A Yeast Two-Hybrid Approach for Probing Cytoskeletal Protein Interactions

Interactions among proteins are fundamental to biological processes. In recent years, molecular biological tools have made it possible to study not only interactions among native proteins, but also among recombinant proteins and protein domains. Such studies often yield considerable insight into the mechanisms by which the biological processes take place. In this chapter, we describe our experience with the yeast two-hybrid system, an approach that has significantly enhanced our laboratory’ efforts to analyze protein-protein interactions that underpin the assembly of cytoskeletal structures. It is not our intention to present an extensive review on the principles and practice of the yeast two-hybrid system. Outstanding reviews already exist, e.g., (1 ). An excellent chapter on the practical aspects of the method recently appeared in Volume 88 of Methods in Molecular Biology (2 ). We strongly encourage the reader to take advantage of this resource. This document provides an excellent primer to the genetics behind the two-hybrid system and detailed information on yeast strains, vectors and other reagents. It is particularly helpful for investigators whose goal is to identify unknown interacting partners of a protein of interest by screening a two-hybrid library. Many vendors of two-hybrid system reagents also provide extensive manuals, both in printed format and on-line at their World Wide Web sites. For example, Clontech, Inc. has separate manuals for all of the two-hybrid systems for which they market kits (http://www.clontech.com/clontech/manuals/ ).