Analysis of Protein Complex Hierarchy in Living Cells

Abstract

The identification and analysis of protein complexes is usually achieved by performing in vitro experiments. We describe a translocation-based method for studying protein complexes in living cells. Annexin A4, a phospholipid- and calcium-binding protein, translocates from the cytoplasm and nucleoplasm to plasma and nuclear membranes in response to elevated intracellular calcium levels. By fusing a bait protein, for instance, a core component of a protein complex or a similarly relevant peptide, to the annexin, translocation of both bait and its interacting target proteins are readily monitored in living cells in response to a single treatment. Proteins of interest are fused to a variety of fluorescent proteins suitable for multiparameter imaging. Using this generally applicable approach, we were able to visualize the formation of protein complexes in their natural environment. Specifically, we detected the hierarchical assembly of four-component protein complexes in single cells.

Signaling efficiency of Gαq through its effectors p63RhoGEF and GEFT depends on their subcellular location

“…To examine whether these proteins interact in intact living cells we performed a co-recruitment assay. Co-recruitment is a strong indicator of a physical interaction or complex formation with the advantage that it can be performed in single, intact, living cells2526. We have recently used this method to demonstrate the interaction between Gαq-Q209L and PLCβ3 in cells25.…”

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

“…Examples of such techniques are FRET-FLIM and splitluciferase assays (Remy and Michnick, 2006;Bayle et al, 2008;Gehl et al, 2011). If the protein-protein interaction results in a subcellular translocation of at least one of the proteins, coexpression and translocation assays can also be applied (Piljić and Schultz, 2008;Schlü cking et al, 2013;Offenborn et al, 2015). Since these techniques use full-length FPs with distinct emission spectra, no FP reconstitution is required for visualization of the protein-protein interaction (hence no artificial stabilization can occur).…”

Direct Monitoring of the Inhibition of Protein–Protein Interactions in Cells by Translocation of PKCδ Fusion Proteins

“…These assays are robust, fast, and flexible; thus, these systems have been considered as an ideal assay for high‐content‐screening approaches to drug discovery 3. 4a Despite these advantages, few experimental applications of translocation‐based cellular assays have been reported. Most of these have been based on regulated transport between the cell nucleus and the cytoplasm using a combination of nuclear localization signals and/or nuclear export signals.…”