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Enzymatic detection of protein translocation

Abstract

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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Figure 1: Design of the minimal fragment.
Figure 2: Nuclear translocation measured using high-affinity β-galactosidase complementation.
Figure 3: Mutation of the α peptide to generate weakly complementing mutants.
Figure 4: Translocation of the AKT PH domain monitored by β-galactosidase complementation.

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Acknowledgements

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.

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Correspondence to Helen M Blau.

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C.L.C. is now employed by KAI Pharmaceuticals.

Supplementary information

Supplementary Fig. 1

Dose response of PDGF assayed using the AKT PH domain CAPT system. (PDF 56 kb)

Supplementary Fig. 2

Limited effect of a peptide fusion on protein expression levels. (PDF 122 kb)

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Wehrman, T., Casipit, C., Gewertz, N. et al. Enzymatic detection of protein translocation. Nat Methods 2, 521–527 (2005). https://doi.org/10.1038/nmeth771

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