Yeast two-hybrid screens to identify Drosophila PP1-binding proteins

Protein phosphatase type 1 (PP1) is one of the major classes of serine/threonine protein phosphatases and has been found in all eukaryotic cells examined to date. Metazoans from Drosophila to humans have multiple genes encoding catalytic subunits of PP1 (PP1c), which are involved in a wide range of biological processes. Studies in the fruit fly Drosophila melanogaster have revealed some of the essential functions of the PP1c genes. However, the PP1c isoforms have pleiotropic and overlapping functions, making it difficult to characterize their many biological roles and identify their specific in vivo substrates. Regulatory subunits of PP1 provide the key to understanding the role of PP1, as they are responsible for directing PP1c to different intracellular locations and/or affecting their activity or substrate specificity. The existence of isoform-specific PP1 regulatory subunits might also help to explain the unique roles of different PP1 catalytic subunits. Drosophila is an excellent model organism in which to characterize the role of PP1 catalytic and regulatory subunits, because it combines molecular and biochemical approaches with powerful genetics, in a well-characterized animal model. In this chapter, we will describe how the two-hybrid system can be used to identify Drosophila PP1c-interacting proteins and study their interactions with different PP1c isoforms and variants. With the appropriate bait and library constructs, this method should also be equally applicable to identifying binding subunits of related phosphatases or PP1c from other organisms.