Integrated Membrane Yeast Two-Hybrid System for the Analysis of Membrane Protein Complexes

Abstract

Protein-protein interactions facilitate cellular functions through the creation of networks and multi-protein complexes. Mapping the interactions within and between protein networks and elucidating the composition of protein complexes provides critical insight into biological processes. Interactions among soluble cytoplasmic proteins have been extensively investigated through the application of immunoaffinity capture as well as conventional nuclear two-hybrid testing. The integrated membrane yeast two-hybrid provides a method to investigate protein-protein interactions between integral membrane proteins in their native membrane environment. This procedure makes use of the ability of the amino-terminal fragment of ubiquitin (Nub) and the carboxyl-terminal fragment of ubiquitin (Cub) to refold reconstituting functional ubiquitin, which can be recognized by a ubiquitin peptidase. Appending a fusion protein composed of Cub fused to LexA and VP16 (CLV) to a candidate "bait" protein and Nub to candidate "prey" proteins allows a test of their interaction. If the two proteins interact closely, the CLV fragment is cleaved and enters the nucleus to activate the expression of reporter genes, signaling the interaction. When the bait and prey proteins are tagged with CLV and NubG, respectively, at their genomic loci, they are only copies of the bait and prey in the cell and are expressed under the regulation of their native promoters. This avoids overexpression artifacts that can occur if the tagged proteins are expressed from plasmids while the untagged chromosomally encoded copies of the bait and prey continue to be expressed. Key features • Allows an in vivo interaction test with integral membrane proteins in the native membrane environment. • Allows integration of NubG tag at the amino or carboxyl-terminus of prey proteins. • Avoids overexpression artifacts that can be caused by expression of CLV-tagged bait and NubG-tagged prey proteins from plasmid-based systems. • Avoids competition from untagged chromosomally encoded bait and prey proteins, as occurs when CLV-tagged bait and NubG-tagged prey are expressed from plasmids.

Keywords: MYTH; Membrane protein; Protein-protein interaction; Split-ubiquitin; Two-hybrid; Yeast; iMYTH.

Figure 1.. Split-ubiquitin membrane yeast two-hybrid (MYTH) systems.

(A) The bait protein of interest is modified by the addition of a sequence coding for the carboxyl-terminus of ubiquitin (Cub) fused to the DNA binding protein LexA and transcriptional activation domain of VP16. This fusion is labeled as CLV in the figure. The prey protein is modified by the addition of a sequence coding for the amino-terminus of ubiquitin (NubG). The tester S. cerevisiae strain is also engineered by the addition of reporter genes under the regulation of LexA operator sites (LexAop) that can be bound by LexA. Commonly employed reporter genes include HIS3 and ADE2 that confer growth on medium lacking histidine and adenine. An additional reporter gene (LacZ) confers blue color development in the presence of X-Gal. In the absence of an interaction between the bait and prey proteins, the reporter genes are not activated, and yeast colonies will remain white in the presence of X-Gal and will not form in the absence of histidine and adenine. (B) In the case that the bait and prey proteins interact with one another, the Cub and NubG fragments will fold into a functional ubiquitin moiety and will be recognized for cleavage by ubiquitin peptidases (Ubp). This cleavage releases the LexA-VP16 fragment of the CLV fusion to migrate into the nucleus and activate the reporter genes, leading to blue colonies in the presence of X-Gal and growth on medium lacking histidine and adenine.

Figure 2.. Vectors for generating integrated bait and prey for iMYTH screening.

(A) pUG-CLVt encodes a MYC-tagged Cub-LexA-VP16 coding sequence and a G418 resistance gene (KanMX). The KanMX cassette is flanked by loxP sequences (blue arrows). The MYC-Cub-LexA-VP16-KanMX DNA fragment can be amplified from the plasmid template by PCR with oligonucleotides F1/R1. These oligonucleotides can be designed such that the 5′ end of F1 has homology to the 3' end of the bait gene coding sequence, and R1 has 5′ homology to the 3′ untranslated region of the bait gene. Transformation of the PCR product into yeast allows the MYC-Cub-LexA-VP16 cassette to recombine into the chromosomal bait gene with G418 for selection. (B) pNAT-ADH-Nx encodes an ADH1 promoter-driven NubG and nourseothricin sulfate resistance gene (NatMX). The cassette can be amplified by oligonucleotides NatNxf/NatNxr. The resulting PCR fragment can integrate the NubG at the amino terminus of a prey gene using nourseothricin sulfate for selection. (C) The vector pNAT-xN encodes the NubG and NatMX sequences that can be amplified with oligonucleotides NatxNf/NatxNr. The resulting PCR product can be used to integrate NubG at the carboxyl-terminus of a prey gene using nourseothricin sulfate for selection. A NubI version of pNAT-xN (not shown) is available for use as positive control.

Figure 3.. SD agar plate assays to assess bait protein expression and interaction specificity.

Tester strain NMY51 harboring an integrated bait OLE1 tagged with the MYC-CLVt gene cassette transformed with the nonspecific prey: ALG5-NubG as a negative control, the high-affinity NubI interaction module ALG5-NubI as a positive control for interaction, and a plasmid expressing NubG-Slc1, which displays physical interaction with Ole1 [28]. Serial dilutions of each culture were prepared and spotted onto (A) plates lacking tryptophan (SD -trp) to select for the bait plasmids. (B) Plates lacking tryptophan, histidine, and adenine and supplemented with 3AT (SD -trp -his -ade + 3AT) to select for bait-prey interaction. (C) Plates lacking tryptophan and supplemented with X-Gal (SD -trp + X-Gal) to detect activation of the lacZ reporter gene.

Figure 4.. iMYTH assay performed with integrated bait and prey.

Tester strain NMY51 harboring an integrated bait OLE1 tagged with the MYC-CLVt gene cassette. The baits are the high-affinity NubI interaction module integrated at the carboxyl-terminus of ALG5 as the positive control for interaction, and the NubG integrated at the carboxyl-terminus of ALG5 as a negative control. The PADH1-NubG cassette was integrated at the amino-terminus of DGA1 to test the interaction of Ole1-CLV and NubG-Dga1. The NubG cassette was integrated at the carboxyl-terminus of SCT1 to test its interaction with Ole1. The upper panel displays cells spotted in 10-fold serial dilutions onto plates lacking histidine and adenine and supplemented with 3AT (SD -his -ade + 3AT) to select for bait–prey interaction. The lower panel displays the same strains spotted onto SD agar plates supplemented with X-Gal (SD X-Gal) to detect activation of the lacZ reporter gene.