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

Takashi Ito, Kosuke Tashiro, Shigeru Muta, Ritsuko Ozawa, Tomoko Chiba, Mayumi Nishizawa, Kiyoshi Yamamoto, Satoru Kuhara, Yoshiyuki Sakaki

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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 MATα 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 ≃4 x 106 different combinations, constituting ≃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.

Original languageEnglish
Pages (from-to)1143-1147
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume97
Issue number3
DOIs
Publication statusPublished - Feb 1 2000

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Protein Interaction Maps
Saccharomycetales
Fungal Proteins
Clone Cells
Saccharomyces cerevisiae Proteins
Complex Mixtures
Reporter Genes
Open Reading Frames
Proteins
Yeasts
DNA

All Science Journal Classification (ASJC) codes

  • General

Cite this

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. / Ito, Takashi; Tashiro, Kosuke; Muta, Shigeru; Ozawa, Ritsuko; Chiba, Tomoko; Nishizawa, Mayumi; Yamamoto, Kiyoshi; Kuhara, Satoru; Sakaki, Yoshiyuki.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 97, No. 3, 01.02.2000, p. 1143-1147.

Research output: Contribution to journalArticle

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