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Directed evolution of APEX2 for electron microscopy and proximity labeling

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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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Figure 1: Yeast-display evolution of APEX2.
Figure 2: APEX2 has improved cellular activity and sensitivity for proteomic tagging and EM.
Figure 3: EM analysis of MICU1 using APEX2.

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  • 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.

References

  1. 1

    Porstmann, B., Porstmann, T., Nugel, E. & Evers, U. J. Immunol. Methods 79, 27–37 (1985).

    CAS  Article  Google Scholar 

  2. 2

    Li, J., Wang, Y., Chiu, S.-L. & Cline, H.T. Front. Neural Circuits 4, 6 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Hopkins, C., Gibson, A., Stinchcombe, J. & Futter, C. Methods Enzymol. 327, 35–45 (2000).

    CAS  Article  Google Scholar 

  4. 4

    Martell, J.D. et al. Nat. Biotechnol. 30, 1143–1148 (2012).

    CAS  Article  Google Scholar 

  5. 5

    Rhee, H.-W. et al. Science 339, 1328–1331 (2013).

    CAS  Article  Google Scholar 

  6. 6

    Hung, V. et al. Mol. Cell 55, 332–341 (2014).

    CAS  Article  Google Scholar 

  7. 7

    Mandelman, D., Schwarz, F.P., Li, H. & Poulos, T.L. Protein Sci. 7, 2089–2098 (1998).

    CAS  Article  Google Scholar 

  8. 8

    Ebert, P.S., Hess, R.A., Frykholm, B.C. & Tschudy, D.P. Biochem. Biophys. Res. Commun. 88, 1382–1390 (1979).

    CAS  Article  Google Scholar 

  9. 9

    Sharp, K.H., Moody, P.C.E., Brown, K.A. & Raven, E.L. Biochemistry 43, 8644–8651 (2004).

    CAS  Article  Google Scholar 

  10. 10

    Nicell, J.A. & Wright, H. Enzyme Microb. Technol. 21, 302–310 (1997).

    CAS  Article  Google Scholar 

  11. 11

    Arnao, M.B., Acosta, M. & del Río, J.A. & García-Cánovas, F. Biochim. Biophys. Acta 1038, 85–89 (1990).

    CAS  Article  Google Scholar 

  12. 12

    Baughman, J.M. et al. Nature 476, 341–345 (2011).

    CAS  Article  Google Scholar 

  13. 13

    De Stefani, D., Raffaello, A., Teardo, E., Szabò, I. & Rizzuto, R. Nature 476, 336–340 (2011).

    CAS  Article  Google Scholar 

  14. 14

    Kamer, K.J. & Mootha, V.K. EMBO Rep. 15, 299–307 (2014).

    CAS  Article  Google Scholar 

  15. 15

    Perocchi, F. et al. Nature 467, 291–296 (2010).

    CAS  Article  Google Scholar 

  16. 16

    Csordás, G. et al. Cell Metab. 17, 976–987 (2013).

    Article  Google Scholar 

  17. 17

    Hoffman, N.E. et al. Cell Rep. 5, 1576–1588 (2013).

    CAS  Article  Google Scholar 

  18. 18

    Vander Heiden, M.G. et al. Proc. Natl. Acad. Sci. USA 97, 4666–4671 (2000).

    CAS  Article  Google Scholar 

  19. 19

    Plovanich, M. et al. PLoS ONE 8, e55785 (2013).

    CAS  Article  Google Scholar 

  20. 20

    Shu, X. et al. PLoS Biol. 9, e1001041 (2011).

    CAS  Article  Google Scholar 

  21. 21

    Gaietta, G. et al. Science 296, 503–507 (2002).

    CAS  Article  Google Scholar 

  22. 22

    Grabenbauer, M. et al. Nat. Methods 2, 857–862 (2005).

    CAS  Article  Google Scholar 

  23. 23

    Horstmann, H., Vasileva, M. & Kuner, T. PLoS ONE 8, e64764 (2013).

    CAS  Article  Google Scholar 

  24. 24

    Lad, L., Mewies, M. & Raven, E.L. Biochemistry 41, 13774–13781 (2002).

    CAS  Article  Google Scholar 

  25. 25

    Hiner, A.N. et al. Biochem. J. 348, 321–328 (2000).

    CAS  Article  Google Scholar 

  26. 26

    Mallilankaraman, K. et al. Cell 151, 630–644 (2012).

    CAS  Article  Google Scholar 

  27. 27

    Pagliarini, D.J. et al. Cell 134, 112–123 (2008).

    CAS  Article  Google Scholar 

  28. 28

    Sanjana, N.E. et al. Nat. Protoc. 7, 171–192 (2012).

    CAS  Article  Google Scholar 

  29. 29

    Sancak, Y. et al. Cell 141, 290–303 (2010).

    CAS  Article  Google Scholar 

  30. 30

    Wen, W., Meinkoth, J.L., Tsien, R.Y. & Taylor, S.S. Cell 82, 463–473 (1995).

    CAS  Article  Google Scholar 

  31. 31

    Seth, R.B., Sun, L., Ea, C.-K. & Chen, Z.J. Cell 122, 669–682 (2005).

    CAS  Article  Google Scholar 

  32. 32

    Szczesna-Skorupa, E. Proc. Natl. Acad. Sci. USA 95, 14793–14798 (1998).

    CAS  Article  Google Scholar 

  33. 33

    Chao, G. et al. Nat. Protoc. 1, 755–768 (2006).

    CAS  Article  Google Scholar 

  34. 34

    Colby, D.W. et al. Methods Enzymol. 388, 348–358 (2004).

    CAS  Article  Google Scholar 

  35. 35

    Liu, D.S., Loh, K.H., Lam, S.S., White, K.A. & Ting, A.Y. PLoS ONE 8, e52823 (2013).

    CAS  Article  Google Scholar 

  36. 36

    Richards, M.K. & Marletta, M.A. Biochemistry 33, 14723–14732 (1994).

    CAS  Article  Google Scholar 

  37. 37

    Kery, V., Elleder, D. & Kraus, J.P. Arch. Biochem. Biophys. 316, 24–29 (1995).

    CAS  Article  Google Scholar 

  38. 38

    Delcarte, J. et al. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 786, 229–236 (2003).

    CAS  Article  Google Scholar 

  39. 39

    Barrows, T.P. & Poulos, T.L. Biochemistry 44, 14062–14068 (2005).

    CAS  Article  Google Scholar 

  40. 40

    Berry, E.A. & Trumpower, B.L. Anal. Biochem. 161, 1–15 (1987).

    CAS  Article  Google Scholar 

  41. 41

    Noble, R.W. & Gibson, Q.H. J. Biol. Chem. 245, 2409–2413 (1970).

    CAS  PubMed  Google Scholar 

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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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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.

Corresponding author

Correspondence to Alice Y Ting.

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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.

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Supplementary Figures 1–16, Supplementary Tables 1 and 2 and Supplementary Discussion (PDF 5389 kb)

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Lam, S., Martell, J., Kamer, K. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods 12, 51–54 (2015). https://doi.org/10.1038/nmeth.3179

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