Abstract
Over the last decades there has been an explosion of new methodologies to study protein complexes. However, most of the approaches currently used are based on in vitro assays (e.g. nuclear magnetic resonance, X-ray, electron microscopy, isothermal titration calorimetry etc). The accurate measurement of parameters that define protein complexes in a physiological context has been largely limited due to technical constrains. Here, we present PICT (Protein interactions from Imaging of Complexes after Translocation), a new method that provides a simple fluorescence microscopy readout for the study of protein complexes in living cells. We take advantage of the inducible dimerization of FK506-binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain to translocate protein assemblies to membrane associated anchoring platforms in yeast. In this assay, GFP-tagged prey proteins interacting with the FRB-tagged bait will co-translocate to the FKBP-tagged anchor sites upon addition of rapamycin. The interactions are thus encoded into localization changes and can be detected by fluorescence live-cell imaging under different physiological conditions or upon perturbations. PICT can be automated for high-throughput studies and can be used to quantify dissociation rates of protein complexes in vivo. In this work we have used PICT to analyze protein-protein interactions from three biological pathways in the yeast Saccharomyces cerevisiae: Mitogen-activated protein kinase cascade (Ste5-Ste11-Ste50), exocytosis (exocyst complex) and endocytosis (Ede1-Syp1).
Highlights
Since specific recognition between proteins governs cellular function, the systematic study of protein-protein interactions (PPIs) has become a central endeavor in the fields of structural and cell biology [1]
PICT is based on the heterodimerization of FKBP-rapamycin binding (FRB) and FK506-binding protein (FKBP) proteins induced by the drug rapamycin [9]
The anchor is a protein that is stably bound to a distinct cellular location and is fused to red fluorescent protein (RFP) and the FKBP domain
Summary
Since specific recognition between proteins governs cellular function, the systematic study of protein-protein interactions (PPIs) has become a central endeavor in the fields of structural and cell biology [1]. On the other hand, allow one to study PPIs in the cellular context, but are usually technically challenging (e.g. Forster resonance energy transfer (FRET) [2] and fluorescence cross-correlation spectroscopy [3]) or are non-quantitative and prone to errors due to indirect readouts from reconstitution of the reporter tags (e.g. yeast two-hybrid [4] and other protein-fragment complementation assays [5,6,7,8]) All these limitations have traditionally restricted the accurate characterization of physiological PPIs. Here we describe a novel method, PICT (Protein interactions from Imaging of Complexes after Translocation), to detect and quantitatively characterize PPIs, both stable and transient, under different physiological states or upon genetic, chemical or environmental perturbations. To illustrate the potential of PICT, we have analyzed three different protein complexes expressed from their genomic loci in yeast: the Ste5-Ste11-Ste complex, the exocyst complex and the Ede1-Syp complex
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
