A toolkit of protein-fragment complementation assays for studying and dissecting large-scale and dynamic protein-protein interactions in living cells.
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. Applications of PCAs in yeast have been limited until recently, simply because appropriate expression plasmids or cassettes had not been developed. However, we have now developed and reported on several PCAs in Saccharomyces cerevisiae that cover the gamut of applications one could envision for studying any aspect of PPIs. Here, 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 a method to study spatial localization and dynamics of PPIs based on fluorescent protein reporter PCAs and finally, two luciferase reporter PCAs that have proved useful for studies of dynamics of PPIs.
Michnick
Stephen W
SW
Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, Québec, Canada.
Ear
Po Hien
PH
Landry
Christian
C
Malleshaiah
Mohan K
MK
Messier
Vincent
V
D043525:Cytosine Deaminase / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism
D011506:Proteins; D013762:Tetrahydrofolate Dehydrogenase; D043525:Cytosine Deaminase