Protocol

Signal Transduction Protocols

Volume 756 of the series Methods in Molecular Biology pp 395-425

Date:

Protein-Fragment Complementation Assays for Large-Scale Analysis, Functional Dissection and Dynamic Studies of Protein–Protein Interactions in Living Cells

  • Stephen W. MichnickAffiliated withDépartement de Biochimie, Université de Montréal Email author 
  • , Po Hien EarAffiliated withDépartement de Biochimie, Université de Montréal
  • , Christian LandryAffiliated withDépartement de Biochimie, Université de Montréal
  • , Mohan K. MalleshaiahAffiliated withDépartement de Biochimie, Université de Montréal
  • , Vincent MessierAffiliated withDépartement de Biochimie, Université de Montréal

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Abstract

Protein-fragment Complementation Assays (PCAs) are a family of assays for detecting protein–protein interactions (PPIs) that have been developed to provide simple and direct ways to study PPIs in any living cell, multicellular organism, or in vitro. PCAs can be used to detect PPI between proteins of any molecular weight and expressed at their endogenous levels. Proteins are expressed in their appropriate cellular compartments and can undergo any posttranslational modification or degradation that, barring effects of the PCA fragment fusion, they would normally undergo. Assays can be performed in any cell type or model organism that can be transformed or transfected with gene expression DNA constructs. Here we focus on recent applications of PCA in the budding yeast, Saccharomyces cerevisiae, that cover the gamut of applications one could envision for studying any aspect of PPIs. We present detailed protocols for large-scale analysis of PPIs with the survival-selection dihydrofolate reductase (DHFR), reporter PCA, and a new PCA based on a yeast cytosine deaminase reporter that allows for both survival and death selection. This PCA should prove a powerful way to dissect PPIs. We then present methods to study spatial localization and dynamics of PPIs based on fluorescent protein reporter PCAs.

Key words

Protein-fragment complementation assay Protein–protein interactions Dihydrofolate reductase Cytosine deaminase Green fluorescent protein Luciferase reporter