Alternative Yeast Two Hybrid Systems

The Interaction Trap and Interaction Mating Erica A. Goiemis and Vladimir Khazak

1. Introduction

Since the original demonstration that for proteins PI and P2 known to interact, it was possible to detect interactions between a DNA-binding domain (DBD) fused PI and activation-domain (AD) fused P2 by assaying transcriptional activation of one or more reporter genes containing cognate DNA binding site for the DBD (1), a number of groups have developed variants of this approach for purposes such as mapping interaction domains on proteins, screening cDNA libraries for novel interacting proteins, and screening novel proteins against predetermined sets of known proteins to identify which pairs interact. We here describe strategies for these purposes utilizing the interaction trap (2), a two-hybrid system variant developed in the laboratory of Roger Brent (Fig. 1).

In the interaction trap, the DBD function is provided by the bacterial protein LexA, and the AD function is provided by bacterial sequences encoding an amphipathic helix ("acid blob" B42) Two reporters are used to score activation: a LexA operator -LacZ plasmid, and a LexA-operator LEU2 gene present in single copy on the chromosome of an appropriate yeast strain. Plasmids required for the interaction trap are shown in Fig. 2, and summarized in the Section 2. The basic principles involved in the interaction trap are essentially the same as those in the two-hybrid system. Therefore, the question arises as to why to use one vs another system variant for studying protein-protein interactions. The interaction trap has several unique advantages. First, because it utilizes the bacterial LexA protein rather than the yeast GAL4 protein as DBD, it is not necessary to use gal4 yeast strains, which are frequently unhealthy. Second, in the case of library screens using yeast proteins, it is not necessary to

From Methods in Molecular Biology, vol 63 Recombinant Protein Protocols Detection and Isolation Edited by R Tuan Humana Press Inc , Totowa, NJ

Yeast Two Hybrid

No or iftle growth on leucine- medium White or pale blue on XGal medium


Moderate to strong growth on leucine- medium Medium to intense blue on XGal medium


No or iftle growth on leucine- medium White or pale blue on XGal medium


Moderate to strong growth on leucine- medium Medium to intense blue on XGal medium

Q Aclrvation Domain Bail Protein -•Strong Interaction Domain LexA —B

LexA operators

Fig. 1. The interaction trap. In the basic application, an EGY48 or EGY191 yeast ccll contains two LexA-operator responsive reporters, the first a chromosomally integrated copy of the LEU2 gene (required for growth on media lacking leucine), and the second a plasmid bearing a GAL1 promoter-LacZ fusion gene (causing yeast to turn blue on media containing X-gal). These yeast additionally contain a plasmid constitu-dvely expressing the DNA-binding domain of LexA-fused to the "bait" protein PI: and a plasmid which is induced by galactose to express an activation domain-fused cDNA library or specific protein P2. On glucose medium (A), the DBD-fused protein is unable to activate expression of either of the two reporters, so yeast are unable to grow on medium lacking leucine, and are white on medium containing X-gal. On galactose medium (B), the AD-fusion protein is induced: a positive interaction is shown in which the DBD and AD-fused proteins interact strongly , resulting in activation of the two reporters, thus causing growth on media lacking leucine, and blue color on media containing X-gal, remove background, owing to recloning of GAL4. Third, the interaction trap utilizes a GAL1 inducible promoter to express the AD fusion, in contrast to the constitutive expression of the AD fusion in the two-hybrid system. The ability to conditionally express one of the fusions assists in eliminating background from library screens, and additionally is helpful in studying interactions in which one of two putative interacting proteins is toxic in yeast. Finally, all two-hybrid systems utilize chimeric fusion proteins to assay interactions. It occasionally happens that an interaction can be scored readily using the two-hybrid system and not at all with the interaction trap, or vice versa, presumably because of particular stenc problems with either the GAL4 or the LexA DBD, or the GAL4 or the B42 AD Having more than one option available increases the likelihood that a protein can be used effectively in a two-hybrid strategy.

The following describes three basic protocols for the interaction trap. The first describes how to detect protein-protein interactions between two predefined protein partners. The second describes how to rapidly screen a predefined panel of proteins against each other or against a novel protein to detect patterns of interaction. The third describes how to screen a library for novel interacting partners for a given protein.

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