APEX Fingerprinting Reveals the Subcellular Localization of Proteins of Interest

• Biotinylating proteins using APEX generates unique molecular patterns
• Molecular patterns provide sub-compartmental information about the APEX-tagged protein
• APEX labeling reveals HMOX1 membrane topology in the ER

SummaryDeciphering the sub-compartmental location of a given protein of interest may help explain its physiological function, but it can be challenging to do using optical or biochemical methods. Imaging with electron microscopy (EM) can provide highly resolved mapping of proteins; however, EM requires complex sample preparation and a specialized facility. Here, we use engineered ascorbate peroxidase (APEX)-generated molecular labeling patterns to provide information regarding intracellular microenvironments in living cells. Using APEX labeling of specific proteins, we uncovered subcellular localization at sub-compartmental resolution and successfully elucidated the membrane protein topology of HMOX1 and sub-mitochondrial localization of recently identified mitochondrial proteins. This method can be expanded to confirm sub-mitochondrial localization and membrane topologies of previously identified mitochondrial proteins.

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