Evaluation and properties of the budding yeast phosphoproteome

Abstract

We have assembled a reliable phosphoproteomic data set for budding yeast Saccharomyces cerevisiae and have investigated its properties. Twelve publicly available phosphoproteome data sets were triaged to obtain a subset of high-confidence phosphorylation sites (p-sites), free of "noisy" phosphorylations. Analysis of this combined data set suggests that the inventory of phosphoproteins in yeast is close to completion, but that these proteins may have many undiscovered p-sites. Proteins involved in budding and protein kinase activity have high numbers of p-sites and are highly over-represented in the vast majority of the yeast phosphoproteome data sets. The yeast phosphoproteome is characterized by a few proteins with many p-sites and many proteins with a few p-sites. We confirm a tendency for p-sites to cluster together and find evidence that kinases may phosphorylate off-target amino acids that are within one or two residues of their cognate target. This suggests that the precise position of the phosphorylated amino acid is not a stringent requirement for regulatory fidelity. Compared with nonphosphorylated proteins, phosphoproteins are more ancient, more abundant, have longer unstructured regions, have more genetic interactions, more protein interactions, and are under tighter post-translational regulation. It appears that phosphoproteins constitute the raw material for pathway rewiring and adaptation at various evolutionary rates.

Fig. 1.

Incremental increase, with time, of the compendium for nonredundant p-sites and phosphoproteins.

Fig. 2.

Boxplot of the length (in amino acids) of regions of intrinsic disorder, for the nonphosphoproteome (denoted with 0) and the 12HQ phosphoproteome (denoted with 1).

Fig. 3.

Boxplot of netphosyeast prediction scores for p-sites with a certain coverage (number of times identified). 0 = the 195,109 ST amino acids in the 12HQ proteins, for which there is no evidence that they are phosphorylated, even when no filters are applied. 1–12 = the number of experiments in the 12HQ set in which an ST amino acid has been detected as phosphorylated. 13 = the 473 ST amino acids known to be phosphorylated and functional according to the PhosphoGrid data set.

Fig. 4.

Cumulative distribution of coverage for p-sites of the 12HQ data set and for the simulation.

Fig. 5.

Distribution of p-sites in proteins, for the 12HQ and 12HQ_3× data sets.

Fig. 6.

Orthology ratio for various fungi (orthologs based on phylogeny and YGOB) for the 12HQ versus negative data set (blue color), for the 12HQ_3x versus negative data set (yellow color) and the MS-detectable 12HQ versus negative data set (red color). A ratio value >1 indicates that phosphoproteins have more yeast orthologs than the negative data set, in that particular fungus.

Fig. 7.

Cumulative distribution of distance among neighboring p-sites.

Fig. 8.

Plot of the Pearson coefficient values for log₂ ratio of netphosyeast score and log₂ ratio of coverage of p-sites. The correlation was calculated for neighboring p-sites with a certain distance. For example, for neighboring p-sites with a distance of two amino acids, we observe that the p-site with the higher netphosyeast score is also found in more experiments than the other p-site and that the correlation is quite high (0.48).