Nic1 Inactivation Enables Stable Isotope Labeling with 13C615N4-Arginine in Schizosaccharomyces pombe

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, ¹³C₆¹⁵N₄-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 ¹³C₆¹⁵N₄-arginine is catabolized by arginase and urease activity to ¹⁵N₁-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni²⁺-dependent urease activity, through deletion of the sole Ni²⁺ transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable ¹³C₆¹⁵N₄-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.Stable Isotope Labeling by Amino acids in Cell culture (SILAC)¹ is one of the most widely used methods in quantitative proteomics (1). It involves in vivo metabolic labeling of cell cultures (or small organisms) with different versions of stable isotope-labeled amino acids (2). To maximize the number of peptides that can be quantified after proteome digestion with trypsin, proteins are usually differentially labeled with different forms of lysine and arginine (3): l-lysine (Lys-0) and l-arginine (Arg-0); ²H₄-lysine(Lys-4) and ¹³C₆-arginine (Arg-6); or ¹³C₆-¹⁵N₂-lysine (Lys-8) and ¹³C₆-¹⁵N₄-arginine (Arg-10). The availability of multiple forms of labeled lysine and arginine support the application of SILAC in duplex (comparison of two states) or triplex (comparison of three states) formats. Efficient anabolic pathways mean that lysine and arginine are not essential for growth of wild type yeast cells. Auxotrophic mutants that are defective in these pathways can be used to switch yeast to an absolute dependence upon the provision of these amino acids in the external medium. Consequently, mutations in arginine and lysine biosynthesis pathways can be used to drive the complete labeling of all tryptic peptides with specific forms of these amino acids (4, 5). SILAC has been used in quantitative proteomics in several yeast species, but most widely in Saccharomyces cerevisiae (6) (budding yeast) and Schizosaccharomyces pombe (fission yeast). S. pombe is extensively exploited to study cell cycle control (7), heterochromatin (8), and differentiation (9) and is increasingly the subject of large-scale quantitative proteomic studies (10, 11).A major challenge that is faced when using SILAC in fission yeast, is metabolic conversion of arginine to other amino acids such as proline, glutamine, and lysine (5). This partial labeling of additional amino acids after the conversion event produces spectra with complex isotope clusters that makes the downstream analysis challenging and error-prone. Inactivation of the “arginine conversion pathway” by removal of the orthinine transferase, Car2, effectively overcomes this problem to support the use of arginine labeling in SILAC-based experiments (5). Although this exploitation of the car2.Δ mutation now enables SILAC technology in fission yeast, the choice of amino acids that can be employed remains limited. Only one form of heavy arginine (R6) is currently used alongside three forms of heavy lysine (Lys-4, Lys-6, and Lys-8) (5, 12). Surprisingly, we could not find any studies that use arginine (Arg-10) in fission yeast, even though this is a widely exploited reagent for labeling other cell types (1).Here, we show that labeling of fission yeast with Arg-10 leads to a general misincorporation of the stable isotope label that prevents the identification of labeled peptides. We hypothesize that successive arginase and urease activities catabolize the guanidinium group of Arg-10 to ¹⁵N₁-labeled ammonia. This labeled ammonium is then used as a general precursor for amino acid biosynthesis. Disruption of Ni²⁺-dependent urease activity through deletion of Ni²⁺ transporter Nic1 in ammonium-supplemented medium, blocked this recycling to support ¹³C₆¹⁵N₄-arginine labeling SILAC strategies. As a proof of principle we employ Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe.