Mapping biochemical networks with protein-fragment complementation assays.
Cellular biochemical machineries, what we call pathways, consist of dynamically assembling and disassembling macromolecular complexes. Although our models for the organization of biochemical machines are derived largely from in vitro experiments, do they reflect their organization in intact, living cells? We have developed a general experimental strategy that addresses this question by allowing the quantitative probing of molecular interactions in intact, living cells. The experimental strategy is based on protein-fragment complementation assays (PCA), a method whereby protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. A biochemical machine or pathway is defined by grouping interacting proteins into those that are perturbed in the same way by common factors (hormones, metabolites, enzyme inhibitors, and so on). In this chapter we review some of the essential principles of PCA and provide details and protocols for applications of PCA, particularly in mammalian cells, based on three PCA reporters, dihydrofolate reductase, green fluorescent protein, and beta-lactamase.
Remy
Ingrid
I
Département de Biochemie, Université de Montréal, Montréal, Québec, Canada.
Michnick
Stephen W
SW
D000818:Animals; D002460:Cell Line; D003124:Colorimetry / Q000379:methods; D006801:Humans; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence / Q000379:methods; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D017510:Protein Folding; D025941:Protein Interaction Mapping / Q000379:methods*
D046911:Macromolecular Substances; D010446:Peptide Fragments