Bimolecular Fluorescence Complementation Assay to Evaluate HSP90-Client Protein Interactions in Cells

Protein-protein interactions (PPI) in cells play a pivotal role in cellular function and dynamics. Cellular proteostasis is maintained by PPI networks between molecular chaperones, co-chaperones, and client proteins. Consequently, strategies to visualize and analyze PPI in cells are useful in understanding protein homeostasis regulation. The Bimolecular Fluorescence Complementation (BiFC) assay has emerged as a useful tool for studying PPI between proteins in live or fixed cells. BiFC is based on the detection of fluorescence generated when interacting protein pairs, produced as fusion proteins with either the N- or C-terminal fragment of a fluorescent protein, are in sufficient proximity to permit reconstitution of the split fluorophore. Here, we describe the application of the BiFC assay to a model of chaperone-client interactions using Hsp90 and the validated client protein CDK4. This assay allows for the distribution and spatiotemporal analysis of HSP90-CDK4 complexes in live or fixed cells and is amenable to studying the effects of inhibitors and mutations on chaperone-client protein networks.