title: A yeast two-hybrid approach for probing protein-protein interactions at the centrosome.
abstract: As a large, nonmembrane bound organelle, the centrosome must rely heavily on protein-protein interactions to assemble itself in the cytoplasm and perform its functions as a microtubule-organizing center. Therefore, to understand how this organelle is built and functions, one must understand the protein-protein interactions made by each centrosome protein. Unfortunately, the highly interconnected nature of the centrosome, combined with its predicted unstructured, coil-rich proteins, has made the use of many standard approaches to studying protein-protein interactions very challenging. The yeast-two hybrid (Y2H) system is well suited for studying the centrosome and is an important complement to other biochemical approaches. In this chapter we describe how to carry out a directed Y2H screen to identify the direct interactions between a given centrosome protein and a library of others. Specifically, we detail using a bioinformatics-based approach (structure prediction programs) to subdivide proteins and screen for interactions using an array-based Y2H approach. We also describe how to use the interaction information garnered from this screen to generate mutations to disrupt specific interactions using mutagenic-PCR and a "reverse" Y2H screen. Finally, we discuss how information from such a screen can be integrated into existing models of centrosome assembly and how it can initiate and guide extensive in vitro and in vivo experimentation to test these models.
authors.affiliation: Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Galletta
authors.affiliation: Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Nasser M
authors.initials: NM
authors.lastname: Rusan
mesh_terms: D018385:Centrosome / Q000502:physiology*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology; D020798:Two-Hybrid System Techniques
chemical_list: D029701:Saccharomyces cerevisiae Proteins
keywords: Centriole; Centrosome; Pericentriolar material; Protein complex; Y2H; Yeast-two hybrid