Metabolic Labeling and Immunoprecipitation of Yeast Proteins

Todd R. Graham1

1 Vanderbilt University, Nashville, Tennessee
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 7.6
DOI:  10.1002/0471143030.cb0706s06
Online Posting Date:  May, 2001
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Abstract

The proteins of Saccharomyces cervsiae can be metabolically labeled, as described here, with 35methionine and 35cysteine or a hydrolysate of E. coli labeled with 35O42−. After the labeling, protocols are provided for the mechanical disruption of the yeast cells or conversion to spheroplasts, with subsequent lysis before immunoprecipitation of the proteins.

     
 
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Table of Contents

  • Basic Protocol 1: Labeling and Immunoprecipitating Yeast Proteins
  • Alternate Protocol 1: Making Yeast Spheroplasts
  • Support Protocol 1: Endo H Treatment of Immunoprecipitates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Labeling and Immunoprecipitating Yeast Proteins

  Materials
  • Yeast
  • Minimal medium containing 2% (w/v) glucose (SD medium), without agar (unit 1.6)
  • Methionine‐free SD medium (unit 1.6)
  • [35S]‐protein hydrolyzate labeling mix (>1000 Ci/mmol)
  • 50× chase solution (see recipe)
  • 50% (w/v) trichloroacetic acid (TCA)
  • Acetone, ice cold
  • SDS/urea buffer (see recipe)
  • Detergent IP buffer (see recipe)
  • Antibody specific for protein of interest
  • Preimmune serum for a negative control
  • Protein A–Sepharose (see recipe)
  • Detergent/urea buffer (see recipe)
  • 50 mM Tris⋅Cl, pH 7.5/1% (w/v) SDS
  • 1× (w/v) SDS sample buffer ( appendix 2A)
  • 125‐ml culture flask
  • Disposable 1.7‐ml centrifuge tubes
  • 0.1‐ to 0.25‐mm glass beads (Glen Mills)
  • Ready‐Caps and vials (Beckman)
  • SpeedVac evaporator
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1)

Alternate Protocol 1: Making Yeast Spheroplasts

  • Radiolabeled yeast cells
  • 2× spheroplast/stop solution (see recipe)
  • Bovine serum albumin (BSA), fatty acid—free Fraction V
  • 10 mg/ml Zymolyase 100T (Seikagako Kogyo)
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Figures

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Literature Cited

Literature Cited
   Byrd, J.C., Tarentino, A.L., Maley, F., Atkinson, P.H., and Trimble, R.B. 1982. Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing. J. Biol. Chem. 257:14657‐14666.
   Cherest, H., Davidian, J.C., Thomas, D., Benes, V., Ansorge, W., and Surdin‐Kerjan, Y. 1997. Molecular characterization of two high‐affinity sulfate transporters in Saccharomyces cerevisiae. Genetics 145: 627‐635.
   Graham, T.R. and Emr, S.D. 1991. Compartmental organization of Golgi‐specific protein modification and vacuolar protein sorting events defined in a sec18(NSF) mutant. J. Cell Biol. 114: 207‐218.
   Graham, T.R., Seeger, M., Payne, G.S., MacKay, V., and Emr, S.D. 1994. Clathrin‐dependent localization of α1,3 mannosyltransferase to the Golgi complex of Saccharomyces cerevisiae. J. Cell. Biol. 127: 667‐678.
   Huffaker, T. and Robbins, P. 1982. Temperature sensitive yeast mutants deficient in asparagine linked glycosylation. J. Biol. Chem. 257: 3203‐3210.
   Klig, L.S., Homann, M.J., Carman, G.M., and Henry, S.A. 1985. Coordinate regulation of phospholipid biosynthesis in Saccharomyces cerevisiae: Pleiotropically constitutive opi1 mutant. J. Bacteriol. 162: 1135‐1141.
   Marzluf, G.A. 1997. Molecular genetics of sulfur assimilation in filamentous fungi and yeast. Annu. Rev. Microbiol 51: 73‐96.
   Reneke, J.E., Blumer, K.J., Courchesne, W.E., and Thorner, J. 1988. The carboxy‐terminal segment of the yeast alpha‐factor receptor is a regulatory domain. Cell 55: 221‐234.
   Steiner, M.R. and Lester, R.L. 1972. In vitro studies of phospholipid biosynthesis in Saccharomyces cerevisiae. Biochim. Biophys. Acta 260: 222‐243.
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