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.

Fig. 1.

Metabolic characterization of S. pombe. A, Growth curve of the SILAC strain in EMMG and EMMS media supplemented with different concentrations of ammonium chloride. B, Growth curve of WT, SILAC, nic1Δ, and SILACn strains in EMMG media at 25 °C. C, Scoring spindle formation defect in, plo1.ts41 cut7.24 SILAC cells during grown in the EMMG and EMMS media. Cultures were grown at 25 °C to a cell density of 1 × 10⁶ before a shift to 37 °C at t = 0 and spindle formation monitored by immunofluorescence. No bipolar spindles formed in either strain and the rate of spindle formation in each medium was identical. D, Metabolism of arginine in S. pombe. For full description of genotype of mutant strains see methods section.

Fig. 2.

Assessment of arginine conversion using MS. A, Isotopic distribution of peptides from a triple-SILAC experiment for an arginine and a lysine containing peptide using the SILAC strain (top) and an arginine and a lysine containing peptide using the SILACn strain (bottom). B, Protein groups (blue bars) and peptide sequences (orange bars) identified in individual experiments using different isotopic versions of lysine and arginine. The lines on the top indicate the media used and the colored background indicates the strain used.

Fig. 3.

The proteome of cell cycle populations generated by the cdc25. 22 arrest-release approach. Log₂ transformed SILAC ratios of protein groups plotted against their respective log₁₀ transformed intensity. On the left a comparison between G1S and G2 while a comparison between M and G2 is on the right. Significantly regulated proteins are marked in red; proteins discussed in the text are circled.