Directed evolution of APEX2 for electron microscopy and proximity labeling

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Directed evolution of APEX2 for electron microscopy and proximity labeling

npg 201 5 Nature America, Inc. All rights reserved.

Stephanie S Lam1, Jeffrey D Martell1,

Kimberli J Kamer2, Thomas J Deerinck3,

Mark H Ellisman3,4, Vamsi K Mootha2 & Alice Y Ting1

APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, we used yeast-display evolution to improve its catalytic efciency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichmentof endogenous mitochondrial and endoplasmic reticulum membrane proteins.

Heme peroxidases are powerful tools for biotechnology owing to the great diversity of reactions that they catalyze. For example, horseradish peroxidase (HRP) is used widely to generate chemiluminescent signals for western blots and chromogenic signals for ELISAs13. Recently, our laboratory engineered a monomeric peroxidase reporter, APEX (28 kDa), derived from dimeric pea4

or soybean5 ascorbate peroxidases (Fig. 1ac). Unlike HRP, APEX lacks disulfides and calcium-binding sites and hence can be expressed in the reducing cytosolic environment of cells without loss of activity4. Consequently, APEX can be used for intracellular specific protein imaging by electron microscopy (EM)4 and spatially resolved proteomic mapping5,6.

For EM (Fig. 1b), APEX is genetically fused to a protein of interest, and the fusion construct is expressed inside cells. The cells are then fixed and overlaid with a solution of diaminobenzidine (DAB) and H2O2. APEX catalyzes the polymerization and local deposition of DAB, which subsequently recruits electron-dense osmium, giving EM contrast. For proteomic mapping (Fig. 1c), APEX is genetically targeted to a cellular organelle or protein complex of interest. Then live cells are treated for 1 min with H2O2 in the presence of biotin-phenol.

APEX catalyzes the oxidation of biotin-phenol to generate a very short-lived biotin-phenoxyl radical. This radical covalently

1Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. 2Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA. 3National Center for Microscopy and Imaging Research, University of California at San Diego, La Jolla, California, USA. 4Department of Neurosciences, University of California at San Diego, La Jolla, California, USA. Correspondence should be addressed to...