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Directed evolution of APEX2 for electron microscopy and proximity labeling

2014; Nature Portfolio; Volume: 12; Issue: 1 Linguagem: Inglês

10.1038/nmeth.3179

1548-7105

Stephanie S Lam, Jeffrey D. Martell, Kimberli J. Kamer, Thomas J. Deerinck, Mark H. Ellisman, Vamsi K. Mootha, Alice Y. Ting,

Mitochondrial Function and Pathology

A genetically encoded peroxidase with improved sensitivity, APEX2, is reported for electron microscopy and proximity labeling at low expression levels. 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 efficiency. 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 enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.