Conserved patterns of protein interaction in multiple species

Abstract

To elucidate cellular machinery on a global scale, we performed a multiple comparison of the recently available protein-protein interaction networks of Caenorhabditis elegans, Drosophila melanogaster, and Saccharomyces cerevisiae. This comparison integrated protein interaction and sequence information to reveal 71 network regions that were conserved across all three species and many exclusive to the metazoans. We used this conservation, and found statistically significant support for 4,645 previously undescribed protein functions and 2,609 previously undescribed protein interactions. We tested 60 interaction predictions for yeast by two-hybrid analysis, confirming approximately half of these. Significantly, many of the predicted functions and interactions would not have been identified from sequence similarity alone, demonstrating that network comparisons provide essential biological information beyond what is gleaned from the genome.

Fig. 1.

Schematic of the multiple network comparison pipeline. Raw data are preprocessed to estimate the reliability of the available protein interactions and identify groups of sequence-similar proteins. A protein group contains one protein from each species and requires that each protein has a significant sequence match to at least one other protein in the group (blast E value < 10^–7; considering the 10 best matches only). Next, protein networks are combined to produce a network alignment that connects protein similarity groups whenever the two proteins within each species directly interact or are connected by a common network neighbor. Conserved paths and clusters identified within the network alignment are compared to those computed from randomized data, and those at a significance level of P < 0.01 are retained. A final filtering step removes paths and clusters with >80% overlap.

Fig. 2.

Representative conserved network regions. Shown are conserved clusters (a–k) and paths (l and m) identified within the networks of yeast, worm, and fly. Each region contains one or more overlapping clusters or paths (see Fig. 3). Proteins from yeast (orange ovals), worm (green rectangles), or fly (blue hexagons) are connected by direct (thick line) or indirect (connection via a common network neighbor; thin line) protein interactions. Horizontal dotted gray links indicate cross-species sequence similarity between proteins (similar proteins are typically placed on the same row of the alignment). Automated layout of network alignments was performed by using a specialized plug-in to the cytoscape software (34) as described in Supporting Text.

Fig. 3.

Modular structure of conserved clusters among yeast, worm, and fly. Multiple network alignment revealed 183 conserved clusters, organized into 71 network regions represented by colored squares. Regions group together clusters that share >15% overlap with at least one other cluster in the group and are all enriched for the same GO cellular process (P < 0.05 with the enriched processes indicated by color). Cluster ID numbers are given within each square; numbers are not sequential because of filtering. Solid links indicate overlaps between different regions, where thickness is proportional to the percentage of shared proteins (intersection/union). Hashed links indicate conserved paths that connect clusters together. Labels a–k and m mark the network regions exemplified in Fig. 2.

Fig. 4.

Verification of predicted interactions by two-hybrid testing. (a) Sixty-five pairs of yeast proteins were tested for physical interaction based on their cooccurrence within the same conserved cluster and the presence of orthologous interactions in worm and fly. Each protein pair is listed along with its position on the agar plates shown in b and c and the outcome of the two-hybrid test. (b) Raw test results are shown, with each protein pair tested in quadruplicate to ensure reproducibility. Protein 1 vs. 2 of each pair was used as prey vs. bait, respectively. (c) This negative control reveals activating baits, which can lead to positive tests without interaction. Protein 2 of each pair was used as bait, and an empty pOAD vector was used as prey. Activating baits are denoted by “a” in the list of predictions shown in a. Positive tests with weak signal (e.g., A1) and control colonies with marginal activation are denoted by “?” in a; colonies D4, E2, and E5 show evidence of possible contamination and are also marked by a “?”. Discarding the activating baits, 31 of 60 predictions tested positive overall. A more conservative tally, disregarding all results marked by a “?,” yields 19 of 48 positive predictions.