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

Protein-protein interactions play pivotal roles in various aspects of the s tructural and functional organization of the cell, and their complete descr iption is indispensable to thorough understanding of the cell. As an approa ch toward this goal, here we report a comprehensive system to examine two-h ybrid 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 d omain fusion ("prey") in a MAT alpha 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 approximate to 4 x 10(6) diff erent combinations, constituting approximate to 10% of the total to be test ed, has revealed 183 independent two-hybrid interactions, more than half of which are entirely novel. Notably, the obtained binary data allow us to ex tract more complex interaction networks, including the one that may explain a currently unsolved mechanism for the connection between distinct steps o f vesicular transport. The approach described here thus will provide many l eads for integration of various cellular functions and serve as a major dri ving force in the completion of the protein-protein interaction map.