Getting more from the two-hybrid system: N-terminal fusions to LexA are efficient and sensitive baits for two-hybrid studies
Getting more from the two-hybrid system: N-terminal fusions to LexA are efficient and sensitive baits for two-hybrid studies Florence Béranger, Sandra Aresta, Jean de Gunzburg and Jacques Camonis*
INSERM U-248, Institut Curie, 26 rue d'Ulm, 75231 Paris cedex 05, France
Received February 3, 1997;Accepted April 2, 1997
ABSTRACT
Two-hybrid methods detect interactions between two proteins fused at the C-termini of, respectively, a DNA-binding domain and the activation domain of a transcriptional activator. Thus the N-terminus of none of these proteins is available for interaction. We have tested whether a bait protein with a reverted polarity (i.e. N-bait-LexA-C) is suitable for two-hybrid interaction. We show that such constructs give a specific interaction signal, and document two cases where the sensitivity is dramatically increased. Such constructs might lead to the identification of partners missed during classical two-hybrid screens.
In deciphering the functions of a protein, screening libraries with the two-hybrid method has become current practice (1 ,2 ). In all cases, a plasmid is used that expresses the protein of interest (the `bait') fused at the C-terminus of a DNA-binding domain protein (DBD). This type of construction potentially jeopardizes interactions that require a `free' N-terminus for a protein to interact with its molecular partners. We have addressed this problem by inverting the polarity of the DBD-bait fusion protein within a LexA-based two-hybrid system that is exquisitely sensitive (3 ). We show here that such bait-LexA fusions are suitable for two-hybrid studies and display positive and specific responses. Even more, a known interaction that is not detected with Lex-bait fusion is detected with a bait-Lex fusion, suggesting that such N-terminal fusions might be more sensitive.
A 1274 bp SphI-SphI fragment of plasmid pVJL10, a derivative of pBTM116, contains a truncated ADH promoter, the LexA coding region, a polylinker and an ADH terminator. SphI sites were blunt-ended and the cassette was inserted between the PvuII sites of plasmid YEplac112 (4 ). The HindIII-PstI fragment containing the LexA ORF and the multicloning site was replaced by a fragment generated by PCR and encoding a multicloning site followed by the LexA ORF and a stop codon. The multicloning site allows the generation of fusions at the N-terminus of LexA according to the sequence: GAA TTC GGA TCC GTC GAC GGT GGT ATG, where ATG is the original LexA initiation codon. Notice that (i) EcoRI, BamHI and SalI are available as cloning sites and (ii) two glycines are used as spacers between the fused proteins. The resulting plasmid was named pFBL23.
We tested the suitability of this plasmid for two-hybrid interactions. Ras and Ral are GTPases of the Ras family that interact with different sets of proteins. Ras (wild type or as its activated V12 allele that exhibits a reduced GTPase activity) interacts with its effectors cRaf1 and RalGDS, whereas the N17 dominant negative mutant that has lost its ability to bind GTP cannot (3 ,5 ,6 ). Conversely, this mutant has increased affinity for the GEF (guanine nucleotide exchange factor) CDC25 as compared to wild type and V12 proteins. Similarly, the Ral GTPase (wild type or the activated V23 allele) interacts with its effector RLIP76, whereas the dominant negative Ral (N28) mutant cannot (7 ,8 ). This allele should interact with RalGDS since it acts as a Ral GEF. No allele of Ral interacts with the Ras effector cRaf1 nor the Ras-GEF CDC25; similarly no Ras allele interacts with RLIP76, the effector of Ral. The activated and dominant negative alleles of H-Ras and RalB proteins were expressed as C-terminal (Fig. 2 A) or N-terminal (Fig. 2 B) fusions with LexA, and their ability to interact with cRaf-1, RalGDS and RLIP76 were assessed. In the following text, plasmids pBTM116 and pVJL10 used to express C-terminal fusion proteins will be referred to generically as pLex plasmids.
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10 Kaiser, C., Michaelis, S. and Mitchell, A. (1994) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press, NY.
* To whom correspondence should be addressed. Tel: +33 1 42 34 66 54; Fax: +33 1 42 34 66 50; Email: jcamonis@curie.fr
