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Updated: Jul 30, 2025
Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics
Published on: June 23, 2022
APEX3 - An Optimized Tool for Rapid and Unbiased Proximity Labeling
Jordan T Becker1, Ashley A Auerbach2, Reuben S Harris3
1Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA; Department of Microbiology and Immunology, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA; Institute for Molecular Virology, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA.
Researchers developed APEX3, an improved proximity labeling tool. This optimized ascorbate peroxidase (APEX2) variant enables unbiased labeling of proteins and interacting factors within the cell nucleus and cytoplasm.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Macromolecular interactions are fundamental to all biological processes.
- Identifying protein complexes is crucial for understanding cellular functions and disease.
- Existing proximity labeling methods like APEX2 have limitations in cellular localization.
Purpose of the Study:
- To address the cytoplasmic-biased localization of APEX2.
- To develop an optimized proximity labeling tool with improved cellular distribution.
- To enable unbiased labeling of interacting proteins in both nucleus and cytoplasm.
Main Methods:
- Identification and functional characterization of a nuclear export signal (NES) in APEX2.
- Site-directed mutagenesis to create APEX2 variants, including the APEX3 mutant (APEX2-L242A).
- Localization studies using fluorescently tagged constructs and confirmation via fusion to RELA.
Main Results:
- APEX2 possesses a functional nuclear export signal (NESAPEX2) causing cytoplasmic bias.
- The APEX3 mutant (APEX2-L242A) exhibits balanced nucleocytoplasmic localization.
- APEX3 retains peroxide-dependent labeling activity while overcoming APEX2's localization defect.
Conclusions:
- APEX3 is a novel, optimized proximity labeling tool with improved cellular localization.
- This tool allows for unbiased labeling of interacting proteins across cellular compartments.
- APEX3 facilitates deeper understanding of macromolecular interactions in various biological contexts.
