title: Directed evolution of APEX2 for electron microscopy and proximity labeling.
abstract: 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.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Stephanie S
authors.initials: SS
authors.lastname: Lam
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Martell
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.
authors.forename: Kimberli J
authors.identifier: 0000000187287793
authors.initials: KJ
authors.lastname: Kamer
authors.affiliation: National Center for Microscopy and Imaging Research, University of California at San Diego, La Jolla, California, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Deerinck
authors.affiliation: 1] National Center for Microscopy and Imaging Research, University of California at San Diego, La Jolla, California, USA. [2] Department of Neurosciences, University of California at San Diego, La Jolla, California, USA.
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.
authors.forename: Vamsi K
authors.initials: VK
authors.lastname: Mootha
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Ting
mesh_terms: D000818:Animals; D060387:Ascorbate Peroxidases / Q000096:biosynthesis* / Q000235:genetics; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000032:analysis; D027682:Cation Transport Proteins / Q000032:analysis; D002522:Chlorocebus aethiops; D019020:Directed Molecular Evolution / Q000379:methods; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000201:enzymology
chemical_list: D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; C116241:MICU1 protein, human; D033681:Mitochondrial Membrane Transport Proteins; D060387:Ascorbate Peroxidases