Conditional Depletion of Nuclear Proteins by the Anchor Away System

Xiaochun Fan1, Joseph V. Geisberg1, Koon Ho Wong1, Yi Jin1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 13.10B
DOI:  10.1002/0471142727.mb1310bs93
Online Posting Date:  January, 2011
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Abstract

Nuclear proteins play key roles in the regulation of many important cellular processes. In Saccharomyces cerevisiae, many genes encoding nuclear proteins are essential. This unit describes a method termed Anchor Away that can be used to conditionally and rapidly deplete nuclear proteins of interest. It involves conditional export of the protein of interest out of the nucleus and its subsequent sequestration in the cytoplasm. This method can be used to simultaneously deplete multiple proteins from the nucleus. Curr. Protoc. Mol. Biol. 93:13.10B.1‐13.10B.8. © 2011 by John Wiley & Sons, Inc.

Keywords: Anchor Away; conditional depletion of nuclear proteins

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: PCR‐Mediated One‐Step Protein Tagging to Construct an Anchor Away Yeast Strain
  • Basic Protocol 2: Conditional Depletion of a Target Nuclear Protein by Addition of Rapamycin in the Anchor Away System
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: PCR‐Mediated One‐Step Protein Tagging to Construct an Anchor Away Yeast Strain

  Materials
  • Template DNA: miniprep‐purified (unit 1.6) plasmid pFA6a‐FRB‐KanMX6 (Haruki et al., ) containing sequence encoding FRB tag and KanMX6 selective marker; available from European Saccharomyces cerevisiae Archive for Functional Analysis (http://web.uni‐frankfurt.de/fb15/mikro/euroscarf/data/laemmli.html)
  • Oligonucleotide primers for tagging (step 1) and colony PCR confirmation (step 7)
  • QIAgen PCR purification kit or equivalent
  • Yeast strains for tagging (HHY168; Haruki et al., ; Mat α tor1‐1 fpr1::NatMX4 RPL13A‐ FKBP12::TRP1); available from European Saccharomyces cerevisiae Archive for Functional Analysis, http://web.uni‐frankfurt.de/fb15/mikro/euroscarf/data/laemmli.html
  • YPD plates (unit 13.1)
  • YPD+Kan plates (unit 13.1, YPD plate containing 200 µg/ml Geneticin from Sigma)
  • YPD+Rapa plates (unit 13.1, YPD plate containing 1 µg/ml rapamycin from Tecoland Corp., http://tecoland.com)
  • 30°C incubator
  • Additional reagents and equipment for amplification of DNA by PCR (unit 15.1), agarose gel electrophoresis (unit 2.5), DNA extraction and precipitation (unit 2.1), purification of DNA (unit 2.6), yeast transformation (unit 13.7), and immunoblotting (unit 10.8)

Basic Protocol 2: Conditional Depletion of a Target Nuclear Protein by Addition of Rapamycin in the Anchor Away System

  Materials
  • YPD medium (unit 13.1)
  • Anchor Away strain (Rpo21‐ FRB; The Struhl Laboratory at Harvard Medical School; available on request) and control strain HHY168 (HHY168 is available from European Saccharomyces cerevisiae Archive for Functional Analysis, http://web.uni‐frankfurt.de/fb15/mikro/euroscarf/data/laemmli.html)
  • 1 mg/ml (1000×) rapamycin (Tecoland Corp., http://tecoland.com) in ethanol (rapamycin dissolved in ethanol is stable for several weeks if stored at −20°C; since poor rapamycin activity is one of the major causes of inefficient Anchor Away, old stocks of rapamycin should be used with caution or avoided altogether)
  • 37% formaldehyde
  • 2.5 M glycine
  • Antibody against the C‐terminal domain of the largest subunit of Pol II such as 8WG16 (Covance)
  • 30°C incubator
  • Spectrophotometer
  • Additional reagents and equipment for chromatin immunoprecipitation (ChIP; unit 21.3)
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Figures

Videos

Literature Cited

   Bartel, B., Wünning, I., and Varshavsky, A. 1990. The recognition component of the N‐end rule pathway. EMBO J. 9:3179‐3189.
   Brown, E.J., Albers, M.W., Shin, T.B., Ichikawa, K., Keith, C.T., Lane, W.S., and Schreiber, S.L. 1994. A mammalian protein targeted by G1‐arresting rapamycin‐receptor complex. Nature 369:756‐758.
   Cafferkey, R., Young, P.R., McLaughlin, M.M., Bergsma, D.J., Koltin, Y., Sathe, G.M., Faucette, L., Eng, W.K., Johnson, R.K., and Livi, G.P. 1993. Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3‐kinase and VPS34 abrogate rapamycin cytotoxicity. Mol. Cell Biol. 13:6012‐6023.
   Chen, J., Zheng, X.F., Brown, E.J., and Schreiber, S.L. 1995. Identification of an 11‐kDa FKBP12‐rapamycin‐binding domain within the 289‐kDa FKBP12‐rapamycin‐associated protein and characterization of a critical serine residue. Proc. Natl. Acad. Sci. U.S.A. 92:4947‐4951.
   Fan, X., Lamarre‐Vincent, N., Wang, Q., and Struhl, K. 2008. Extensive chromatin fragmentation improves enrichment of protein binding sites in chromatin immunoprecipitation experiments. Nucleic Acids Res. 36:e125.
   Haruki, H., Nishikawa, J., and Laemmli, U.K. 2008. The anchor‐away technique: Rapid, conditional establishment of yeast mutant phenotypes. Mol. Cell 31:925‐932.
   Heitman, J., Movva, N.R., and Hall, M.N. 1991. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253:905‐909.
   Longtine, M.S., McKenzie, A. 3rd, Demarini, D.J., Shah, N.G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J.R. 1998. Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14:953‐961.
   Lorenz, M.C. and Heitman, J. 1995. TOR mutations confer rapamycin resistance by preventing interaction with FKBP12‐rapamycin. J. Biol. Chem. 270:27531‐27537.
   Moqtaderi, Z., Bai, Y., Poon, D., Weil, P.A., and Struhl, K. 1996. TBP‐associated factors are not generally required for transcriptional activation in yeast. Nature 383:188‐191.
Internet Resources
  http://www.sequence.stanford.edu/group/yeast_deletion_project/Deletion_primers_PCR_sizes.txt
  Provides information on primer sequences that can be used to tag yeast genes with FRB.
  http://www.uni‐frankfurt.de/fb15/mikro/euroscarf/data/laemmli.html
  Description of Anchor Away system, original reference. Includes sequence information of plasmids and genotypes of yeast strains.
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