Introduction

In recent years, the discovery and characterization of protein interactions on a large scale has helped to elucidate many novel biological pathways (1). Due to their relative speed and ease of use, genetic screening systems, such as the yeast two-hybrid (Y2H) system (2), have been especially successful in finding novel protein interactions. However, the Y2H system is biased against certain classes of proteins, for example, transcription factors (3). When fused to a DNA binding domain, these proteins autonomously activate transcription and therefore cannot be used in a Y2H assay. Alternative methods have emerged, such as the repressed transactivator (RTA) system (4) or the RUra3-based split-ubiquitin assay (5). Although capable of circumventing the limitations of the Y2H system with regard to self-activating proteins, these assays have their own particular drawbacks and have not seen frequent use to date.

In order to create a broadly applicable and flexible screening system for transactivating proteins, we have modified the well-established split-ubiquitin system (6). The split-ubiquitin system is one of the most widely used protein complementation systems and has been applied successfully to the identification of protein interactions involving integral membrane proteins, membrane-associated proteins, and soluble proteins (7). By combining the split-ubiquitin assay with a stringent transcriptional output using multiple independent reporter genes, we have established a screening system that can be used to detect interactions between transcriptionally active proteins and to identify novel interactors of a protein of interest using cDNA library screening.

Materials and Methods

Bait and Prey Constructs

To construct the bait vector pCOWT, the cDNA encoding the entire open reading frame (ORF) of the yeast endoplasmatic reticulum (ER) OST4 was amplified by PCR from a genomic Saccharomyces cerevisiae library (Dualsystems Biotech, Zürich, Switzerland) using a forward primer with an XbaI site and a reverse primer with an SfiI restriction site. The OST4 cDNA was inserted upstream of the multiple cloning site into the vector pCCW (Dualsystems Biotech). The following bait proteins were cloned via SfiI sites downstream of the OST4 sequence into pCOWT: (i) N-terminally truncated Simian virus 40 (SV40) large T antigen (amino acids 84–708), (ii) full-length human p53, (iii) fulllength S. cerevisiae Urilp (systematic gene name YFL023W), and (iv) full-length human IκB-α. The following prey proteins were cloned downstream of the NubG cDNA into the prey vector pDSL-Nx (Dualsystems Biotech): (i) truncated human p53 (amino acids 72–390), (ii) full-length human p53, (iii) full-length human NF-κB subunit p65 and truncated p65 (amino acids 303–551), and (iv) full-length S. cerevisiae Pex4p, Pfd2p, Pfd6p, and Rpb5p. All constructs were verified by sequencing, and the expression of all baits and preys was verified by Western blot analysis (data not shown). Sequences of all constructs and details of the construction process are available upon request.

Yeast Transformation and Spotting

The S. cerevisiae strain NMY32 [MATa trp1 leu2 his3 ade2 LYS2:: lexA-HIS3 ade2::lexA-ADE2 URA3::lexA-lacZ] (Dualsystems Biotech) was cotransformed with bait and prey plasmids using the lithium acetate method (8). Transformants were selected for the presence of bait and prey plasmids during 3 days of growth at 30°C on SD-trp-leu medium [minimal medium containing 2% glucose (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland), 0.67% yeast nitrogen base (BD Biosciences, Basel, Switzerland), complete amino acid mixture lacking leucine and tryptophan (Qbiogene, Basel, Switzerland), and 2% bacto agar (BD Biosciences)]. Several colonies were transferred to liquid SD-trp-leu medium and grown overnight to an optical density (OD₅₄₆) of 1.0. Five microliters of different dilutions (1:10, 1:100, and 1:1000) were spotted onto SD-trp-leu and SD-trp-leu-his-ade media (minimal medium as described above, but lacking tryptophan, leucine, histidine, and adenine) and grown for 2 days at 30°C.

β-Galactosidase Assay

β-galactosidase activity was analyzed by the pellet X-gal assay as described previously (9).

Cytosolic Yeast Two-Hybrid Screen

A yeast cDNA library (S. cerevisiae, strain Jel1) in the prey vector pNubGx (Dualsystems Biotech) was transformed into the yeast reporter strain NMY32 harboring pCOWT-Uri1p using the lithium acetate protocol (8). Transformants were selected on SD-trp-leu-his-ade medium supplemented with 15 mM 3-aminotriazole (3-AT; Applichem, Darmstadt, Germany) for bait-prey interaction. Library plasmids were isolated from 63 positive clones, amplified in Escherichia coli, and analyzed by restriction analysis for insert sizes. The plasmids that contained an insert and were shown to be expressed in Western blot anaylses were further processed by a bait-prey interaction test. For this purpose, the individual prey plasmids were cotransformed with the Uri1p bait into NMY32 and spotted as described in the section entitled Yeast Transformation and Spotting on medium selective for the plasmids or for protein interaction. Positive clones were sequenced and retested for bait dependency by carrying out a bait-prey interaction test with the Uri1p bait and unrelated baits such as Gal4p or IκB-α. The identity of cDNA inserts was determined by performing Basic Local Alignment Search Tool (BLAST) searches (10) against GenBank®.