The Pivotal Role of Protein Phosphorylation in the Control of Yeast Central Metabolism

Abstract

Protein phosphorylation is the most frequent eukaryotic post-translational modification and can act as either a molecular switch or rheostat for protein functions. The deliberate manipulation of protein phosphorylation has great potential for regulating specific protein functions with surgical precision, rather than the gross effects gained by the over/underexpression or complete deletion of a protein-encoding gene. In order to assess the impact of phosphorylation on central metabolism, and thus its potential for biotechnological and medical exploitation, a compendium of highly confident protein phosphorylation sites (p-sites) for the model organism Saccharomyces cerevisiae has been analyzed together with two more datasets from the fungal pathogen Candida albicans Our analysis highlights the global properties of the regulation of yeast central metabolism by protein phosphorylation, where almost half of the enzymes involved are subject to this sort of post-translational modification. These phosphorylated enzymes, compared to the nonphosphorylated ones, are more abundant, regulate more reactions, have more protein-protein interactions, and a higher fraction of them are ubiquitinated. The p-sites of metabolic enzymes are also more conserved than the background p-sites, and hundreds of them have the potential for regulating metabolite production. All this integrated information has allowed us to prioritize thousands of p-sites in terms of their potential phenotypic impact. This multi-source compendium should enable the design of future high-throughput (HTP) mutation studies to identify key molecular switches/rheostats for the manipulation of not only the metabolism of yeast, but also that of many other biotechnologically and medically important fungi and eukaryotes.

Keywords: comparative phosphoproteomics; metabolism; phosphorylation; yeast.

Figure 1

Protein phosphorylation is likely to exert significant control over S. cerevisiae central metabolism. Nodes represent metabolites and lines represent reactions in the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic map. Blue color is for reactions that are controlled by at least one enzyme that undergoes phosphorylation. Red color is for reactions that are controlled by at least one enzyme that contains High Confidence (HC) p-site/s. Mapping was performed with the KEGG mapper tool (Kanehisa et al. 2012), using the Uniprot identifiers of the yeast phosphorylated enzymes.

Figure 2

The general properties of the phosphoproteome, compared to the negative phosphoproteome. The bars show which properties of the phosphoproteome are higher/lower (% difference), compared to the negative phosphoproteome. Only statistically significant differences are shown. This is estimated for various datasets. HC, High Confidence subset of the phosphoproteome; MetPhos_vs_MetNegPhos, metabolic proteins of the phosphoproteome vs. metabolic proteins of the negative phosphoproteome set. PAC, Protein Abundance Controlled dataset; Phos_vs_NegPhos: phosphoproteome vs. negative phosphoproteome.

Figure 3

Conservation of p-sites in various ascomycete ancestors. Conservation was inferred by two different methods: pairwise comparison of yeast with other extant species (red boxes in the phylogenetic tree) and ancestral sequence reconstruction. Dark blue bars show % conservation of all metabolic p-sites. Light blue bars show % conservation of all nonmetabolic p-sites. Red bars show % conservation of High Confidence (HC) metabolic p-sites. Orange bars show conservation of HC nonmetabolic p-sites.

Figure 4

Molecular representations of two p-sites examined with molecular dynamic simulations in (A) the yeast phosphoglycerate mutase (Gpm1p) and (B) aspartyl-tRNA (transfer RNA) synthetase (Dps1p). The X-ray crystal structures of the enzymes are illustrated with cartoons colored by secondary structure and the p-site serine residues are shown with spheres (green C, red O, and blue N atoms). Distances between the p-sites and the catalytic active sites are indicated with dashed lines between Ser116 and a sulfate ion in Gpm1p [ Protein Data Bank identifier (PDB ID): 5pgm], and between Ser301 and adenine-1 (A-1) of tRNA in Dps1p (PDB ID: 1asy). The red arrows indicate regions close to the active sites of the enzymes that display altered dynamics upon phosphorylation. (C and D) Plots of the atomic fluctuations of the backbone Cα atoms extracted from 100-ns MD simulations of the native and phosphorylated enzymes.