title: Nic1 inactivation enables stable isotope labeling with 13C615N4-arginine in Schizosaccharomyces pombe.
abstract: Stable Isotope Labeling by Amino Acids (SILAC) is a commonly used method in quantitative proteomics. Because of compatibility with trypsin digestion, arginine and lysine are the most widely used amino acids for SILAC labeling. We observed that Schizosaccharomyces pombe (fission yeast) cannot be labeled with a specific form of arginine, (13)C(6) (15)N(4)-arginine (Arg-10), which limits the exploitation of SILAC technology in this model organism. We hypothesized that in the fission yeast the guanidinium group of (13)C(6) (15)N(4)-arginine is catabolized by arginase and urease activity to (15)N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni(2+)-dependent urease activity, through deletion of the sole Ni(2+) transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable (13)C(6) (15)N(4)-arginine labeling for SILAC strategies in S. pombe. Finally, we employed Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe.
authors.affiliation: From the ‡Proteome Center Tuebingen, University of Tuebingen, Tuebingen, 72076, Germany;
authors.forename: Alejandro
authors.initials: A
authors.lastname: Carpy
authors.affiliation: §CRUK Cell Division Group, CRUK Manchester Research Institute, University of Manchester, Manchester, M20 4BX, UK.
authors.forename: Avinash
authors.initials: A
authors.lastname: Patel
authors.affiliation: §CRUK Cell Division Group, CRUK Manchester Research Institute, University of Manchester, Manchester, M20 4BX, UK.
authors.forename: Ye Dee
authors.initials: YD
authors.lastname: Tay
authors.affiliation: §CRUK Cell Division Group, CRUK Manchester Research Institute, University of Manchester, Manchester, M20 4BX, UK.
authors.forename: Iain M
authors.initials: IM
authors.lastname: Hagan
authors.affiliation: From the ‡Proteome Center Tuebingen, University of Tuebingen, Tuebingen, 72076, Germany; boris.macek@uni-tuebingen.de.
authors.forename: Boris
authors.initials: B
authors.lastname: Macek
mesh_terms: D001120:Arginine / Q000378:metabolism*; D002247:Carbon Isotopes; D027682:Cation Transport Proteins / Q000235:genetics*; D002453:Cell Cycle; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes; D040901:Proteomics / Q000379:methods; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*
chemical_list: D002247:Carbon Isotopes; D027682:Cation Transport Proteins; C410739:Nic1 protein, S pombe; D009587:Nitrogen Isotopes; D029702:Schizosaccharomyces pombe Proteins; D001120:Arginine