Cross-species protein interactome mapping reveals species-specific wiring of stress response pathways

Abstract

The fission yeast Schizosaccharomyces pombe has more metazoan-like features than the budding yeast Saccharomyces cerevisiae, yet it has similarly facile genetics. We present a large-scale verified binary protein-protein interactome network, "StressNet," based on high-throughput yeast two-hybrid screens of interacting proteins classified as part of stress response and signal transduction pathways in S. pombe. We performed systematic, cross-species interactome mapping using StressNet and a protein interactome network of orthologous proteins in S. cerevisiae. With cross-species comparative network studies, we detected a previously unidentified component (Snr1) of the S. pombe mitogen-activated protein kinase Sty1 pathway. Coimmunoprecipitation experiments showed that Snr1 interacted with Sty1 and that deletion of snr1 increased the sensitivity of S. pombe cells to stress. Comparison of StressNet with the interactome network of orthologous proteins in S. cerevisiae showed that most of the interactions among these stress response and signaling proteins are not conserved between species but are "rewired"; orthologous proteins have different binding partners in both species. In particular, transient interactions connecting proteins in different functional modules were more likely to be rewired than conserved. By directly testing interactions between proteins in one yeast species and their corresponding binding partners in the other yeast species with yeast two-hybrid assays, we found that about half of the interactions that are traditionally considered "conserved" form modified interaction interfaces that may potentially accommodate novel functions.

Figure 1

S. pombe stress-response binary interactome network, StressNet. (A) Functional classification of the proteins included in our high-quality high-coverage HT-Y2H screen. (B) Network view of the stress-response binary interactome network in S. pombe. (C) Fraction of protein pairs in PRS, RRS, and StressNet that tested positive using Y2H, PCA, and wNAPPA. Data are shown as measurements + statistical error (SE). (D) Degree distribution of StressNet. P(k) is the probability that a protein has a degree = k.

Figure 2

Biological properties of StressNet interactions. (A) Pearson correlation coefficient (PCC) distribution of expression profiles of interacting and random protein pairs (dashed line corresponds to PCC cutoff above which pairs are considered to be significantly coexpressed; inset shows the fraction of significantly coexpressed pairs). (B) PCC distribution of genetic interaction profiles of interacting and random protein pairs (dashed line corresponds to PCC cutoff above which pairs are considered to be significantly similar; inset shows the fraction of pairs with significantly similar interaction profiles). (C) Enrichment of colocalized protein pairs. (D) Enrichment of protein pairs sharing similar functions. For each panel, the random set is constructed by considering all pairwise combinations of genes or proteins in the corresponding space. All P values represent comparisons between StressNet interactions and random pairs using a cumulative binomial test. Inset graphs and data in C and D are shown as measurements + SE.

Figure 3

Evolutionary analysis of interactions. (A) Schematic of conserved and rewired interactions between the two yeast species. S.p., S. pombe; S.c., S. cerevisiae (B) Conservation rate (fraction of conserved interactions) in our interactome calculated in two different ways. Measured represents the value calculated using a Bayesian framework that incorporates the precision and recall of our assay. Literature represents the value estimated using budding yeast interactions reported in the literature. (C) Fraction of conserved interactions involving essential and non-essential proteins. The differences in B and C are not significant based on a cumulative binomial test. (D) Distribution of the fraction of conserved interactions as a function of overall sequence similarity. (E) Distribution of the fraction of conserved interactions as a function of sequence similarity of interaction interfaces. For D and E, P values are used to test whether there is a significant difference (using a cumulative binomial test) in conservation percentage between the groups corresponding to the lowest and highest similarity percentages. R² (coefficient of determination) represents the significance of the correlation between conservation and similarity percentages. (F) Distribution of dN/dS [ratio of the number of non-synonymous substitutions per non-synonymous site (dN) to the number of synonymous substitutions per synonymous site (dS)] as a function of number of rewired interactions. The differences are not significant based on a two-sided Kolmogorov-Smirnov test. Data are shown as the measurements + SE.

Figure 4

Functional analysis of conserved and rewired interactions in S. pombe and S. cerevisiae. (A) Fraction of globally coexpressed pairs (as measured by PCC) among conserved and rewired interactions. (B) Fraction of locally coexpressed pairs (as measured by LES) among conserved and rewired interactions (C) Fraction of functionally similar pairs among conserved and rewired interactions. For each panel, the random set is constructed by considering all pairwise combinations of genes/proteins in the corresponding space. All P values represent comparisons between rewired interactions and random pairs using a cumulative binomial test. Data are shown as measurements + SE.

Figure 5

Analysis of intact and co-evolved interactions. (A) Schematic of intact and co-evolved interactions. (B) Fraction of intact and co-evolved interactions in our interactome. Data are shown as measurements + SE. No significant difference detected using a cumulative binomial test. (C) The MAPK Sty1 stress response pathway. All undirected lines represent interactions detected in our interactome. The black arrow represents transcriptional regulation. (D) Sty1-Snr1 interaction validated in S. pombe using co-immunoprecipitation (N = 3 blots). (E) Y2H analysis of the ability of Hog1 and Ehd3 to interact and Sty1 and Snr1 to interact (N = 3 experiments). (F) Sensitivity assays for different deletion strains of S. cerevisiae and S. pombe under various stress conditions (N = 3 experiments).