A comprehensive two-hybrid analysis to explore the yeast protein interactome - PubMed (original) (raw)

A comprehensive two-hybrid analysis to explore the yeast protein interactome

T Ito et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Protein-protein interactions play crucial roles in the execution of various biological functions. Accordingly, their comprehensive description would contribute considerably to the functional interpretation of fully sequenced genomes, which are flooded with novel genes of unpredictable functions. We previously developed a system to examine two-hybrid interactions in all possible combinations between the approximately 6,000 proteins of the budding yeast Saccharomyces cerevisiae. Here we have completed the comprehensive analysis using this system to identify 4,549 two-hybrid interactions among 3,278 proteins. Unexpectedly, these data do not largely overlap with those obtained by the other project [Uetz, P., et al. (2000) Nature (London) 403, 623-627] and hence have substantially expanded our knowledge on the protein interaction space or interactome of the yeast. Cumulative connection of these binary interactions generates a single huge network linking the vast majority of the proteins. Bioinformatics-aided selection of biologically relevant interactions highlights various intriguing subnetworks. They include, for instance, the one that had successfully foreseen the involvement of a novel protein in spindle pole body function as well as the one that may uncover a hitherto unidentified multiprotein complex potentially participating in the process of vesicular transport. Our data would thus significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system.

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Figures

Figure 1

Figure 1

Outline of the comprehensive two-hybrid analysis. We cloned almost all yeast ORFs individually as a DNA-binding domain fusion (bait) in a_MAT_a strain and as an activation domain fusion (prey) in a MAT_α strain, and subsequently divided them into pools, each containing 96 clones. These bait and prey clone pools were systematically mated with each other, and the diploid cells formed were selected for the simultaneous activation of three reporter genes (ADE2, HIS3, and_URA3) followed by sequence tagging to obtain ISTs.

Figure 2

Figure 2

Overlap among the results of large-scale two-hybrid projects. The Venn diagram indicates the overlap among the core data of our analysis and those obtained by the high-throughput IST analysis and the protein array approach by Uetz et al. (11).

Figure 3

Figure 3

Biologically intriguing interaction networks. Three subnetworks consisting of proteins involved in autophagy (A), spindle pole body function (B), and vesicular transport (C) are shown. The arrows indicate the orientation of each two-hybrid interaction, beginning from the bait to the prey. Hypothetical proteins of unknown functions are indicated by black ovals with white letters.

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