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Directed evolution of APEX2 for electron microscopy and proximity labeling

Nature Methods volume 12, pages 5154 (2015) | Download Citation

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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.

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Change history

  • 10 December 2014

    In the version of this article initially published online, the asterisks in Figure 2a were incorrectly depicted as exclamation marks, and some ordinate labels in the left graph of Figure 2e were obscured. The errors have been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We acknowledge funding from the US National Institutes of Health (DP1 OD003961 to A.Y.T.; P41 GM103412, R01GM086197 to M.H.E.; 5R01GM077465-08 to V.K.M.), the Howard Hughes Medical Institute (V.K.M.), and the Howard Hughes Medical Institute Collaborative Initiative Award (A.Y.T.). S.S.L. and J.D.M. were supported by US National Science Foundation Graduate Research Fellowships and National Defense Science and Engineering Graduate Fellowships. N. Watson acquired EM images of MICU1-APEX2. V. Hung provided APEX2-Stx17 and ATP5J-APEX2 EM images. K. Cox (Massachusetts Institute of Technology) provided some plasmids. FACS experiments were performed at the Koch Institute Swanson Biotechnology Center Flow Cytometry Facility. Color bright-field imaging was performed at the Koch Institute Microcopy Core Facility. We thank C. Drennan for use of her Cary300 spectrophotometer. D. McSwiggen assisted with enzyme purification. We thank T. Poulos, K. White, and the laboratory of D. Wittrup for advice.

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Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Stephanie S Lam
    • , Jeffrey D Martell
    •  & Alice Y Ting
  2. Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Kimberli J Kamer
    •  & Vamsi K Mootha
  3. National Center for Microscopy and Imaging Research, University of California at San Diego, La Jolla, California, USA.

    • Thomas J Deerinck
    •  & Mark H Ellisman
  4. Department of Neurosciences, University of California at San Diego, La Jolla, California, USA.

    • Mark H Ellisman

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Contributions

S.S.L., J.D.M. and A.Y.T. designed the research, analyzed the data and wrote the paper. All authors edited the paper. K.J.K. and V.K.M. prepared MICU1 stable cells and performed calcium uptake assays. S.S.L. and J.D.M. performed EM sample preparation, and T.J.D. and M.H.E. performed EM imaging. J.D.M. performed enzyme kinetic assays and analysis. S.S.L. performed all other experiments.

Competing interests

The Massachusetts Institute of Technology has submitted a patent application on the peroxidase technology. J.D.M. and A.Y.T. are authors on this patent.

Corresponding author

Correspondence to Alice Y Ting.

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DOI

https://doi.org/10.1038/nmeth.3179

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