrossRefGoogle Scholar
- 13.Hudson JJR, Bednarova K, Kozakova L, Liao C, Guerineau M, Colnaghi R, Vidot S, Marek J, Bathula SR, Lehmann AR, Palecek J (2011) Interactions between the Nse3 and Nse4 components of the SMC5-6 complex identify evolutionarily conserved interactions between MAGE and EID families. PLoS One 6:e17270CrossRefGoogle Scholar
- 15.van der Crabben SN, Hennus MP, McGregor GA, Ritter DI, Nagamani SCS, Wells OS, Harakalova M, Chinn IK, Alt A, Vondrova L, Hochstenbach R, van Montfrans JM, Terheggen-Lagro SW, van Lieshout S, van Roosmalen MJ, Renkens I, Duran K, Nijman IJ, Kloosterman WP, Hennekam E, Orange JS, van Hasselt PM, Wheeler DA, Palecek JJ, Lehmann AR, Oliver AW, Pearl LH, Plon SE, Murray JM, van Haaften G (2016) Destabilized SMC5/6 complex leads to chromosome breakage syndrome with severe lung disease. J Clin Investig 126:2881–2892CrossRefGoogle Scholar
- 16.Kozakova L, Vondrova L, Stejskal K, Charalabous P, Kolesar P, Lehmann AR, Uldrijan S, Sanderson CM, Zdrahal Z, Palecek JJ (2015) The melanoma-associated antigen 1 (MAGEA1) protein stimulates the E3 ubiquitin-ligase activity of TRIM31 within a TRIM31-MAGEA1-NSE4 complex. Cell Cycle 14:920–930CrossRefGoogle Scholar
- 20.Vondrova L, Nociar M, Adamus M, and Palecek, JJ (2018) Kite proteins regulate Nse4 bridge in SMC5/6 complex. Manuscript in preparationGoogle Scholar