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Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells
Published on: March 3, 2015
Dissecting the Contingent Interactions of Protein Complexes with the Optimized Yeast Cytosine Deaminase
Po Hien Ear1,2, Jacqueline Kowarzyk1, Stephen W Michnick1
1Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
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.
Insights
This study introduces the optimized yeast cytosine deaminase protein-fragment complementation assay (OyCD PCA) for studying protein interactions in yeast. This method uses a cell-death reporter to analyze enzyme-substrate and protein-protein interactions, enabling high-throughput screening.
Area of Science:
- Molecular Biology
- Biochemistry
- Yeast Genetics
Background:
- Protein-protein interactions are crucial for cellular functions, including enzyme activity and regulation.
- Studying these interactions in vivo, especially in complex systems like multisubunit enzymes, presents significant challenges.
- Existing methods may lack the throughput or specificity required for comprehensive analysis of interaction dynamics.
Purpose of the Study:
- To present a detailed protocol for the optimized yeast cytosine deaminase protein-fragment complementation assay (OyCD PCA).
- To demonstrate the utility of OyCD PCA in studying contingent interactions between enzyme complexes, substrates, and regulatory subunits.
- To enable high-throughput screening of protein-protein interactions and functional analysis of protein mutants.
Main Methods:
- Utilized a death selection technique based on engineered yeast cytosine deaminase (OyCD) for maximum activity.
- OyCD converts the pro-drug 5-fluorocytosine (5-FC) to the toxic compound 5-fluorouracil (5-FU), leading to cell death.
- Developed OyCD PCA vectors compatible with Gateway technology for efficient generation of fusion genes.
Main Results:
- The OyCD PCA effectively measures contingent interactions between enzyme catalytic subunits, regulatory subunits, and substrates.
- The assay can differentiate functions of mutant proteins by assessing their interaction disruptions with specific partners.
- Demonstrated scalability for medium- to high-throughput analysis of pairwise protein-protein interactions in yeast deletion strains.
Conclusions:
- OyCD PCA is a powerful and scalable tool for dissecting complex protein interactions in yeast.
- The assay facilitates the study of enzyme regulation, substrate binding, and the functional impact of protein mutations.
- This method significantly advances the capacity for high-throughput investigation of molecular interactions in biological systems.
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