APEX3 – An Optimized Tool for Rapid and Unbiased Proximity Labeling

Macromolecular interactions regulate all aspects of biology. The identification of interacting partners and complexes is important for understanding cellular processes, host-pathogen conflicts, and organismal development. Multiple methods exist to label and enrich interacting proteins in living cells. Notably, the soybean ascorbate peroxidase, APEX2, rapidly biotinylates adjacent biomolecules in the presence of biotin-phenol and hydrogen peroxide. However, during initial experiments with this system, we found that APEX2 exhibits a cytoplasmic-biased localization and is sensitive to the nuclear export inhibitor leptomycin B (LMB). This led us to identify a putative nuclear export signal (NES) at the carboxy-terminus of APEX2 (NES_APEX2), structurally adjacent to the conserved heme binding site. This putative NES is functional as evidenced by cytoplasmic localization and LMB sensitivity of a mCherry-NES_APEX2 chimeric construct. Single amino acid substitutions of multiple hydrophobic residues within NES_APEX2 eliminate cytoplasm-biased localization of both mCherry-NES_APEX2 as well as full-length APEX2. However, all but one of these NES substitutions also compromises peroxide-dependent labeling. This unique separation-of-function mutant, APEX2-L242A, is termed APEX3. Localization and functionality of APEX3 are confirmed by fusion to the nucleocytoplasmic shuttling transcriptional factor, RELA. APEX3 is therefore an optimized tool for unbiased proximity labeling of cellular proteins and interacting factors.