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Plant Physiology 132:1870-1883 (2003) © 2003 American Society of Plant Biologists MPB2C, a Microtubule-Associated Plant Protein Binds to and Interferes with Cell-to-Cell Transport of Tobacco Mosaic Virus Movement Protein1Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Institute of Medical Biochemistry, University of Vienna, Dr. Bohr-Gasse. 9, A-1030 Vienna, Austria
The movement protein of tobacco mosaic virus, MP30, mediates viral cell-to-cell transport via plasmodesmata. The complex MP30 intra- and intercellular distribution pattern includes localization to the endoplasmic reticulum, cytoplasmic bodies, microtubules, and plasmodesmata and likely requires interaction with plant endogenous factors. We have identified and analyzed an MP30-interacting protein, MPB2C, from the host plant Nicotiana tabacum. MPB2C constitutes a previously uncharacterized microtubule-associated protein that binds to and colocalizes with MP30 at microtubules. In vivo studies indicate that MPB2C mediates accumulation of MP30 at microtubules and interferes with MP30 cell-to-cell movement. In contrast, intercellular transport of a functionally enhanced MP30 mutant, which does not accumulate and colocalize with MP30 at microtubules, is not impaired by MPB2C. Together, these data support the concept that MPB2C is not required for MP30 cell-to-cell movement but may act as a negative effector of MP30 cell-to-cell transport activity.
Plant viruses produce movement proteins required to mediate cell-to-cell spread of the viral genomic information via plasmodesmata (Lucas, 1995  ;
Lazarowitz and Beachy, 1999;
McLean and Zambryski, 2000;
Tzfira et al., 2000;
Haywood et al., 2002;
Heinlein, 2002). Tobacco
mosaic virus (TMV), a virus with a (+) single-stranded RNA genome encodes one
of the most extensively studied movement proteins, MP30. Several biochemical
and cell biological features of MP30 were revealed: MP30 binds cooperatively
single-stranded nucleic acids (Citovsky et
al., 1990), interacts with the tubulin- and actin-based
cytoskeleton (Heinlein et al.,
1995; McLean et al.,
1995), associates to the endoplasmic reticulum (ER;
Heinlein et al., 1998;
Reichel and Beachy, 1998;
Mas and Beachy, 1999;
Gillespie et al., 2002), and
increases the size exclusion limit of plasmodesmata
(Wolf et al., 1989;
Waigmann et al., 1994). In the
current model, MP30 is proposed to form complexes with genomic TMV-RNA (vRNA)
and to move these vRNA-protein complexes from ER-associated sites of synthesis
throughout the cell via the cytoskeletal network toward plasmodesmata. There,
MP30 induces an increase in plasmodesmal size exclusion limit and facilitates
the transport of the vRNA-MP30 complex through the enlarged plasmodesmal
channels into adjacent cells (Lazarowitz
and Beachy, 1999; Tzfira et
al., 2000; Haywood et al.,
2002; Heinlein,
2002). In line with this model, microtubules have been
functionally implicated in guiding vRNA-MP30 complexes toward plasmodesmata
(Mas and Beachy, 2000; Boyko
et al., 2000a,
2000b). However, an alternate,
non-movement-promoting role of microtubules in providing a route toward an
MP30 degradation site was suggested
(Padgett et al., 1996;
Mas and Beachy, 1999;
Reichel and Beachy, 2000).
This model is supported by the observation that TMV was able to spread in
tissues with functionally impaired microtubules and by studies performed with
a mutant variant of MP30, MPR3
(Toth et al., 2002) with
strongly decreased affinity to microtubules but increased cell-to-cell
movement capacity (Gillespie et al.,
2002).
The complex intra- and intercellular transport processes likely require
interaction of MP30 with cellular factors that assist in subcellular
distribution and cell-to-cell movement. So far, a cell wall-associated pectin
methyl esterase (PME; Dorokhov et al.,
1999
For a number of movement proteins from other viruses, interacting cellular
factors such as a rab receptor-like protein, Hsp40 (DnaK) family members, a
homeodomain protein, and a transcriptional coactivator were recently
identified (Soellick et al.,
2000 Here, we report the identification of a previously uncharacterized MP30-interacting plant protein, MPB2C, from the TMV host plant Nicotiana tabacum. MPB2C associates with microtubules in a punctuate manner and colocalizes with MP30. In functional studies, MPB2C mediates MP30 subcellular redistribution to microtubule-associated sites and selectively inhibits MP30 cell-to-cell movement. A potential role of MPB2C as a negative effector of MP30 transport activity is discussed.
Identification of MP30-Interacting Factor MPB2C via the Yeast Sos Recruitment System (SRS)
The partially hydrophobic nature of MP30
(Citovsky et al., 1990
In the SRS, the bait protein is fused to the human Ras guanyl nucleotide
exchange factor (hSos), which complements the temperature-sensitive (ts)
cdc25-2 yeast growth phenotype upon recruitment to the plasma membrane. Prey
library proteins are N-terminally linked to a myristoylation signal providing
plasma membrane localization. Bait-prey interaction results in recruitment of
hSos to the plasma membrane and leads to growth of yeast colonies at the
restrictive temperature (Aronheim et al.,
1997 First, a bait MP30-Sos fusion protein expressed by the yeast shuttle vector ADNS-MP (Fig. 1A, top scheme) was constructed. To ensure that MP30-Sos by itself was not able to complement, or to interfere with complementation of, the cdc25-2 ts phenotype, cells were cotransformed with ADNS-MP30 and empty library vector YESMpolyA. No yeast colonies were formed at the restrictive temperature (Fig. 1B, top panels). Thus, MP30 did not mediate hSos localization to the plasma membrane in yeast. Selective Gal-dependent growth was observed when cells were cotransformed with positive control vector YESM7 encoding an hSos-interacting protein and MP30-Sos-expressing bait plasmid (Fig. 1B, panels in second row). This functional test indicates that the MP30-hSos fusion protein is stable, can be translocated to the plasma membrane, and is able to activate the Ras pathway. Thus, MP30 fused to hSos was suitable to be used as bait in a SRS screen.
Next, directional cDNA derived from poly(A+)-RNA obtained from leaf, stem, shoot apex, and flower tissue of the natural TMV host plant N. tabacum cv Turkish was used to construct a myristoylation signal-tagged expression library (Fig. 1A, bottom scheme). To isolate cDNAs, which encode interaction partners of MP30, approximately 106 independent cDNA clones were screened. A novel cDNA named movement protein binding 2C, library (MPB2Clib) coding for a 124-amino acid protein (Fig. 3A) was selected for further analysis.
Specific interaction between MPB2Clib and MP30-Sos was apparent
by Gal-dependent growth of cells co-expressing MPB2Clib and
MP30-Sos at the restrictive temperature
(Fig. 1B, panels in third row).
In contrast, upon co-expression of MPB2Clib with a nonrelevant bait
protein, p110-Sos, no growth of yeast colonies was observed
(Fig. 1B, bottom panels). These
complementation tests confirmed the specificity of interaction between MP30
and MPB2Clib. Interestingly, MPB2Clib interacted with
Specificity of the yeast-based interaction studies was confirmed in vitro
by overlay assays. First, an Anti-Xpress-tag-MPB2Clib fusion
protein of approximately 20 kD, and, as a negative control, tag protein alone
with approximately 8 kD were produced in Escherichia coli and
detected by western blotting using Anti-Xpress-tag antibody
(Fig. 2A, lanes 1 and 2). For
overlay assays (Fig. 2B), purified MP30 was blotted onto nitrocellulose, and MP30 presence and size were
verified with an antibody directed against MP30
(Fig. 2A, lane 3). In parallel,
part of the blot was renatured and incubated with either total E.
coli lysate containing tag-MPB2Clib or tag protein alone.
Addition of tagged MPB2Clib resulted in strong binding, which could
be competed by the addition of an excess of MP30 to the overlay mixture
(Fig. 2B). In contrast, no
signal was detected in the control reaction with tag protein alone
(Fig. 2B). To determine whether
MPB2C is capable of interacting with a viral movement protein functionally
related to MP30, an immobilized cucumber mosaic virus movement protein (CMV3a;
Li and Palukaitis, 1996
By RACE techniques, full-length MPB2C cDNA with a size of 1.2 kb was isolated. Upon northern-blot analysis of poly(A+)-RNA from green tissue of N. tabacum with an MPB2C-specific probe (Fig. 3B, lane 1), a band correlating to the calculated size of MPB2C cDNA was observed. As a positive control for RNA quality, a ubiquitin-specific probe was used (Fig. 3B, lane 2). Together, RACE and northern results indicate that the full-length MPB2C cDNA was isolated. Full-length MPB2C cDNA translates into a 321-amino acid protein (Fig. 3A) with a calculated molecular mass of 36 kD. MPB2C protein expression was confirmed by western blotting with an anti-MPB2C antibody raised against purified E. coli-generated MPB2C. When total protein extract derived from leaf tissue was analyzed with the anti-MPB2C antibody, a single MPB2C-specific band with a molecular mass of approximately 40 kD was detected (Fig. 3C, lane 1) that was not present upon incubation of the blot with preimmune serum (Fig. 3C, lane 2), indicating that the MPB2C protein is constitutively expressed in leaf tissue. Note that the aberrantly high molecular mass of the MPB2C protein in denaturing polyacrylamide gels was also observed for the recombinant E. coli MPB2C protein (data not shown).
Structural analysis of full-length MPB2C protein revealed a large
coiled-coil region typical for cytoskeletal-associated factors
(Lupas et al., 1991
To evaluate intracellular localization, full-length and N-terminally
truncated variants of MPB2C lacking either the hydrophobic region
(
The filamentous appearance of the Furthermore, to determine the subcellular localization of endogenous MPB2C, protoplasts derived from leaf tissue were analyzed by immunofluorescence. Fixed protoplasts were incubated with anti-tubulin antibody and anti-MPB2C antibody (Fig. 5, A and B) or, as a negative control, with anti-tubulin antibody and preimmune serum (Fig. 5C). Intracellular localization patterns were evaluated by confocal microscopy. The MPB2C-specific signal was detected in the green channel and manifested as punctae that generally aligned to or were in close contact with microtubular filaments detected in the red channel (Fig. 5, A and B, arrows). In contrast, in protoplasts incubated with preimmune serum, microtubule-associated punctae were not detected (Fig. 5C). For comparison, a high-resolution image of transiently co-expressed MPB2C-RFP and MAP4-GFP is provided. Note that MPB2C-RFP punctae (Fig. 5D, arrows) align to MAP4-GFP-decorated microtubules highly similar to endogenous MPB2C.
Collectively, these studies indicate that endogenous MPB2C localizes in discrete punctae to microtubular filaments and that this punctuate localization pattern is correctly reflected by transiently expressed MPB2C-RFP. However, in transient expression experiments, we additionally observe large MPB2C-RFP-decorated bodies that do not correctly reflect the localization of endogenous MPB2C and are likely a consequence of the high expression level achieved in the transient assay.
As a necessary prerequisite for interaction between MPB2C and MP30 in plant
cells in vivo, both proteins should be present in the same subcellular
compartment(s). In line, the filament and body-like appearance of MPB2C
resembles described intracellular localization patterns of MP30 (Heinlein et
al., 1995
Upon transient expression in epidermal cells of N. tabacum source
leaves, MPB2C-RFP-decorated bodies and punctae
(Fig. 6, A and D) colocalize to
MP30-GFP (Fig. 6, B and E) in
the merged image (Fig. 6, C and
F; arrowheads in A, B, and C point to some of the colocalizing
punctae). In addition, an MP30-GFP signal is also observed in small regular
cortical punctae (Fig. 6B) reflecting ER-associated MP30-GFP
(Gillespie et al., 2002
Interestingly, in the presence of transiently expressed MPB2C-RFP fusion
proteins, MP30-GFP was nearly exclusively localized to microtubule-associated
sites and to the ER (Fig. 6, B, E, and
H) and was localized in only 4% of co-expressing cells to the
characteristic cell wall-associated punctae, reflecting plasmodesmal
association of MP30 (compare Fig.
7A; Crawford and Zambryski,
2000
Following biolistic bombardment of epidermal cells, MP30-GFP fusion
proteins are known to move from cell to cell via plasmodesmata
(Fig. 7A; Crawford and
Zambryski, 2000
Inhibition of intercellular transport of MP30-GFP in cells expressing
MPB2C-RFP proteins could be due to specific interaction between MPB2C and MP30
or could result from general down-regulation of plasmodesmal transport
potentially induced by overexpression of MPB2C proteins. To discriminate
between these possibilities, the viral movement protein CMV3a, which is
functionally related to MP30 but does not interact with MPB2C in vitro
(compare Fig. 2B), was
employed. Like MP30-GFP, a GFP-tagged CMV3a fusion protein moves between cells
(Fig. 7G;
Itaya et al., 1997
The negative effect on cell-to-cell transport of MP30-GFP might be due to
improper amounts of fluorescently tagged MPB2C proteins and/or the RFP tag
which might mask a potential positive involvement of MPB2C in MP30
cell-to-cell transport. To further clarify the function of MPB2C in
intercellular transport of MP30, we employed a L72V point mutant of MP30
(MPR3; Gillespie et al.,
2002
When MPR3-GFP was co-expressed with full-length MPB2C-RFP or
We further tested whether in vitro binding between MPB2C and MPR3 would be reduced as compared with binding between MPB2C and MP30. For this purpose, MPR3 and MP30 were overexpressed and purified from E. coli (Fig. 8A). Equal amounts of purified MPR3 and MP30 protein were blotted onto nitrocellulose, renatured, and overlayed with purified MPB2C protein. Binding of MPB2C to MP30 or MPR3 was detected with an antibody directed against the MPB2C protein. Several molar ratios of MPB2C to MPR3 and MP30 were tested. When the molar amount of MPB2C exceeded that of MPR3 or MP30 2-fold, MPR3 showed slightly reduced binding to MPB2C as compared with MP30 (Fig. 8B, compare lanes MPR3 and MP30). When MPB2C was present at a 2.5-fold lower molar amount than MPR3 or MP30, binding of MPB2C to MPR3 was clearly reduced as compared with MP30 (Fig. 8C, compare lanes MPR3 and MP30). Together, these results indicate that in vitro MPB2C binds with lower affinity to the MPR3 protein than to the MP30 protein.
The frequently observed localization patterns of MP30 comprising accumulation at ER-associated punctae, microtubules, large bodies, and cell wall-associated punctae have been combined in a model for TMV cell-to-cell transport that involves transfer of MP30 and viral RNA from the ER via a microtubular delivery mechanism toward and through plasmodesmata (Lazarowitz and Beachy, 1999 ;
Tzfira et al., 2000;
Haywood et al., 2002;
Heinlein, 2002), thereby
indicating a movement promoting role of microtubules in transport of the
MP30-vRNA complex (Heinlein et al.,
1995,
1998;
McLean et al., 1995;
Mas and Beachy, 2000; Boyko et
al., 2000a,
2000b;
Kotlizky et al., 2001). On the
other hand, microtubules have also been implicated in MP30 degradation
(Padgett et al., 1996;
Mas and Beachy, 1999;
Reichel and Beachy, 2000). In
line, an MP30 mutant that escapes accumulation at microtubules but displays
enhanced cell-to-cell movement was recently described (MPR3;
Gillespie et al., 2002),
indicating that accumulation at microtubules is detrimental for MP30
movement. The complex MP30 localization pattern likely requires interaction with several plant endogenous factors. Here, we describe the isolation and characterization of a previously uncharacterized, plant-specific MP30-binding protein, MPB2C, that localizes in discrete punctae to microtubules. The C-terminal one-third of the 321-amino acid-long MPB2C is sufficient to bind MP30. The N-terminal part of MPB2C harbors a hydrophobic region and a coiled-coil domain involved in association to microtubules. As shown by northern and western analysis, MPB2C is constitutively expressed in N. tabacum leaf tissue. Massively Parallel Signature Sequencing data on the Arabidopsis homolog of MPB2C also demonstrate constitutive expression at low levels throughout plant tissues (http://allometra.com/mpss.shtml), indicating a general housekeeping function of MPB2C.
The localization of MPB2C to discrete punctae along microtubules points to
the presence of specific target sites on microtubules and is reminiscent of
patterns observed for other microtubule-associated proteins such as a
kinesin-related motor protein (Cai et al.,
2000
What may be the nature of large bodies that are detected in addition to
microtubule-associated punctae upon transient expression of MPB2C? In
co-expression experiments, MPB2C bodies are also tagged by MP30-GFP and
MAP4-GFP and form in close proximity to the microtubular network (compare
Fig. 4, D–F), suggesting
that these bodies may contain a mixture of microtubule-associated proteins.
Interestingly, microtubule-connected bodies are also observed upon
overexpression of CLIP-170-GFP (Perez et
al., 1999
In transient expression studies, both full-length MPB2C and
Functional analysis by transient expression studies revealed a specific,
MPB2C-mediated decrease in MP30 cell-to-cell movement, suggesting a negative
impact of MPB2C on MP30 transport activity. The negative effect of MPB2C on
MP30 transport activity was analyzed in two different host plants and varied
from a complete block in N. tabacum to a more than 50% reduction in
N. benthamiana. This host-dependent difference may be correlated to
the well-known high susceptibility of N. benthamiana for viral
infection (Gibbs et al., 1997
Further support for a non-movement-promoting role of MPB2C was derived from
analyzing the impact of MPB2C proteins on intercellular transport activity of
MPR3, an L72V point mutant of MP30 with improved cell-to-cell
movement capacity and decreased affinity for microtubules
(Gillespie et al., 2002 Considering that the MPR3 protein and wild-type MP30 are distinguished only by a point mutation, it is likely that the two proteins move cell-to-cell via a similar mechanism, and that interaction with MPB2C might also not be required for cell-to-cell movement of wild-type MP30. Because the MPR3 protein is characterized by enhanced transport activity and because we observed an MPB2C-mediated decrease of MP30 transport activity in transient expression studies, we presently favor the hypothesis that endogenous MPB2C serves a negative role in MP30 cell-to-cell transport. However, to further elucidate and define the function of MPB2C in cell-to-cell transport of TMV-MP and in intercellular spread of TMV, it will be necessary to generate plants with reduced endogenous MPB2C levels such as anti-sense or silenced plants.
In line with our hypothesis, we propose a model wherein endogenous MPB2C
encounters and binds to MP30 at microtubules and acts in a pathway that
promotes MP30 accumulation at microtubules. This pathway is not involved in
MP30 cell-to-cell transport but might be assigned for degradation as
previously suggested (Padgett et al.,
1996 In summary, we report the identification and characterization of MPB2C, a plant endogenous, microtubule-localized MP30 interaction partner. Future studies will be directed toward a detailed evaluation of the impact of MPB2C on local and systemic spread of TMV and to reveal the as yet unknown endogenous function of the MPB2C protein.
Plant Growth Conditions Six- to 8-week-old Nicotiana tabacum cv Turkish or Nicotiana benthamiana plants were used. Plants were grown in a controlled environment with a cycle of 16 h of light at 22°C and 8 h of darkness at 20°C.
Conventional PCR cloning techniques were employed to construct the MP30-Sos
expressing bait plasmids ADNS-MP30 and ADNS
Compositions of yeast (Saccharomyces cerevisiae) Gal and Glc
containing growth media and transformation reaction techniques and growth
conditions were employed as described
(Ausubel et al., 1987
Poly(A+)-RNA was isolated as described above, and northern
analysis was carried out using the NorthernMax-Gly Kit according to the
manufacturer's protocol (Ambion, Austin, TX). An MPB2C-specific probe was
produced by PCR of the library plasmid YES-MPB2Clib with primers
FK76 and FK94. The ubiquitin-specific probe was prepared as described
(Sablowski and Meyerowitz,
1998
A glutathionine S-transferase (GST)-MPB2C expression construct was
obtained by PCR amplification of MPB2C from 35S::MPB2C-RFP using primers
5'-GCAGGATCCGATGACGATGACAAGATGGCGCTTGCATTTG-3' and
5'-GCAGAATTCTTATGTTCTCAGAACAAG-3'. The resulting fragment was
digested with BamHI/EcoRI and was cloned into
BamHI/EcoRI-digested vector pGEX-6P-1 encoding a GST moiety
followed by a PreScission Protease cleavage site (Amersham Biosciences,
Uppsala) to yield construct pGEX-MPB2C. To obtain soluble GST-MPB2C,
BL21-CodonPlus bacteria transformed with pGEX-MPB2C were grown at 30°C
until optical density = 0.6. Induction was performed with 0.1 mM
isopropyl-
N. tabacum leaf tissue was frozen and ground to powder in
liquid N2. Proteins were extracted by shaking the powder in the
same volume of 2x Laemmli sample buffer
(Ausubel et al., 1987
The MPB2C cDNA fragment isolated in the SRS-screen was PCR amplified with
specific primers FK76
(5'-CCGGCTCGAGAGTTAAGTTAGCTGACAAGAAAGCAGC-3') and FK94
(5'-CGGAATTCCGTTAAGTTAGCTGACAAGAAAGCAGCGGTAG-3') and was cloned as
an XhoI/EcoRI fragment into
XhoI/EcoRI-digested pRSETA to obtain the expression plasmid
pRSETA-
Protoplasts were prepared as described previously
(Koop et al., 1996
The pUC18-based vectors pZY226 and pZY260 containing a 10x Ala linker
at the C or N terminus of a 35S driven mGFP6, respectively, were a generous
gift to F.K. by D. Jackson (Cold Spring Harbor Laboratories, Cold Spring
Harbor, NY). GFP expression vectors producing soluble smGFP (CD3–326;
Arabidopsis Biological Resource Center, Ohio State University, Columbus),
ER-GFP (Haseloff et al.,
1997
To obtain MPB2C-RFP fusion proteins with a separating Ala linker between
the two protein moieties, a 35S::GFP-RFP plasmid was constructed first by
cloning a SalI/XbaI-digested RFP PCR fragment amplified from
pDsRed1-N1 (BD Biosciences Clontech) using primers FK111
(5'-GCCGTCGACATGGTGCGCTCCTCC-3') and FK112
(5'-CGCCTCTAGACTACAGGAACAGGTGG-3') into
SalI/XbaI-digested pZY260. A BamHI/XbaI
fragment of the 35S::GFP-RFP plasmid containing the Ala linker and RFP was
ligated with BsaI(NcoI)/BamHI-digested MPB2C PCR fragments
(primer combinations: FK131 (5'-GCCGGTCTCCCATGGCGCTTGCATTTGAC-3')
or FK 114 (5'-GCCGGTCTCCCATGACCTATACCAAA-3') with FK110) into
NcoI/XbaI-digested pZY226 resulting in expression plasmids
35S::MPB2C-RFP or 35S::
DNA was coated onto 1-µm gold particles, and N. tabacum or N. benthamiana source leaves (>20 cm) were bombarded using a Helios Gene Gun following the manufacturer's protocol (Bio-Rad Laboratories, Hercules, CA). Each bombardment experiment was repeated at least five times with the total number of analyzed cells ranging from 40 to 100. Images were obtained with a TCS-SP confocal microscope (Leica Microsystems, Heidelberg). GFP and RFP were excited with a ArKr laser at 476 and 568 nm, respectively. GFP was detected at 500 to 520 nm, and RFP was detected at 600 to 620 nm. Micrographs were assembled from serial optical sections and were prepared for presentation by Photoshop 5.1 software (Adobe Systems, San Jose, CA).
The cDNA sequence of MPB2C has been deposited at GenBank under accession number AF326729.
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes with the exception of the anti-MPB2C antibodies, which are available only in limited amounts.
We thank A. Aronheim for providing plasmids and the yeast strain required for the SRS screen. We are also indebted to Dave Jackson for providing vectors pZY260 and pZY226, to Peter Palukaitis for providing vector pET-CMV3a, to Vitaly Citovsky for providing vector 35S::RFP, to Biao Ding for providing vector pRTL-CMV3a-GFP, to Richard Cyr for providing a MAP4-GFP expression construct, and to Karl Oparka for providing MPR3-GFP expression construct. E.W. is thankful to Andrea Barta for her invaluable support throughout the whole project. Received March 5, 2003; returned for revision March 21, 2003; accepted April 24, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.022269.
1 This work was supported by the Austrian Science Foundation (grant nos.
P12614–MOB and Sfb17, project part 08 to E.W.). E.W. was supported by an
Austrian Programme for Advanced Research and Technology fellowship (APART 441)
from the Austrian Academy of Science.
2 Present address: Section of Plant Science, University of California Davis,
One Shields Ave, Davis, CA 95616.
3 These authors contributed equally to this paper. * Corresponding author; e-mail waigmann{at}bch.univie.ac.at; fax 43–1–4277–9616.
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TOP
ABSTRACT
RESULTS
1-58MP30-Sos (
, ura3-5,
his3-200, ade2-101, lys2-801, trp1-109, leu2-3, cdc25-2, and Gal-) strains
transformed with library plasmid and various bait plasmids were first
incubated in a 21°C incubation chamber on selectable Glc plates
(non-restrictive conditions). Subsequently, colonies of transformed cells were
replica plated in parallel onto plates containing Gal- or Glc-selective
medium. Plates were incubated at 36C° (restrictive conditions) for >5 d
to evaluate bait-prey interaction-mediated growth complementation of the
cdc25-2 ts phenotype (Aronheim et al.,
1997
-D-thiogalactopyranoside for 1 h at 15°C.
Bacteria were collected by centrifugation and were lysed by sonication in
phosphate-buffered saline (PBS; Ausubel et
al., 1987
