Metabolic Labeling and Immunoprecipitation of Yeast Proteins

互联网2013-12-31

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Abstract
Table of Contents
Materials
Literature Cited

Abstract

The proteins of Saccharomyces cervsiae can be metabolically labeled, as described here, with ³⁵ methionine and ³⁵ cysteine or a hydrolysate of E. coli labeled with ³⁵ O₄ ^2? . 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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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 )
[³⁵ S]‐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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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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Metabolic Labeling and Immunoprecipitation of Yeast Proteins

Abstract

Table of Contents

Materials

Basic Protocol 1: Labeling and Immunoprecipitating Yeast Proteins

Alternate Protocol 1: Making Yeast Spheroplasts

Figures

Videos

Literature Cited