Rapid Depletion of Budding Yeast Proteins via the Fusion of an Auxin‐Inducible Degron (AID)

Kohei Nishimura1, Masato T. Kanemaki2

1 Center of Frontier Research, National Institute of Genetics, Research Organization of Information and Systems, Shizuoka, 2 JST, PRESTO, Saitama
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 20.9
DOI:  10.1002/0471143030.cb2009s64
Online Posting Date:  September, 2014
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Abstract

The auxin‐inducible degron (AID) system allows the rapid and reversible proteolysis of proteins of interest, and enables the generation of conditional mutants of budding yeast. The construction of budding yeast AID mutants is simple, and the effect of depletion of essential proteins on proliferation can be confirmed by analyzing their phenotype. In this protocol, we describe a procedure to generate AID mutants of budding yeast via a simple transformation using PCR‐amplified DNA. We also describe methods to confirm the depletion of proteins of interest that are required for proliferation by serial‐dilution and liquid‐culture assays. Curr. Protoc. Cell Biol. 64:20.9.1‐20.9.16. © 2014 by John Wiley & Sons, Inc.

Keywords: auxin; degron; protein degradation; conditional mutant; ubiquitin–proteasome pathway

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Construction of Yeast Strains Expressing a Degron Fusion Protein
  • Basic Protocol 2: Degradation of Degron Fusion Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Construction of Yeast Strains Expressing a Degron Fusion Protein

  Materials
  • Template DNA (see Fig. B)
  • Phusion High‐fidelity DNA polymerase (NEB, cat. no. M0530) and 5× Phusion HF buffer
  • 2.5 mM dNTPs (Takara, cat. no. 4030)
  • 0.8% agarose gel (Voytas, )
  • PCR purification kit (e.g., Qiagen QIAquick PCR purification kit)
  • TE buffer, pH 7.5 (see recipe)
  • Budding yeast strain shown in Figure C freshly grown on a YPD plate (see recipe for YPD plates)
  • YPD medium (see recipe)
  • TE/LiAc (see recipe)
  • 10 mg/ml salmon sperm DNA (Trevigen, cat. no. 9610‐5‐D)
  • TE/LiAc/PEG (see recipe)
  • 100% dimethylsulfoxide (DMSO)
  • Selection plates (see recipe)
  • 100 mM CuSO 4 stock solution (see recipe)
  • Lysis buffer (see recipe)
  • Phenol/chloroform/isoamyl alcohol (25:24:1)
  • 100% and 70% ethanol
  • 10 mg/ml RNase stock solution: dissolve RNase power (Sigma, cat. no. R6513) at a 10 mM concentration in 10 mM Tris·Cl, pH 7.5, containing 15 mM NaCl, and then boil for 15 min to inactivate DNase (store in aliquots at −20°C)
  • EX‐Taq (Takara, cat. no. RR001) and 10× EX‐Taq buffer
  • Oligonucleotide sequencing facility
  • Thermal cycler
  • 25°C shaking incubator
  • 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Centrifuge
  • 42°C water bath or heat block
  • 2‐ml screw‐cap microcentrifuge tubes
  • 0.5‐mm glass beads (Sigma, cat. no. G8772)
  • Additional reagents and equipment for oligonucleotide synthesis (Egli et al., ), the polymerase chain reaction (PCR; Kramer and Coen, ), agarose gel electrophoresis (Voytas, ), and culture of yeast, including determining cell density (UNTI )

Basic Protocol 2: Degradation of Degron Fusion Proteins

  Materials
  • Degron‐mutant yeast cells glycerol stock (see protocol 1, step 24)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • YPD or YPG plates with or without IAA (see recipe)
  • 100 mM CuSO 4 stock solution (see recipe)
  • YPR and YPG media (see recipe)
  • 70% ethanol
  • Liquid N 2
  • 0.5 M IAA (see recipe)
  • Hemacytometer (UNTI )
  • Centrifuge
  • 25°C bacteriological incubator
  • Additional reagents and equipment for counting cells using a hemacytometer (UNTI ), processing of fixed cell suspensions for flow cytometry (Labib et al., ), and extraction of whole proteins for western blotting (Foiani et al., )
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Figures

Videos

Literature Cited

Literature Cited
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  Labib, K., Diffley, J.F., and Kearsey, S.E. 1999. G1‐phase and B‐type cyclins exclude the DNA‐replication factor Mcm4 from the nucleus. Nat. Cell Biol. 1:415‐422.
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  Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T., and Kanemaki, M. 2009. An auxin‐based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods 6:917‐922.
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