title: SILAC labeling of yeast for the study of membrane protein complexes.
abstract: Despite their simplicity compared to multicellular organisms, single-celled yeasts such as the baker's yeast Saccharomyces cerevisiae are widely recognized as model organisms for the study of eukaryotic cell biology. To gain deeper insights into the molecular mechanisms underlying cellular processes, it is of utmost interest to establish the interactome of distinct proteins and to thoroughly analyze the composition of individual protein complexes and their dynamics. Combining affinity purification of epitope-tagged proteins with high-resolution mass spectrometry and quantitative proteomics strategies, in particular stable isotope labeling by amino acids in cell culture (SILAC), represents an unbiased and powerful approach for a most accurate characterization of protein complexes. In this chapter, we provide detailed protocols for the generation of yeast strains (S. cerevisiae) amenable to SILAC-labeling, for epitope tagging of a protein of interest for affinity purification, and for the SILAC-based characterization of membrane protein complexes including the identification of stable core components and transient interaction partners.
authors: Department of Biochemistry and Functional Proteomics, Faculty of Biology, and BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schänzlestr. 1, Freiburg, 79104, Germany.
authors: Silke
authors: S
authors: Oeljeklaus
authors: Andreas
authors: A
authors: Schummer
authors: Ida
authors: I
authors: Suppanz
authors: Bettina
authors: B
authors: Warscheid
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D053000:Analytic Sample Preparation Methods; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D046508:Culture Techniques; D023281:Genomics; D007553:Isotope Labeling / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
chemical_list: D000596:Amino Acids; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D014357:Trypsin