Fundamental to eukaryotic cell signaling is the regulation of protein function by directed localization. Detection of these events has been largely qualitative owing to the limitations of existing technologies. Here we describe a method for quantitatively assessing protein translocation using proximity-induced enzyme complementation. The complementation assay for protein translocation (CAPT) is derived from β-galactosidase and comprises one enzyme fragment, ω, which is localized to a particular subcellular region, and a small complementing peptide, α, which is fused to the protein of interest. The concentration of α in the immediate vicinity of ω correlates with the amount of enzyme activity obtained in a dose- and time-dependent manner, thus acting as a genetically encoded biosensor for local protein concentration. Using CAPT, inducible protein movement from the cytosol to the nucleus or plasma membrane was quantitatively monitored in multiwell format and in live mammalian cells by flow cytometry.
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We thank T. Meyer for the GFP-tagged AKT and C1A constructs, and A. Banfi for the MFG-IRES-CD8 vector. We also thank T. Byun, K. Marks and J. Jones for helpful discussion in design of the system. This work was supported by US National Institutes of Health grants AG09521, AG20961, HL65572, HD18179, Ellison Medical Foundation Grant AG-33-0817, and a grant from the Baxter Foundation to H.M.B.
C.L.C. is now employed by KAI Pharmaceuticals.
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A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes
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