Enzymatic detection of protein translocation.
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 beta-galactosidase and comprises one enzyme fragment, omega, which is localized to a particular subcellular region, and a small complementing peptide, alpha, which is fused to the protein of interest. The concentration of alpha in the immediate vicinity of omega 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.
Baxter Laboratory for Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA.
Tom S
TS
Wehrman
Clayton L
CL
Casipit
Nevin M
NM
Gewertz
Helen M
HM
Blau
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D021381:Protein Transport / Q000502:physiology*; D011506:Proteins / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism*
D011506:Proteins; D001616:beta-Galactosidase