Dev. Cell 35, 513–525 (2015)

Credit: ELSEVIER

Protein tagging and protein detection with antibodies are mainstays in the toolbox for visualizing the localization of a protein of interest (POI), but the requirement to either tag each POI or use specific antibody reagents is a bottleneck for the general application of the techniques. Based on the recent demonstration of a peroxidase-based detection strategy (APEX tagging), Ariotti et al. developed a modular system for enzyme-based protein tagging. In APEX tagging, a peroxidase is appended to a POI to allow detection by light microscopy and EM of an electron-dense polymer formed by the enzyme. In the current work, the authors generated a construct containing APEX attached to a green fluorescent protein (GFP)-binding peptide (APEX-GBP) and then used this to label cells that express GFP-tagged POIs, verifying their known localization to various subcellular compartments, such as endosomes and nuclei. The method also tracks protein redistribution over time, as demonstrated for a protein that progressively moves from lipid droplets to multivesicular bodies, and in large-scale screening applications, as shown using a library of phosphoinositide-binding peptides. A linescan analysis could quantitatively assess protein enrichment at various subcellular sites. Electron tomography could also be integrated with the approach, enabling high-resolution three-dimensional analyses. These advances, as well as the application of APEX-GBP for in vivo localization of zebrafish proteins, suggest that the system could find widespread utility in rapid protein localization efforts.