Dissecting the Contingent Interactions of Protein Complexes with the Optimized Yeast Cytosine Deaminase Protein-Fragment Complementation Assay

Abstract

Here, we present a detailed protocol for studying in yeast cells the contingent interaction between a substrate and its multisubunit enzyme complex by using a death selection technique known as the optimized yeast cytosine deaminase protein-fragment complementation assay (OyCD PCA). In yeast, the enzyme cytosine deaminase (encoded by FCY1) is involved in pyrimidine metabolism. The PCA is based on an engineered form of yeast cytosine deaminase optimized by directed evolution for maximum activity (OyCD), which acts as a reporter converting the pro-drug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a toxic compound that kills the cell. Cells that have OyCD PCA activity convert 5-FC to 5-FU and die. Using this assay, it is possible to assess how regulatory subunits of an enzyme contribute to the overall interaction between the catalytic subunit and the potential substrates. Furthermore, OyCD PCA can be used to dissect different functions of mutant forms of a protein as a mutant can disrupt interaction with one partner, while retaining interaction with others. As it is scalable to a medium- or high-throughput format, OyCD PCA can be used to study hundreds to thousands of pairwise protein-protein interactions in different deletion strains. In addition, OyCD PCA vectors (pAG413GAL1-ccdB-OyCD-F[1] and pAG415GAL1-ccdB-OyCD-F[2]) have been designed to be compatible with the proprietary Gateway technology. It is therefore easy to generate fusion genes with the OyCD reporter fragments. As an example, we will focus on the yeast cyclin-dependent protein kinase 1 (Cdk1, encoded by CDC28), its regulatory cyclin subunits, and its substrates or binding partners.