DUALhunter Application Note
Abstract
The identification of protein interactions is a central task in
biology today, which has been greatly simplified by the invention of
yeast based screening assays such as the yeast two-hybrid system.
However, yeast two-hybrid systems exclude several important protein
classes, such as integral membrane proteins or transcription factors.
Here, we demonstrate the ability of the DUALhunter system to detect
interactions between trancriptionally active proteins and to identify
novel protein interactions by library screening.
While traditionally, biochemical methods such as
co-immunoprecipitation or affinity purification have been used to
characterize protein complexes, the invention of yeast based screening
assays such as the yeast two-hybrid system 1 has resulted
in a dramatic increase in the number of novel protein interactions
described in the literature since they enable the quick identification
of novel protein interactions by virtue of cDNA library screening. A
protein of interest is assayed against a complex mixture of full-length
proteins, protein domains and protein fragments expressed from a cDNA
library and those proteins or fragments interacting specifically with
it are isolated. The fact that no prior knowledge of the interaction
partner is needed makes these systems extremely powerful tools for
protein interaction discovery. However, the yeast two-hybrid system is
limited to proteins which are able to translocate to the nucleus and
which do not display any autonomous capacity for activating
transcription 2. This excludes several important protein
classes, such as integral membrane proteins, transcription factors or
strongly acidic proteins. To circumvent these limitations we set out
to develop a flexible screening system which would allow the use
of potentially any cytoplasmic protein as a bait for screening, called
the “DUALhunter system”.
Principle of the DUALhunter system
The DUALhunter system is based on the well-known split-ubiquitin complementation assay 3-5.
To screen a protein of interest for novel interactors, the protein of
interest (the bait) is fused at its N-terminus to the small membrane
protein Ost4p and at its C-terminus to a reporter module encompassing
the C-terminal half of ubiquitin (Cub) followed by a transcription
factor (Figure 1A). Ost4p serves to anchor the bait at the membrane,
whereas Cub and the attached transcription factor are needed to detect
the protein interaction. Potential interactors (preys) are expressed
from a cDNA library as fusions to a modified N-terminal half of
ubiquitin (NubG, Figure 1B). Interaction between the bait and a prey
results in formation of split-ubiquitin from Cub and NubG. Ubiquitin
specific proteases (UBPs) present in the yeast cell recognize the
split-ubiquitin and release the attached transcription factor by
cleavage of the polypeptide chain after Cub. The transcription factor
then translocates to the yeast nucleus and activates a set of cognate
reporter genes, thereby converting the protein interaction into a
transcriptional readout which is easily measured, for example as growth
on a selective minimal medium (Figure 1C).
Figure 1. (A) Principle of the DUALhunter system. A
protein of interest (the bait) is inserted between the membrane protein
Ost4p and the C-terminal half of ubiquitin (Cub) followed by the
artificial transcription factor LexA-VP16. (B) A second protein (the
prey) is fused to the mutated N-terminal half of ubiquitin (NubG). (C)
If bait and prey interact, Cub and NubG complement to form
split-ubiquitin, followed by cleavage and translocation of the
transcription factor to the nucleus and transcriptional activation of
endogenous reporter genes.
Pairwise interaction assays between known proteins
We chose Uri1p 6, a protein with proposed roles in
protein translation and folding, as a model protein since our previous
experiments have shown that Uri1p cannot be used in a classical yeast
two-hybrid assay due to the presence of a long acidic region which
autonomously activate transcription. Using a classcial two-hybrid
system, the only remaining choice is to divide the protein into
subdomains or fragments and to screen those in isolation. However, this
may result in a loss of potential interactors. In contrast, the
DUALhunter system allows the use of full-length Uri1p. Figure 2
demonstrates pairwise interactions between Uri1p and two of its known
interaction partners, Pfd6p and Rbp5p. Yeast was transformed with bait
and prey plasmids expressing Uri1p and Pfd6p or Rbp5p respectively, and
the interactions were assayed by growth on selective minimal medium.
Several unrelated control preys were used to demonstrate the specficity
of the interaction.
Figure 2. Pairwise interactions in the DUALhunter system. Yeast co-expressing the indicated baits and preys was plated either on medium selecting for the presence of both bait and prey (left column) or on medium selecting for a protein interaction (right column). NubG: empty vector expressing only NubG. Gal80p, Pex4p and Pfd2p: non-cognate proteins used as negative controls.
Screening cDNA library to identify novel interactors
We then screened the Uri1p bait against a S. cerevisiae cDNA library to identify novel interaction partners. Seven million independent clones were screened, resulting in 63 putative interactors. These clones were analyzed in detail, and clones encoding the same protein were subgrouped. In total, we identified 21 different putative interactors of Uri1p in this screen. Of those, 15 clones interacted reproducibly with Uri1p in a bait-dependency test and did not interact with several unrelated control baits. The 15 novel Uri1p interactors are shown in Table 1. Several interactors were found to be involved in protein translation, supporting a previously suggested role for Uri1p in this process. Co-immunoprecipitation assays confirmed the interactions between Uri1p and several of the newly identified proteins (data not shown). In summary, the results of the cDNA library screen show that Uri1p interacts with several members of the translation and protein folding machinery, confirming the proposed role of Uri1p in protein translation and suggesting a novel role for Uri1p in protein folding. We have also used several other transcriptionally active proteins in the DUALhunter assay, including p53 and its binding partners, and several members of the NF-KkB complex (data not shown). The results show that the DUALhunter system is very flexible and can be used to quickly identify novel interaction partners of those proteins which are unsuitable for use in classical yeast two-hybrid systems.
Table 1. Novel interactors of Uri1p identified by DUALhunter screening. Confirmed: confirmation of screening results by co-immunoprecipitation. ND: not done.
References
1. Fields, S. & Sternglanz, R. Trends Genet. 10, 286-292 (1994).
2. Fashena, S.J. et al. Gene 250, 1-14 (2000).
3. Johnsson, N. & Varshavsky, A. Proc.Natl.Acad.Sci.U.S.A. 91, 10340-10344 (1994).
4. Stagljar, I. et al. Proc. Natl. Acad. Sci. U. S. A. 95, 5187-92 (1998).
5. Thaminy, S. et al. Genome Res 13, 1744-53 (2003).
6. Gstaiger, M. et al. Science 302, 1208-12 (2003).
DUALhunter kit from Dualsystems Biotech AG
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