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 (NESAPEX2),
structurally adjacent to the conserved heme binding site. This putative NES is
functional as evidenced by cytoplasmic localization and LMB sensitivity of a
mCherry-NESAPEX2chimeric construct. Single amino acid
substitutions of multiple hydrophobic residues within NESAPEX2eliminate cytoplasm-biased localization of both mCherry-NESAPEX2as
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.