Mapping Biochemical Networks with Protein Fragment Complementation Assays

Abstract

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, etc.). 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 β-lactamase.

Detection of protein–protein interactions at the septin collar inSaccharomyces cerevisiaeusing a tripartite split-GFP system

“…A variety of methods have been developed to assess the physical encounter of two molecules in the cell, from Förster resonance energy transfer (FRET; Padilla-Parra and Tramier, 2012) to a number of protein-fragment complementation assays (PCAs; Remy and Michnick, 2015), with readouts as diverse as drug resistance, enzyme activity, colorimetric changes, and a fluorescence signal. Among the PCA approaches, bimolecular fluorescence complementation (BiFC) has gained rather wide acceptance (Magliery et al ., 2005; Kerppola, 2009; Ohashi and Mizuno, 2014; Miller et al ., 2015) because it can detect even weak or transient interactions for the reason that once two associating proteins bring the two halves of the fluorescent reporter protein together, reconstitution of the reporter stabilizes the complex.…”

Lighting the Way to Protein-Protein Interactions: Recommendations on Best Practices for Bimolecular Fluorescence Complementation Analyses

“…Since genetic methods (e.g., two-hybrid systems, protein-fragment complementation assays; Remy and Michnick, 2015) facilitate the detection of protein interactions in vivo and do not require special reagents (such as specific antibodies for coimmunoprecipitation or affinity purification), they have quickly become popular tools to (1) screen for novel interaction partners of a given protein, (2) verify suspected interactions between proteins, and (3) characterize structural and/ or sequence motifs involved in known protein interactions. However, although these methods are seemingly straightforward and potentially powerful, not all interactions detected with them are physiologically relevant.…”

Protein Kinase C ζ Interacts with a Novel Binding Region of Gαq to Act as a Functional Effector

“…Cloning and Mutagenesis-Venus-YFP expression constructs for the protein complementation assay (PCA) were obtained by sub-cloning Gnaq (mouse, accession number NM_ 002072) and Prkcz (rat, accession number NM_022507.1) into the 5Ј-and 3Ј-ends of the Venus YFP PCA fragments, referred to here as N-terminal fragment (1-158 aa; F [1]) and the C-terminal fragment (159 -239 aa; F [2]), respectively, as previously described (10). PKC binding-deficient mutants, G␣q bindingdeficient mutants and G␣q constitutively active mutants were prepared using the QuickChange site-directed mutagenesis kit (Stratagene) following manufacturer's instructions.…”

“…If the proteins interact the fragments of the reporter protein will be brought into proximity where they can spontaneously fold together and reconstitute enzymatic activity or fluorescence (10). Venus YFP-based PCA: Cells were co-transfected with the Venus YFP PCA expression vectors coding for prey-F [1] and/or bait-F [2].…”