Molecular dissection of yeast spindle pole bodies by two hybrid, in vitro binding, and co-purification

Abstract

Publisher Summary This chapter focuses on three approaches that proved to be powerful tools in the molecular dissection of the budding yeast spindle pole bodies (SPB): (1) the yeast two hybrid system, (2) immuno- and affinity precipitation of SPB subcomplexes, and (3) in vitro binding of SPB components. The yeast two-hybrid system is based on the observation that the transcriptional activator Gal4p is composed of two distinct domains: a DNA-binding, Gal4pDNA, and an acidic transactivation domain, Gal4pTA. A functional transcription factor is restored when the separated Gal4p domains are joined together by interacting proteins: one of them fused to Gal4pDNA and the other to Gal4pTA. The reconstituted Gal4p activates reporter genes under the control of a GALUAS. Immuno- and affinity precipitation have been used in the field of SPB research to identify new interactors of known SPB components and to confirm interactions found by the yeast two-hybrid system. The principal of immunoprecipitation is to enrich proteins via an antibody antigen binding reaction. In contrast to the two-hybrid approach and coimmunoprecipitation, in vitro binding of SPB proteins method demonstrates that two proteins interact directly with each other.