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. 2000 Feb 1;97(3):1143-7.
doi: 10.1073/pnas.97.3.1143.

Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins

T Ito  1 , K Tashiro, S Muta, R Ozawa, T Chiba, M Nishizawa, K Yamamoto, S Kuhara, Y Sakaki
Affiliations

Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins

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

Abstract

Protein-protein interactions play pivotal roles in various aspects of the structural and functional organization of the cell, and their complete description is indispensable to thorough understanding of the cell. As an approach toward this goal, here we report a comprehensive system to examine two-hybrid interactions in all of the possible combinations between proteins of Saccharomyces cerevisiae. We cloned all of the yeast ORFs individually as a DNA-binding domain fusion ("bait") in a MATa strain and as an activation domain fusion ("prey") in a MATalpha 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 transformants were subjected to strict selection for the activation of three reporter genes followed by sequence tagging. Our initial examination of approximately 4 x 10(6) different combinations, constituting approximately 10% of the total to be tested, has revealed 183 independent two-hybrid interactions, more than half of which are entirely novel. Notably, the obtained binary data allow us to extract more complex interaction networks, including the one that may explain a currently unsolved mechanism for the connection between distinct steps of vesicular transport. The approach described here thus will provide many leads for integration of various cellular functions and serve as a major driving force in the completion of the protein-protein interaction map.

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Figures

Figure 1
Figure 1
Outline of the strategy for comprehensive two-hybrid screening for mutually interacting proteins. The genotypes of the two strains used in the system are also shown. See text for details.
Figure 2
Figure 2
Complex two-hybrid interaction networks. Two-hybrid interaction networks for proteins related to spindle pole body (A) and vesicular transport (B) are shown. Red arrows indicate two-hybrid interactions, beginning from the bait and ending at the prey. Double-headed arrows mean that the interactions were detected bidirectionally. Note that arrows indicate the direction of two-hybrid interactions but not any biological orientation. Solid blue lines indicate known interactions recorded in the Yeast Proteome Database (14) but not yet detected by our two-hybrid screening. Shaded boxes are complexes or networks that were described in previous studies. Proteins of unknown function are indicated as black circles with white letters.
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