Genome Res. 13:1744-1753, 2003
©2003 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/03 $5.00
Methods
Identification of Novel ErbB3-Interacting Factors Using the Split-Ubiquitin Membrane Yeast Two-Hybrid System
Safia Thaminy1,
Daniel Auerbach2,
Anthony Arnoldo1 and
Igor Stagljar1,3
1 Institute of Veterinary Biochemistry and Molecular Biology, University of
Zurich-Irchel, CH-8057 Zurich, Switzerland
2 Dualsystems Biotech Inc., CH-8057 Zurich, Switzerland
 |
ABSTRACT
|
|---|
Analysis of membrane protein interactions is difficult because of the
hydrophobic nature of these proteins, which often renders conventional
biochemical and genetic assays fruitless. This is a substantial problem
because proteins that are integral or associated with membranes represent
approximately one-third of all proteins in a typical eukaryotic cell. We have
shown previously that the modified split-ubiquitin system can be used as a
genetic assay for the in vivo detection of interactions between the two
characterized yeast transmembrane proteins, Ost1p and Wbp1p. This so-called
split-ubiquitin membrane yeast two-hybrid (YTH) system uses the
split-ubiquitin approach in which reconstitution of two ubiquitin halves is
mediated by a protein–protein interaction. Here we converted the
split-ubiquitin membrane YTH system into a generally applicable in vivo
screening approach to identify interacting partners of a particular mammalian
transmembrane protein. We have demonstrated the effectiveness of this approach
by using the mammalian ErbB3 receptor as bait and have identified three
previously unknown ErbB3-interacting proteins. In addition, we have confirmed
one of the newly found interactions between ErbB3 and the membrane-associated
RGS4 protein by coimmunoprecipitating the two proteins from human cells. We
expect the split-ubiquitin membrane YTH technology to be valuable for the
identification of potential interacting partners of integral membrane proteins
from many model organisms.
The recent advances in analyses of completely sequenced genomes of numerous
model organisms, and also the human genome, have revealed that approximately
one-third of all predicted gene products of a given organism are likely to be
associated with membranes (Auerbach et al.
2002b ). These proteins execute a variety of essential cellular
tasks, which include cell signaling, transport of membrane-impermeable
molecules, and cell adhesion.
The central position of membrane proteins in mediating signaling across the
membrane makes them one of the most important pharmacological targets today.
Receptor tyrosine kinases, such as the epidermal growth factor (EGF) family
(also known as the ErbB receptor family;
Riese II and Stern 1998),
mediate signals influencing essential cellular responses such as
differentiation, proliferation, and survival. Currently, there are four
members of the ErbB family: ErbB1 (also termed EGFR or HER1), ErbB2 (also
termed HER2 or Neu), ErbB3 (also termed HER3), and ErbB4 (also termed HER4).
The importance of ErbB receptors, in particular ErbB1, ErbB2, and ErbB3, in
the development and malignancy of human cancer has been amply demonstrated
(Olayioye et al. 2000).
Interactions among proteins are essential for proper cellular functioning.
By associating an uncharacterized protein with other proteins of known
function, deductions about its potential role in the cell can often be made
(von Mering et al. 2002).
Traditionally, biochemical methods such as coimmunoprecipitation,
cross-linking, and copurification by chromatography have been used to
investigate the composition of protein complexes. However, these biochemical
methods require harsh treatments for cell disruption and therefore may not
preserve weak and/or transient interactions.
To address technical difficulties associated with the biochemical
characterization of physical protein–protein interactions, alternative
genetic methods have been developed. The most powerful genetic method for the
study of protein–protein interactions is the yeast two-hybrid (YTH)
system, which is based on reconstitution of a functional transcription factor
through a defined protein–protein interaction
(Fields and Song 1989). Since
its description, various modifications of the YTH system have been described,
which include the SOS-recruitment system
(Aronheim et al. 1994),
Ras-recruitment system (RRS; Broder et al.
1998), reverse RRS (Hubsman et
al. 2001), split-ubiquitin assay
(Johnsson and Varshavsky
1994), and a G protein fusion method
(Ehrhard et al. 2000).
Despite significant progress in development of the YTH system, the analysis
of interactions between membrane proteins remained a significant challenge
because of the hydrophobic nature of these proteins
(Auerbach et al. 2002a;
Stagljar and Fields 2002). In
addition, integral and membrane-associated proteins often undergo
posttranslational modifications or oligomerize via interactions between their
transmembrane domains, all of which is unfavorable for a traditional YTH
assay. Consistent with these problems, two independently performed genome-wide
YTH screens have shown that the coverage of membrane protein interactions is
poor (Uetz et al. 2000;
Ito et al. 2001).
We have shown previously that the modified split-ubiquitin system can be
used as a genetic assay for the in vivo detection of interactions between two
essential subunits of the yeast oligosaccharyl transferase membrane protein
complex, Ost1p and Wbp1p (Stagljar et al.
1998; Stagljar and te Heesen
2000). This so-called split-ubiquitin membrane YTH system takes
advantage of the split-ubiquitin approach first described by Johnsson and
Varshavsky (1994). It is based
on the ability of the N- and C-terminal halves of ubiquitin, Nub and Cub, to
reassemble into a quasi-native ubiquitin (split-ubiquitin). Ubiquitin-specific
proteases (UBPs), present in the cytosol and nucleus of all eukaryotic cells,
recognize such reconstituted ubiquitin but not its halves and cleave off a
reporter protein that is linked to the C terminus of Cub. The assay is
designed in such a way that the association of Nub and Cub is only efficient
if the ubiquitin halves are linked to two proteins that interact in vivo. In
the split-ubiquitin membrane YTH system, one protein of interest, the bait X,
is fused to the Cub domain, followed by an artificial transcription factor
(TF), and the other, the prey Y, is fused to NubG
(Fig. 1). To detect a potential
interaction between these two proteins X and Y, the two plasmids are
introduced into the yeast reporter strain. Interaction of both proteins
results in the assembly of split-ubiquitin and the proteolytic release of TF
by UBPs, allowing TF to enter the nucleus, leading to the activation of the
yeast reporter genes (Stagljar et al.
1998; Thaminy and Stagljar
2002). The split-ubiquitin membrane YTH technology has also been
used to study the interaction between the yeast  1,2-mannosidase Mns1p
and Rer1p in the ER (Massaad and
Herscovics 2001), to investigate the influence of mutations on the
assembly of fragments of presenilin
(Cervantes et al. 2001), and
with plant proteins to study intra- and intermolecular interactions between
sucrose transporters (Reinders et al.
2002) as well as between TOM2A and TOM1 transmembrane proteins
(Tsujimoto et al. 2003).
View larger version (20K):
[in this window]
[in a new window]
|
Figure 1 Outline of the split-ubiquitin membrane yeast two-hybrid system.
(A) A membrane bait protein of interest X is fused to Cub followed by
an artificial transcription factor (TF), while another membrane (or
cytoplasmic) protein Y is fused to the NubG domain (Y-NubG). On interaction of
the X and Y proteins, ubiquitin reconstitution occurs, leading to proteolytic
cleavage by UBPs, and the subsequent release of the transcription factor. This
factor activates reporter genes to result in
HIS3+/lacZ+ yeast cells. (B)
If X and Y do not interact, there is no ubiquitin reconstitution and thus no
UBP-mediated cleavage, resulting in
HIS-/lacZ- yeast cells.
|
|
In this study, we have generated a new set of reagents for the expression
of heterologous proteins in the split-ubiquitin membrane YTH system and
converted the system into a generally applicable in vivo screening approach
for identification of interacting partners of a particular mammalian
transmembrane protein. We have demonstrated the effectiveness of this approach
using the mammalian ErbB3 receptor as bait, and have identified three
previously unknown ErbB3-interacting proteins. We expect the split-ubiquitin
membrane YTH technology to be valuable for the identification of interacting
partners of many integral membrane proteins from any model organism, and it
may also prove useful for drug discovery purposes.
|
RESULTS
|
|---|
New Bait and Prey Vectors for the Split-Ubiquitin Membrane Yeast
Two-Hybrid System
Our central goal was to use the split-ubiquitin membrane YTH technology to
identify proteins that interact with a given mammalian membrane target
protein. To this end, we have constructed a new set of bait and prey vectors
that allow the constitutive expression of heterologous membrane proteins in
yeast (Fig. 2). The bait vector
pCYC-BAIT-Cub-TF is a centromeric vector containing a CEN/ARS origin of
replication, a Cytochrome-C oxidase (CYC1) promoter, which provides
low-level expression of the bait fusion protein, a multicloning site (MCS),
the C-terminal (Cub) domain of yeast ubiquitin fused in frame to an artificial
transcription factor consisting of Staphylococcus aureus Protein A,
Escherichia coli LexA, and the Herpes simplex virus
transactivator VP16 (collectively called TF), as well as the LEU2
gene for selection in yeast (Fig.
2A). The prey vector pADH-PREY-2HA-NubG carries a 2 µm origin
of replication, the ADH1 promoter, a MCS followed by two
hemagglutinin (HA) epitope tags fused in frame to the mutated N-terminal
(NubG) domain of yeast ubiquitin, the ADH terminator, and the TRP1
gene for selection in yeast (Fig.
2B). Both pCYC-BAIT-Cub-TF and pADH-PREY-2HA-NubG vectors are
suitable for the expression of Type I transmembrane protein in the
split-ubiquitin membrane YTH system. We have also generated novel bait
(pCYC-TF-Cub-BAIT) and prey (pADH-NubG-HA-PREY) vectors for the analysis of
Type II transmembrane proteins. The construction and application of these
vectors in the split-ubiquitin membrane YTH system will be described elsewhere
(S. Thaminy and I. Stagljar, unpubl.).
View larger version (28K):
[in this window]
[in a new window]
|
Figure 2 Maps of novel vectors for the expression of Type I transmembrane bait and
prey proteins in the split-ubiquitin membrane yeast two-hybrid system.
(A) The bait vector pCYC-BAIT-Cub-TF is a LEU2-based low
copy number (CEN/ARS) vector bearing a weak yeast CYC1 promoter, the
MCS, and the Cub domain followed by the TF. The foreign cDNA sequence encoding
a transmembrane bait protein of interest is introduced into the MCS in frame
to Cub-TF portion. Also shown is the MCS sequence upstream of the Cub-TF
fusion containing the unique XbaI, SpeI, PstI, and
HindIII restriction sites. (B) The prey vector pADH-PREY-2HA-NubG is a TRP1-based
multicopy (2µ) vector bearing a strong yeast ADH1 promoter, the
MCS, and two HA tags followed by the NubG domain. The cDNA or a library of
genomic or cDNA fragments is fused in frame to the NubG cassette. Also shown
is the MCS sequence upstream of the two HA-NubG cassettes containing the
unique restriction sites NdeI, NcoI, SmaI, and
BamHI. Both bait and prey vectors were constructed as described in
the Methods section.
|
|
Expression of ErbB3 Bait Within the Yeast Membrane
Mammalian ErbB3 protein was the first heterologous bait to be tested in the
split-ubiquitin membrane YTH system because it has an important function in
cell signaling and is an interesting drug target
(Olayioye et al. 2000;
de Bono and Rowinsky 2002). To
generate an ErbB3 bait, we fused the full-length rat ErbB3 protein (aa
1–1339) N terminally to Cub-TF, thus generating ErbB3-Cub-TF
(Fig. 3A). We then examined
whether coexpression of ErbB3-Cub-TF with either an empty prey vector or a
noninteracting yeast membrane protein Ost1p
(Stagljar et al. 1998) fused
to the NubI or NubG domains would activate the yeast gene reporter system.
Expression of ErbB3-Cub-TF with a noninteracting Ost1-NubG and empty prey
vector resulted in HIS-/lacZ- cells,
indicating that the ErbB3-Cub-TF bait is not self-activating
(Fig. 3B). In contrast,
coexpression of ErbB3-Cub-TF and a control plasmid pADH-Ost1–2HA-NubI,
containing the wild-type Nub sequence (NubI), resulted in the split-ubiquitin
formation and activation of the yeast reporter system. This occurs because any
NubI protein fusion associates with Cub independent of additional
protein–protein interactions
(Johnsson and Varshavsky,
1994; Stagljar et al.
1998).
View larger version (20K):
[in this window]
[in a new window]
|
Figure 3 (A) The structure of the ErbB3-Cub-TF bait protein used in this
study. Like other members of the ErbB family, ErbB3 is a type I transmembrane
protein consisting of an extracellular ligand binding domain (blue dotted
box), a single membrane-spanning region (striped box), and a cytoplasmic
protein tyrosine kinase domain (blue open box). The ErbB3 bait was fused to
Cub (red box), followed by an artificial transcription factor (TF; green box).
The number of amino acids of ErbB3, Cub, and TF portions are indicated.
(B) Growth of yeast cells expressing ErbB3-Cub-TF bait with various
Nub-fusions on agar plates lacking tryptophan and leucine (left), and
tryptophan, leucine, and histidine containing 10 mM 3-aminotriazole (3-AT;
middle). The L40 yeast reporter strain was cotransformed with the
ErbB3-Cub-TF bait and indicated prey plasmids, and three independent colonies
were grown on Leu-Trp- and
Leu-Trp-His-selective plates prior to
assessment of  -galactosidase activity using X-gal filter test
(right). (C) ErbB3 is localized within the yeast membrane.
Cytosolic (lanes 1 and 3) and membrane (lanes 2 and
4) fractions of yeast cells expressing the ErbB3-Cub-TF bait were
subjected to SDS-PAGE. The insoluble fraction (lane 2) was treated
with 1% Triton X-100 to solubilize the proteins, and centrifuged to separate
the soluble (lane 3) from insoluble proteins (lane 4). The
ErbB3-Cub-TF bait and a control endogenous yeast membrane protein Sec61p were
detected by immunoblot analysis using a mouse monoclonal anti-ErbB3 antibody
(upper panel), and an anti-Sec61 polyclonal antibody (lower
panel). The positions of molecular markers are indicated.
|
|
To demonstrate that the bait is targeted to the yeast membrane,
ErbB3-Cub-TF was expressed in the L40 yeast reporter strain. Subsequently,
yeast cells were lysed, then centrifuged to resolve the membrane and cytosolic
fractions, and separated by SDS-PAGE. A protein of approximately 200 kD
corresponding to ErbB3-Cub-TF fusion was detected within the membrane fraction
(Fig. 3C, lane 2, upper panel)
but not in the cytosolic fraction (Fig.
3C, lane 1, upper panel). To control our fractionation experiment,
we examined the distribution of an endogenous yeast ER membrane protein
Sec61p. Similar to the ErbB3-Cub-TF bait, Sec61p was found exclusively within
the membrane fraction (Fig. 3C,
lane 2, lower panel) and not in the cytosolic fraction
(Fig. 3C, lane 1, lower panel).
We then treated the membrane fraction (containing the insoluble proteins) with
the detergent TritonX-100, and centrifuged the extract to separate soluble and
insoluble fractions. After detergent treatment, both the ErbB3-Cub-TF bait and
Sec61p were only detected within the soluble fraction
(Fig. 3C, lane 3, upper and
lower panels) and not within the insoluble fraction
(Fig. 3C, lanes 4, upper and
lower panels). In conclusion, these biochemical experiments indicate that the
ErbB3-Cub-TF bait is expressed and correctly localized within the yeast
membrane.
Screening of a Human Brain cDNA Library Fused to NubG
To identify novel ErbB3 interacting partners, we constructed a human brain
cDNA library. The cDNAs were inserted N terminally to two HA tags followed by
the NubG sequence, thus generating the library in Y-NubG orientation (where Y
is an insert cDNA). This human brain cDNA library was transformed into the L40
yeast reporter strain (Vojtek et al.
1993) that expressed ErbB3-Cub-TF as bait. From 1.5 x
107 transformants, 170 clones displayed histidine prototrophy and
-galactosidase activity. The library plasmids from these
HIS3+/lacZ+ colonies were recovered
and transformed into E. coli for amplification. They were then
reintroduced into L40, expressing either the ErbB3-Cub-TF bait or a control
bait consisting of the yeast oligosaccharyl transferase subunit Wbp1 fused to
Cub-TF (Stagljar et al. 1998).
One hundred and forty-five independent clones failed to display a
HIS3+/lacZ+ phenotype with either the
ErbB3-Cub-TF bait or the Wbp1-Cub-TF control bait. These clones represent
false positives that most probably arise from several distinct mechanisms
during the initial growth selection process. Such false positives have also
been observed in other genetic systems such as the traditional YTH system
(Serebriiskii et al. 2000).
Possible reasons for the occurrence of this class of false positives in our
screen are given in the Discussion section. Twenty of the 170 independent
clones interacted with both the ErbB3-Cub-TF and Wbp1-Cub-TF control bait.
These plasmids encode a class of false positives that apparently bind to the
TF portion of the bait proteins. Importantly, five of the 170 independent
clones interacted specifically with the ErbB3-Cub-TF but not with the
Wbp1-Cub-TF control bait (Table
1). Two cDNAs encoded RGS4, a member of a protein family termed
Regulators of G-protein Signaling (RGS) that
act as negative regulators within G protein pathways
(Hollinger and Hepler 2002).
RGS4 was shown to exist as a membrane-bound protein
(Srinivasa et al. 1998), but
it was also found in the cytosol of human neuronal NG108 cells
(Druey et al. 1998). Recently,
it was reported that RGS16, another member of the RGS family, interacts with
the epidermal growth factor (EGFR) receptor
(Derrien and Druey 2001), a
member of the ErbB family. Two additional clones encoded the hypothetical zinc
finger protein ZNF207. So far, the function and localization of ZNF207 in
human cells remain elusive (Pahl et al.
1998). The last clone identified encoded Egr-1 (Early
Growth Response protein-1). Egr-1 is a zinc-finger
transcription factor that can be localized both to the nucleus and cytosol of
human cells (Matheny et al.
1994). Many biological functions have been attributed to Egr-1,
which include neurite outgrowth, wound repair, growth control, and apoptosis
(Beckmann and Wilce 1997).
Furthermore, recent studies have shown that Egr-1 is a regulator of the
platelet-derived growth factor receptor
(Khachigian et al. 1995) and
EGF receptor signaling pathways (Amorino et
al. 2002).
ErbB3 and RGS4 Form a Complex in Human Cells
Given the fact that ErbB3 interacts with the membrane-associated RGS4
protein in the split-ubiquitin membrane YTH system, we wanted to test whether
ErbB3 forms a complex with RGS4 in vivo. To this end, ErbB3 and RGS4 were
transiently overexpressed in HEK293T cells and coimmunoprecipitation
experiments were performed (Fig.
4). ErbB3 was immunoprecipitated with RGS4 using the anti-RGS4
antibody (lower panel, lanes 4 and 5), but not with the control IgG antibody
(lower panel, lanes 2 and 3). Moreover, the interaction between ErbB3 and RGS4
was not affected by increasing the salt concentration from 100 mM to 150 mM
(lower panel, lanes 4 and 5). We conclude from the split-ubiquitin membrane
YTH system and coimmunoprecipitation assays that ErbB3 interacts with RGS4
both in yeast and human cells.
View larger version (28K):
[in this window]
[in a new window]
|
Figure 4 ErbB3 and RGS4 form a complex in human cells. Cell lysates of HEK293T cells
coexpressing ErbB3 and RGS4 were immunoprecipitated with either IgG antibody
(lanes 2 and 3) or goat polyclonal anti-RGS4 antibody (lanes
4 and 5) using either 100 mM (lanes 2 and
4) or 150 mM NaCl (lanes 3 and 5), and analyzed by
immunoblotting with either rabbit polyclonal anti-RGS4 antibody (upper
panel) or mouse monoclonal anti-ErbB3 antibody (lower panel).
One-tenth of the same extract was used as the input control (lane
1).
|
|
Mapping of the ErbB3 Interaction Region Within RGS4
To investigate which region of RGS4 is required for binding to ErbB3,
several RGS4 truncations were generated and tested for their ability to
interact with ErbB3-Cub-TF in the split-ubiquitin membrane YTH assay
(Fig. 5A). Binding to the
ErbB3-Cub-TF bait was assessed using a quantitative -galactosidase assay
(Fig. 5B), and the correct
expression and localization of all RGS4 deletion mutants were confirmed by
immunoblotting analysis using an anti-HA antibody
(Fig. 5C;
Fig. 1 Supplementary Material,
available online at
www.genome.org).
The results of our interaction region mapping experiment indicate that the
RGS4-NubG clone (aa 1–178) identified in the split-ubiquitin membrane
YTH assay, as well as the full-length RGS4-NubG (aa 1–205), strongly
interacts with ErbB3-Cub-TF. Deletion of the N-terminal 33 amino acids of RGS4
(aa 34–205), known to be necessary for membrane localization of the
protein in yeast (Srinivasa et al.
1998), slightly decreased the binding of RGS4 to ErbB3. Immunoblot
analysis of membrane and cytosolic fractions isolated from yeast cells
expressing RGS4-NubG (aa 34–205) showed that this deletion mutant was
localized both in the cytosol and membrane fractions, whereas the full-length
RGS4-NubG was exclusively expressed in the membrane fraction
(Fig. 1 Supplementary
material). The RGS4 core domain alone (aa 58–178), which is shared by
all RGS family members (Srinivasa et al.
1998), also showed reduced binding to ErbB3 compared with
full-length RGS4. However, a deletion affecting the C-terminal 30 amino acids
of the core domain (amino acids 1–148 and amino acids 58–148)
strongly decreased the interaction with ErbB3. These results indicate that
residues important for the interaction of RGS4-NubG with ErbB3-Cub-TF are
located in the C-terminal region of the RGS box.
|
DISCUSSION
|
|---|
In this study, we describe a novel yeast-based screening technology, the
split-ubiquitin membrane YTH system, designed for the study of membrane
protein interactions. We have demonstrated the utility of this technology to
detect three novel proteins associated with the mammalian ErbB3 tyrosine
kinase receptor. In addition, we have confirmed one of the newly found
interactions between ErbB3 and membrane-associated RGS4 protein by
coimmunoprecipitating the two proteins from human cells. We have also shown
that the split-ubiquitin membrane YTH technology can be used to map the region
of RGS4 that mediates the interaction with the ErbB3 receptor. However, the
functional importance of the ErbB3/RGS4 and other two interactions identified
in our ErbB3 screen still remains to be determined.
The split-ubiquitin membrane YTH technology has several advantages over the
conventional YTH (Fields and Song
1989). Unlike the YTH, the split-ubiquitin membrane YTH is not
limited to the analysis of soluble proteins or subdomains of membrane
proteins; thus, screening with full-length integral membrane proteins or
membrane-associated proteins offers the opportunity to identify
protein–protein interactions as they take place in their natural
setting. In addition, the split-ubiquitin membrane YTH system can be applied
to any transmembrane bait protein as long as the Cub-TF and NubG modules that
are fused to the protein of interest are located in the cytoplasm. Moreover,
in the split-ubiquitin membrane YTH system genomic or cDNA libraries can be
screened in both orientations (Y-NubG or NubG-Y). In this way, it is possible
to identify both Type I (Y-NubG orientation) and Type II (NubG-Y orientation)
transmembrane proteins that interact with a particular membrane bait protein.
To test the feasibility of this approach, we have recently generated a NubG-Y
human brain cDNA library and screened it for proteins interacting with the
human -2 adrenergic receptor, a G-protein coupled receptor (D. Auerbach
and I. Stagljar, unpubl.). We have also extended the split-ubiquitin membrane
YTH approach to detect novel protein interactors of the yeast oligosaccharyl
transferase subunit Wbp1 by screening the yeast random genomic NubG-fused
libraries (D. Auerbach and I. Stagljar, unpubl.).
Like other genetic screening systems, a major drawback of the
split-ubiquitin membrane YTH is a high number of false positives that arise
during the selection process. Several independent factors may account for such
a high rate of false positives in the split-ubiquitin membrane YTH system.
Promoter mutations may activate either the HIS3 or lacZ
reporter genes, leading to growth on selective medium in the absence of a
protein–protein interaction. In addition, certain proteins, when
overexpressed in yeast, may unspecifically activate the HIS3 and
lacZ genes (Serebriiskii et al.
2000). Furthermore, interactions between integral membrane
proteins in our system are especially sensitive to protein levels and
transiently increased expression levels, which, for example, may occur because
of copy number variations of the library plasmids, thus producing unspecific
interactions (D. Auerbach. and I. Stagljar, unpubl.). However, these false
positives can be eliminated using a set of genetic criteria that can be
rapidly tested: plasmids encoding putative interactors are rapidly recovered
from yeast and transformed back into the original yeast reporter strain,
together with a plasmid encoding the bait protein-Cub-TF fusion, or with a
plasmid encoding a noncognate bait protein-Cub-TF fusion. Putative interactors
that are positive with the original bait, but negative with the noncognate
bait, are considered as true positives and are selected for further study.
Thus, when using automated procedures that facilitate the recovery of library
plasmids from yeast, false positives from a split-ubiquitin membrane YTH
screen can be eliminated quickly.
Despite the fact that we expressed the full-length ErbB3 receptor in its
natural setting, we did not identify any of the components associated with the
ErbB signaling cascade. ErbB homo- or heterodimerization is induced by binding
of extracellular ligands to their respective receptors, which then leads to
phosphorylation of the cytosolic kinase domains and recruitment of adaptor
proteins (Yarden 2001).
However, ErbB3 is an exception because it possesses no intrinsic kinase
activity and, consequently, signaling via ErbB3 requires heterodimerization
with another ErbB family member. As all adaptor proteins that have been
identified as components of ErbB signaling cascades so far bind exclusively to
activated (e.g., phosphorylated) receptor tails
(Olayioye et al. 2000), this
may explain why we did not find those proteins in our screen.
Nrdp1 (also called FLRF) is a ubiquitin ligase involved in regulation of
steady-state ErbB receptor levels
(Diamonti et al. 2002;
Qiu and Goldberg 2002). Nrdp1
has been shown to interact with the nonactivated ErbB3 receptor cytoplasmic
tail in a YTH assay (Diamonti et al.
2002). For this reason, we would expect Nrdp1 to bind to the ErbB3
receptor when expressed in yeast. However, we failed to identify Nrdp1 in our
library screen. The reason for this is presently unclear but may be connected
to an underrepresentation or absence of Nrdp1 in the cDNA library we used.
Like the YTH system, the split-ubiquitin membrane YTH system is prone to
false positives and thus, interactions that have been identified in a screen
should be confirmed in an independent system. Coimmunoprecipitation or
pull-down assays using recombinantly expressed proteins or protein fragments
have traditionally been used most often to confirm YTH results. However, both
assays are problematic when used with integral membrane proteins. In a first
step, it may therefore be advantageous to directly assess newly found
interactions in the split-ubiquitin membrane YTH system by means of a
competition experiment, in which untagged bait is overexpressed together with
the original bait-Cub-TF/prey-NubG combination. The activation of reporter
genes is then expected to be abolished by overexpression of the competing
partner. Alternatively, methods that are more suitable for use with membrane
proteins, such as colocalization experiments or fluorescence resonance energy
transfer, may also be used to confirm an interaction.
Among alternative yeast-based screening approaches developed for membrane
proteins, the reverse RRS is also capable of detecting interactions involving
a membrane protein as bait (Hubsman et al.
2001). However, interactions involving two integral membrane
proteins cannot be detected in the RRS system, because the use of a prey
membrane protein would activate the system in the absence of any
protein–protein interaction. Recently, another split-ubiquitin-based
genetic approach has been described
(Wittke et al. 1999), which
uses a destabilized version of the yeast Ura3 protein (termed rUra3) as a
reporter moiety. The interaction of two proteins fused to Cub-rUra3 and NubG,
respectively, leads to the cleavage of rUra3, followed by its degradation by
enzymes of the N-end rule pathway
(Varshavsky 1996). Use of the
counterselectable compound 5-fluororotic acid allows the selection of clones
where a protein–protein interaction has led to the complete degradation
of the rUra3 protein. In the past, the rUra3 based split-ubiquitin assay has
been used to analyze changes in protein conformation and stability of the
Saccharomyces cerevisiae ER membrane protein Sec62
(Dunnwald et al. 1999) and to
map the interactions between several S. cerevisiae integral membrane
proteins (Wittke et al. 1999).
Furthermore, this system has recently been used in a screening format to
identify proteins interacting with Gal4p and Tup1p, two yeast transcription
factors (Laser et al. 2000).
However, to our knowledge, the rUra3-based split-ubiquitin assay has not yet
been reported to work as a screening system for membrane proteins.
The successful application of the split-ubiquitin membrane YTH system as a
screening system for membrane proteins, a class of proteins that to date has
been difficult to analyze using conventional biochemical and genetic assays,
represents a step forward in the analysis of physical protein–protein
interactions. The next challenge lies in the adaptation of the split-ubiquitin
membrane YTH technology for pharmacological purposes. Using the reagents
described in this study, it may be possible to design selection systems that
identify peptides, single-chain antibodies, or small molecules that
specifically inhibit the interaction between two particular transmembrane
proteins. Last but not least, the further development of a split-ubiquitin
membrane YTH system, combined with more sophisticated vectors, libraries, and
reporter genes, will also enable its adaptation to a high-throughput format to
elucidate interactions between membrane and cytosolic proteins on a
genome-wide scale. Taken together, these studies will undoubtedly broaden our
knowledge of how membrane proteins interact in a cell.
|
METHODS
|
|---|
Construction of Plasmids
Plasmid sequences and detailed construction schemes of all constructs used
in this paper are available on request. All plasmids were verified by
sequencing, and the expression of all constructs was checked by Western blot
analysis using suitable antibodies.
pCYC-BAIT-Cub-TF
A DNA fragment encoding the Cub-TF portion was amplified by PCR from
pY-Cub-PLV (Reinders et al.
2002) and cloned in the multiple cloning site of the
HindIII- and NaeI-digested p415CYC1 vector
(Mumberg et al. 1995).
pCYC-ErbB3-Cub-TF
The cDNA encoding the rat ErbB3 was amplified by PCR from pBS-ErbB3 (kindly
provided by John Koland) and cloned in frame to Cub-TF portion of the
SpeI- and HindIII-digested pCYC-BAIT-Cub-TF.
pADH-PREY-2HA-NubG
Two oligos, X-HA-NubG-up
(5'-GATCCAGTACCCATACGATGTTCCAGATTACGCTCA-3') and X-HA-NubG-low
(5'-GATCTGAGCGTAATCTGGAACATCGTATGGGTACTG-3'), were ligated to each
other and inserted into the BamHI site of the pX-NubG vector. To
construct the pX-NubG vector, the DNA sequence encoding the NubG portion was
amplified by PCR from pOST1-NubG (Stagljar
et al. 1998) and inserted into the PstI site of the
Gal4-DNA binding-domain-less pAS2–1 vector (BD Biosciences).
pADH-Ost1–2HA-NubG and pADH-Ost1–2HA-NubI
The entire open reading frame encoding the Ost1p was amplified directly
from yeast genomic DNA and cloned into the NcoI and BamHI
sites of pADH-PREY-2HA-NubG and pADH-PREY-2HA-NubI, respectively, to yield
pADH-Ost1–2HA-NubG and pADH-Ost1–2HA-NubI.
pADH-RGS4–2HA-NubG Deletion Mutants
These constructs were generated by PCR amplification of different portions
of RGS4 and subsequent cloning by in vivo recombination in yeast in the
Nde I-digested pADH-PREY-2HA-NubG.
Construction of the NubG-Fused Random Human Brain cDNA Library
The random human brain cDNAs were generated using the SuperScriptII RNase
H-Reverse Transcriptase (INVITROGEN) from 5 µg of total human fetal brain
RNA (AMBION). The primary library was introduced in pADH-PREY-2HA-NubG vector
(Y-NubG orientation, where Y is an insert cDNA) using the Gateway system and
had a complexity of 1 x 107 clones. The subsequent random
analysis of 23 E. coli colonies showed that the percentage of
recombinants is 87% and that the average insert size is 1.1 kb. Requests for
the Y-NubG-fused random human brain cDNA library should be directed to
info{at}dualsystems.com.
ErbB3 Split-Ubiquitin Membrane Yeast Two-Hybrid Screen
Two hundred micrograms of the human brain cDNA library fused N terminally
to NubG (Y-NubG orientation) were transformed into the yeast reporter strain
L40(MATa trp1 leu2 his3 LYS2::lexA-HIS3 URA3::lexA-lacZ) expressing
ErbB3-Cub-TF bait using the lithium acetate protocol. Approximately 1.5
x 107 TRP+ LEU+
transformants were selected on SD
Leu-Trp-His- medium containing 10 mM
3-aminotriazole (3-AT). Library plasmids were isolated from 170 initial
positive HIS3+/lacZ+ yeast colonies
and rescued into E. coli XL1-Blue according to standard procedures.
Isolated library plasmids were retransformed into L40 expressing either
ErbB3-Cub-TF or the yeast Wbp1-Cub-TF control bait. Three individual colonies
of each transformant were tested for the activation of the lacZ
reporter by X-gal filter lift-off assay after incubation for 5 h at room
temperature. Only the plasmids that activated the HIS3 and
lacZ reporters in combination with the ErbB3-Cub-TF bait, but not
with the Wbp1-Cub-TF control bait, were selected for sequencing and further
studies.
Protein Purification and Immunoblotting
Protein extracts and membrane fractionations were performed as described
previously (David et al. 1997).
Briefly, cells equivalent to A600 = 100 were resuspended
in 50 mM Tris-HCl (pH 7.9) supplemented with protease inhibitors (Roche
Molecular Biochemicals) and lysed by vortexing with glass beads (Sigma).
Unlysed cells were removed by centrifugation at 700g, and the
membrane fraction was collected by centrifugation at 150,000g. To
solubilize the membrane fraction, we resuspended the pellet in the lysis
buffer containing 1% Triton X-100, followed by centrifugation at
150,000g. Rabbit anti-VP16 polyclonal antibody (1:1000; Clontech),
mouse anti-HA monoclonal antibody (1:1000; Babco), mouse monoclonal anti-ErbB3
antibody (1:5000; Neomarkers), rabbit polyclonal anti-RGS4 antibody (1:1000;
kindly provided by Kirk Druey), rabbit polyclonal Sec61p antibody, and Yrb1p
antibody (respectively 1:2000 and 1:5000; kindly provided by Claude Jakob)
were used.
Immunoprecipitation
The full-length ErbB3 cDNA was amplified by PCR and inserted into the
XbaI and HindIII sites of pcDNA3 (Invitrogen). HA-tagged
full-length human RGS4 in pcDNA3 was kindly provided by Kirk Druey. HEK 293T
cells were grown and transiently transfected as previously described
(Derrien and Druey 2001). Two
days after transfection, cells were washed with PBS and lysed in a buffer
containing 100 or 150 mM Nacl, 50 mM Tris (pH 7.5), 5 mM EDTA, 1%
Nonidet-P-40, 2 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and a
mixture of protease inhibitors (Roche Molecular Biochemicals). Lysed cells
were incubated for 1 h at 4 °C and centrifuged at 13,000g for 15
min. The supernatant was designated as cell lysate. RGS4 was
immunoprecipitated with 10µg goat polyclonal anti-RGS4 N-16 antibody
(1:1000; Santa Cruz Biotechnology) or control IgG and then incubated with
Protein G sepharose beads (Amersham Pharmacia Biotechnology). The beads were
washed three times with lysis buffer containing 0.5% Nonidet-P-40 prior to
addition of Laemmli buffer and SDS-PAGE. Immunoblottings were performed using
the appropriate antibodies.
|
Acknowledgements
|
|---|
We thank Michael Fetchko, Stan Fields, Michael Hottiger, and John Miller
for helpful discussions; John Koland, Kirk Druey, and Claude Jakob for
reagents; and Ulrich Hübscher for his support. We also thank Stephan te
Heesen for his generous help during the initial phase of the split-ubiquitin
membrane YTH project. The I.S. group is supported by Zürcher Krebsliga,
Gebert-Rüf Foundation, Walter Honegger Foundation, Bonizzi-Theler
Foundation, EMDO Foundation, Novartis Foundation, Olga Mayenfisch Foundation,
Sassella Foundation, Fonds für medizinische Forschung, Kommission
für Technische Inovation (KTI, Nr. 5343.2 SUS), and the Swiss National
Science Foundation (31–58798.99 and 3100A0–100256/1).
The publication costs of this article were defrayed in part by payment of
page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 USC section 1734 solely to
indicate this fact.
|
Footnotes
|
|---|
Article and publication are at
http://www.genome.org/cgi/doi/10.1101/gr.1276503.
3 Corresponding author.
E-MAIL
stagljar{at}vetbio.unizh.ch;
FAX41-1-635 68 40. 
[Supplementary material is available online at www.genome.org. The
following individuals kindly provided reagents, samples, or unpublished
information as indicated in the paper: J. Koland, K. Druey, and C. Jakob.]
|
REFERENCES
|
|---|
Amorino, G.P., Hamilton, V.M., Valerie, K., Dent, P., Lammering,
G., and Schmidt-Ullrich, R.K. 2002. Epidermal growth factor
receptor dependence of radiation-induced transcription factor activation in
human breast carcinoma cells. Mol. Biol. Cell
13:
2233-2244.[Abstract/Free Full Text]
Aronheim, A., Engelberg, D., Li, N., al-Alawi, N., Schlessinger,
J., and Karin, M. 1994. Membrane targeting of the nucleotide
exchange factor Sos is sufficient for activating the Ras signaling pathway.
Cell 78:
949-961.[CrossRef][Medline]
Auerbach, D., Galeuchet-Schenk, B., Hottiger, M.O., and Stagljar,
I. 2002a. Genetic approaches to the identification of
interactions between membrane proteins in yeast. J. Recept. Signal
Transduct. Res. 22:
471-481.[CrossRef][Medline]
Auerbach, D., Thaminy, S., Hottiger, M.O., and Stagljar, I.
2002b. The post-genomic era of interactive proteomics: Facts and
perspectives. Proteomics
2: 611-623.[CrossRef][Medline]
Beckmann, A.M. and Wilce, P.A. 1997. Egr transcription
factors in the nervous system. Neurochem. Int.
31:
477-510.[CrossRef][Medline]
Broder, Y.C., Katz, S., and Aronheim, A. 1998. The ras
recruitment system, a novel approach to the study of protein–protein
interactions. Curr. Biol.
8:
1121-1124.[CrossRef][Medline]
Cervantes, S., Gonzalez-Duarte, R., and Marfany, G.
2001. Homodimerization of presenilin N-terminal fragments is
affected by mutations linked to Alzheimer's disease. FEBS
Lett. 505:
81-86.[CrossRef][Medline]
David, N.E., Gee, M., Andersen, B., Naider, F., Thorner, J., and
Stevens, R.C. 1997. Expression and purification of the
Saccharomyces cerevisiae -factor receptor (Ste2p), a
7-transmembrane-segment G protein-coupled receptor. J. Biol.
Chem. 272:
15553-15561.[Abstract/Free Full Text]
de Bono, J.S. and Rowinsky, E.K. 2002. The ErbB
receptor-family: A therapeutic target for cancer. Trends Mol.
Med. 8:
S19-26.[CrossRef][Medline]
Derrien, A. and Druey, K.M. 2001. RGS16 function is
regulated by epidermal growth factor receptor-mediated tyrosine
phosphorylation. J. Biol. Chem.
276:
48532-48538.[Abstract/Free Full Text]
Diamonti, A.J., Guy, P.M., Ivanof, C., Wong, K., Sweeney, C., and
Carraway, 3rd, K.L. 2002. An RBCC protein implicated in
maintenance of steady-state neuregulin receptor levels. Proc. Natl.
Acad. Sci 99:
2866-2871.[Abstract/Free Full Text]
Druey, K.M., Sullivan, B.M., Brown, D., Fischer, E.R., Watson, N.,
Blumer, K.J., Gerfen, C.R., Scheschonka, A., and Kehrl, J.H.
1998. Expression of GTPase-deficient Gi2 results in
translocation of cytoplasmic RGS4 to the plasma membrane. J. Biol.
Chem. 273:
18405-18410.[Abstract/Free Full Text]
Dunnwald, M., Varshavsky, A., and Johnsson, N. 1999.
Detection of transient in vivo interactions between substrate and transporter
during protein translocation into the endoplasmic reticulum. Mol.
Biol. Cell 10:
329-344.[Abstract/Free Full Text]
Ehrhard, K.N., Jacoby, J.J., Fu, X.Y., Jahn, R., and Dohlman, H.G.
2000. Use of G-protein fusions to monitor integral membrane
protein–protein interactions in yeast. Nat.
Biotechnol. 18:
1075-1079.[CrossRef][Medline]
Fields, S. and Song, O. 1989. A novel genetic system
to detect protein–protein interactions. Nature
340:
245-246.[CrossRef][Medline]
Hollinger, S. and Hepler, J.R. 2002. Cellular
regulation of RGS proteins: Modulators and integrators of G protein signaling.
Pharmacol. Rev. 54:
527-559.[Abstract/Free Full Text]
Hubsman, M., Yudkovsky, G., and Aronheim, A. 2001. A
novel approach for the identification of protein–protein interaction
with integral membrane proteins. Nucleic Acids Res.
29: E18.
Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M., and
Sakaki, Y. 2001. A comprehensive two-hybrid analysis to explore
the yeast protein interactome. Proc. Natl. Acad. Sci.
98:
4569-4574.[Abstract/Free Full Text]
Johnsson, N. and Varshavsky, A. 1994. Split ubiquitin
as a sensor of protein interactions in vivo. Proc. Natl. Acad.
Sci. 91:
10340-10344.[Abstract/Free Full Text]
Khachigian, L.M., Williams, A.J., and Collins, T.
1995. Interplay of Sp1 and Egr-1 in the proximal platelet-derived
growth factor A-chain promoter in cultured vascular endothelial cells.
J. Biol. Chem. 270:
27679-27686.[Abstract/Free Full Text]
Laser, H., Bongards, C., Schuller, J., Heck, S., Johnsson, N., and
Lehming, N. 2000. A new screen for protein interactions reveals
that the Saccharomyces cerevisiae high mobility group proteins
Nhp6A/B are involved in the regulation of the GAL1 promoter. Proc.
Natl. Acad. Sci. 97:
13732-13737.[Abstract/Free Full Text]
Massaad, M.J. and Herscovics, A. 2001. Interaction of
the endoplasmic reticulum 1,2-mannosidase Mns1p with Rer1p using the
split-ubiquitin system. J. Cell Sci.
114:
4629-4635.
Matheny, C., Day, M.L., and Milbrandt, J. 1994. The
nuclear localization signal of NGFI-A is located within the zinc finger DNA
binding domain. J. Biol. Chem.
269:
8176-8181.[Abstract/Free Full Text]
Mumberg, D., Muller, R., and Funk, M. 1995. Yeast
vectors for the controlled expression of heterologous proteins in different
genetic backgrounds. Gene
156:
119-122.[CrossRef][Medline]
Olayioye, M.A., Neve, R.M., Lane, H.A., and Hynes, N.E.
2000. The ErbB signaling network: Receptor heterodimerization in
development and cancer. EMBO J.
19:
3159-3167.[CrossRef][Medline]
Pahl, P.M., Hodges, Y.K., Meltesen, L., Perryman, M.B., Horwitz,
K.B., and Horwitz, L.D. 1998. ZNF207, a ubiquitously expressed
zinc finger gene on chromosome 6p21.3. Genomics
53:
410-412.[CrossRef][Medline]
Qiu, X.B. and Goldberg, A.L. 2002. Nrdp1/FLRF is a
ubiquitin ligase promoting ubiquitination and degradation of the epidermal
growth factor receptor family member, ErbB3. Proc. Natl. Acad.
Sci. 99:
14843-14848.[Abstract/Free Full Text]
Reinders, A., Schulze, W., Thaminy, S., Stagljar, I., Frommer,
W.B., and Ward, J.M. 2002. Intra- and intermolecular interactions
in sucrose transporters at the plasma membrane detected by the split-ubiquitin
system and functional assays. Structure
10:
763-772.[Medline]
Riese II, D.J. and Stern, D.F. 1998. Specificity
within the EGF family/ErbB receptor family signaling network.
Bioessays 20:
41-48.[CrossRef][Medline]
Serebriiskii, I., Estojak, J., Berman, M., and Golemis, E.A.
2000. Approaches to detecting false positives in yeast two-hybrid
systems. Biotechniques
28: 328-330,
332-326.[Medline]
Srinivasa, S.P., Bernstein, L.S., Blumer, K.J., and Linder, M.E.
1998. Plasma membrane localization is required for RGS4 function
in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci.
95:
5584-5589.[Abstract/Free Full Text]
Stagljar, I. and Fields, S. 2002. Analysis of membrane
protein interactions using yeast-based technologies. Trends
Biochem. Sci. 27:
559-563.[CrossRef][Medline]
Stagljar, I. and te Heesen, S. 2000. Detecting
interactions between membrane proteins in vivo using chimeras.
Methods Enzymol. 327:
190-198.[Medline]
Stagljar, I., Korostensky, C., Johnsson, N., and te Heesen, S.
1998. A genetic system based on split-ubiquitin for the analysis
of interactions between membrane proteins in vivo. Proc. Natl.
Acad. Sci. 95:
5187-5192.[Abstract/Free Full Text]
Thaminy, S. and Stagljar, I. 2002. The membrane-based
yeast two-hybrid system. In Protein–protein
interactions (ed. E. Golemis), pp.
395-405. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY.
Tsujimoto, Y., Numaga, T., Ohshima, K., Yano, M.A., Ohsawa, R.,
Goto, D., Naito, S., and Ishikawa, M. 2003. Arabidopsis
tobomavirus multiplication (TOM) 2 locus encodes a transmembrane protein that
interacts with TOM1. EMBO J.
22:
335-343.[CrossRef][Medline]
Uetz, P., Giot, L., Cagney, G., Mansfield, T.A., Judson, R.S.,
Knight, J.R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., et al.
2000. A comprehensive analysis of protein–protein
interactions in Saccharomyces cerevisiae. Nature
403:
623-627.[CrossRef][Medline]
Varshavsky, A. 1996. The N-end rule: Functions,
mysteries, uses. Proc. Natl. Acad. Sci.
93:
12142-12149.[Abstract/Free Full Text]
Vojtek, A.B., Hollenberg, S.M., and Cooper, J.A. 1993.
Mammalian Ras interacts directly with the serine/threonine kinase Raf.
Cell 74:
205-214.[CrossRef][Medline]
von Mering, C., Krause, R., Snel, B., Cornell, M., Oliver, S.G.,
Fields, S., and Bork, P. 2002. Comparative assessment of
large-scale data sets of protein–protein interactions.
Nature 417:
399-403.[Medline]
Wittke, S., Lewke, N., Muller, S., and Johnsson, N.
1999. Probing the molecular environment of membrane proteins in
vivo. Mol. Biol. Cell
10:
2519-2530.[Abstract/Free Full Text]
Yarden, Y. 2001. Biology of HER2 and its importance in
breast cancer. Oncology
61: 1-13.
Received February 17, 2003;
accepted in revised format May 12, 2003.
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
S. M. Gisler, S. Kittanakom, D. Fuster, V. Wong, M. Bertic, T. Radanovic, R. A. Hall, H. Murer, J. Biber, D. Markovich, et al.
Monitoring Protein-Protein Interactions between the Mammalian Integral Membrane Transporters and PDZ-interacting Partners Using a Modified Split-ubiquitin Membrane Yeast Two-hybrid System
Mol. Cell. Proteomics,
July 1, 2008;
7(7):
1362 - 1377.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C.J. Carter
Schizophrenia Susceptibility Genes Directly Implicated in the Life Cycles of Pathogens: Cytomegalovirus, Influenza, Herpes simplex, Rubella, and Toxoplasma gondii
Schizophr Bull,
June 13, 2008;
(2008)
sbn054v1.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. M. Sanderson
A new way to explore the world of extracellular protein interactions
Genome Res.,
April 1, 2008;
18(4):
517 - 520.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Suter, M. J. Fetchko, R. Imhof, C. I. Graham, I. Stoffel-Studer, C. Zbinden, M. Raghavan, L. Lopez, L. Beneti, J. Hort, et al.
Examining protein protein interactions using endogenously tagged yeast arrays: The Cross-and-Capture system
Genome Res.,
December 1, 2007;
17(12):
1774 - 1782.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C.-J. Cheng, X.-c. Ye, F. Vakar-Lopez, J. Kim, S.-M. Tu, D.-T. Chen, N. M. Navone, L.-Y. Yu-Lee, S.-H. Lin, and M. C-T. Hu
Bone Microenvironment and Androgen Status Modulate Subcellular Localization of ErbB3 in Prostate Cancer Cells
Mol. Cancer Res.,
July 1, 2007;
5(7):
675 - 684.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Kim, H. R. Kim, and K.-H. Paek
Arabidopsis tonoplast proteins TIP1 and TIP2 interact with the cucumber mosaic virus 1a replication protein.
J. Gen. Virol.,
November 1, 2006;
87(Pt 11):
3425 - 3431.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Shire, P. Kapoor, K. Jiang, M. N. T. Hing, N. Sivachandran, T. Nguyen, and L. Frappier
Regulation of the EBNA1 Epstein-Barr Virus Protein by Serine Phosphorylation and Arginine Methylation.
J. Virol.,
June 1, 2006;
80(11):
5261 - 5272.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Mo and M. Bard
Erg28p is a key protein in the yeast sterol biosynthetic enzyme complex
J. Lipid Res.,
September 1, 2005;
46(9):
1991 - 1998.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Chavan, A. Yan, and W. J. Lennarz
Subunits of the Translocon Interact with Components of the Oligosaccharyl Transferase Complex
J. Biol. Chem.,
June 17, 2005;
280(24):
22917 - 22924.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
ABSTRACT
RESULTS
