Native SILAC: Metabolic Labeling of Proteins in Prototroph Microorganisms Based on Lysine Synthesis Regulation

Florian Fröhlich,Romain Christiano,Tobias C. Walther

doi:10.1074/mcp.m112.025742

Abstract

Mass spectrometry (MS)-based quantitative proteomics has matured into a methodology able to detect and quantitate essentially all proteins of model microorganisms, allowing for unprecedented depth in systematic protein analyses. The most accurate quantitation approaches currently require lysine auxotrophic strains, which precludes analysis of most existing mutants, strain collections, or commercially important strains (e.g. those used for brewing or for the biotechnological production of metabolites). Here, we used MS-based proteomics to determine the global response of prototrophic yeast and bacteria to exogenous lysine. Unexpectedly, down-regulation of lysine synthesis in the presence of exogenous lysine is achieved via different mechanisms in different yeast strains. In each case, however, lysine in the medium down-regulates its biosynthesis, allowing for metabolic proteome labeling with heavy-isotope-containing lysine. This strategy of native stable isotope labeling by amino acids in cell culture (nSILAC) overcomes the limitations of previous approaches and can be used for the efficient production of protein standards for absolute SILAC quantitation in model microorganisms. As proof of principle, we have used nSILAC to globally analyze yeast proteome changes during salt stress.

Highlights

Breakthroughs in proteomics [1,2,3,4] open up new possibilities for biological systems analysis
Lys20, Lys21, Lys4, Lys12, Lys1, Lys2, and Lys9 were strongly down-regulated in cells grown in the presence of lysine (Fig. 1B). This represents all enzymes in the ␣-aminoadipate pathway of lysine synthesis in S. cerevisiae except Aro8, which might function in other amino acid metabolism pathways [26] (Fig. 1C)
Because S. pombe is separated from S. cerevisiae by ϳ400 million years of evolution [13] and is widely used as a model system for biological research, we investigated the regulation of lysine biosynthesis enzymes and the possibility of nSILAC labeling in this organism

Summary

Metabolic Labeling of Lysine Prototroph Microorganisms

Bacterial lysine synthesis via the diaminopimelate pathway is regulated by a combination of transcriptional and post-transcriptional mechanisms. Our results reveal important differences in lysine regulation between S. cerevisiae and S. pombe. Despite these differences, both organisms allowed us to exploit the regulation of lysine biosynthesis for the development of a novel strategy for labeling prototrophic microorganisms. Both organisms allowed us to exploit the regulation of lysine biosynthesis for the development of a novel strategy for labeling prototrophic microorganisms This strategy avoids complications of studying amino acid prototrophic mutant strains (e.g. when studying cellular metabolism), and enables analysis of the large arsenal of mutants and systematic strain collections available for these model systems of cell biology, as well as industrially or medically important yeast and bacterial strains

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION