Rapid Inactivation of Proteins by Knocksideways

Margaret S. Robinson1, Jennifer Hirst1

1 University of Cambridge, Cambridge, United Kingdom
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.20
DOI:  10.1002/0471143030.cb1520s61
Online Posting Date:  December, 2013
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Abstract

The knocksideways system inactivates proteins by using a small molecule to trap them onto mitochondria. It is typically ∼3 to 4 orders of magnitude faster than a knockdown. To get the best results out of a knocksideways, five parameters need to be optimized: the bait, the prey, the small molecule, the cell or organism, and the assay. Curr. Protoc. Cell Biol. 61:15.20.1‐15.20.7. © 2013 by John Wiley & Sons, Inc.

Keywords: clathrin; AP‐1; AP‐2; rapamycin; AP21967

     
 
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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Cells (e.g., HeLa cells) expressing both bait and prey, depleted of the endogenous protein of interest, and growing in a suitable medium at the appropriate temperature
  • Control cells (i.e., not expressing bait and/or prey)
  • Small molecule dissolved in ethanol at 2× concentration: either 1 mM rapamycin (Sigma cat. no. R0395‐1MG) or 1 mM AP21967 (Clontech, cat. no. 635057)
  • Centrifuge
  • Timer
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Figures

Videos

Literature Cited

Literature Cited
  Bayle, J.H., Grimley, J.S., Stankunas, K., Gestwicki, J.E., Wandless, T.J., and Crabtree, G.R. 2006. Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity. Chem. Biol. 13:99‐107.
  Cheeseman, L.P., Harry, E.F., McAinsh, A.D., Prior, I.A., and Royle, S.J. 2013. Specific removal of TACC3/ch‐TOG/clathrin at metaphase deregulates kinetochore fiber tension. J. Cell Sci. 126:2102‐2113.
  Hirst, J., Borner, G.H.H., Antrobus, R., Peden, A.A., Hodson, N.A., Sahlender, D.A., and Robinson, M.S. 2012. Distinct and overlapping roles for AP‐1 and GGAs revealed by the “knocksideways” system. Curr. Biol. 22:1711‐1716.
  Hosoi, H., Dilling, M.B., Shikata, T., Liu, L.N., Shu, L., Ashmun, R.A., Germain, G.S., Abraham, R.T., and Houghton, P.J. 1999. Rapamycin causes poorly reversible inhibition of mTOR and induces p53‐independent apoptosis in human rhabdomyosarcoma cells. Cancer Res. 59:886‐894.
  Robinson, M.S., Sahlender, D.A., and Foster, S.D. 2010. Rapid inactivation of proteins by rapamycin‐induced rerouting to mitochondria. Dev. Cell 18:324‐331.
  Willox, A.K. and Royle, S.J. 2012. Stonin 2 is a major adaptor protein for clathrin‐mediated synaptic vesicle retrieval. Curr. Biol. 22:1435‐1439.
  Wu, X., Zhao, X., Puertollano, R., Bonifacino, J.S., Eisenberg, E., and Greene, L.E. 2003. Adaptor and clathrin exchange at the plasma membrane and trans‐Golgi network. Mol. Biol. Cell 14:516‐528.
  Zizioli, D., Meyer, C., Guhde, G., Saftig, P., von Figura, K., and Schu, P. 1999. Early embryonic death of mice deficient in g‐adaptin. J. Biol. Chem. 274:5385‐5390.
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