The Plant Journal (2010) 61, 259–270
doi: 10.1111/j.1365-313X.2009.04048.x
Arabidopsis homolog of the yeast TREX-2 mRNA export
complex: components and anchoring nucleoporin
Qing Lu1,2, Xurong Tang1,3, Gang Tian1,4, Fang Wang1,5, Kede Liu1,5, Vi Nguyen1, Susanne E. Kohalmi4, Wilfred A. Keller3,
Edward W.T. Tsang3, John J. Harada6, Steven J. Rothstein2 and Yuhai Cui1,*
1
Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, London, ON N5V 4T3, Canada,
2
Department of Molecular and Cellular Biology, University of Guelph, ON N1G 2W1, Canada,
3
Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, SK S7N 0W9, Canada,
4
Department of Biology, University of Western Ontario, London, ON N6A 5B7, Canada,
5
National Key Laboratory of Crop Genetic Improvement, Huazhong Agriculture University, Wuhan 430070, China, and
6
Section of Plant Biology, College of Biological Sciences, University of California, Davis, CA 95616, USA
Received 23 July 2009; revised 22 September 2009; accepted 5 October 2009; published online 9 November 2009.
*
For correspondence (fax +1 519 457 3997; e-mail yuhai.cui@agr.gc.ca).
SUMMARY
Nuclear pore complexes (NPCs) are vital to nuclear–cytoplasmic communication in eukaryotes. The yeast NPCassociated TREX-2 complex, also known as the Thp1–Sac3–Cdc31–Sus1 complex, is anchored on the NPC via
the nucleoporin Nup1, and is essential for mRNA export. Here we report the identification and characterization
of the putative Arabidopsis thaliana TREX-2 complex and its anchoring nucleoporin. Physical and functional
evidence support the identification of the Arabidopsis orthologs of yeast Thp1 and Nup1. Of three Arabidopsis
homologs of yeast Sac3, two are putative TREX-2 components, but, surprisingly, none are required for mRNA
export as they are in yeast. Physical association of the two Cdc31 homologs, but not the Sus1 homolog, with
the TREX-2 complex was observed. In addition to identification of these TREX-2 components, direct
interactions of the Arabidopsis homolog of DSS1, which is an established proteasome component in yeast and
animals, with both the TREX-2 complex and the proteasome were observed. This suggests the possibility of a
link between the two complexes. Thus this work has identified the putative Arabidopsis TREX-2 complex and
provides a foundation for future studies of nuclear export in Arabidopsis.
Keywords: TREX-2 mRNA export complex, nucleoporin NUP1, THP1, SAC3, DSS1.
INTRODUCTION
The nuclear and cytoplasmic compartmentation of eukaryotes physically separates transcription and translation,
necessitating nucleo-cytoplasmic trafficking. The exchange
of molecules between the nucleus and cytoplasm is mediated by nuclear pore complexes (NPCs) embedded in the
nuclear envelope. The structure and composition of the NPC
and the associated transport machineries have been well
characterized in yeast and mammalian systems (Cole and
Scarcelli, 2006; Tran and Wente, 2006; Akhtar and Gasser,
2007; Kohler and Hurt, 2007; D’Angelo and Hetzer, 2008;
Iglesias and Stutz, 2008), but little is known of these structures in plants (Chinnusamy et al., 2008; Xu and Meier, 2008;
Meier and Brkljacic, 2009). In this paper, we focus on one
NPC-associated mRNA export complex in Arabidopsis
thaliana.
The overall structure of NPCs is evolutionarily conserved
from yeasts to mammals. The NPC has an eight-fold
ª 2009 Her Majesty the Queen in Right of Canada
Journal compilation ª 2009 Blackwell Publishing Ltd
symmetrical structure comprising a central transport channel and two rings – the cytoplasmic and nuclear rings – to
which eight filaments are attached. The cytoplasmic filaments have loose ends, while the nuclear filaments are
joined in a distal ring, forming a nuclear basket. NPCs
typically contain approximately 30 different nucleoporin
proteins. Most of the nucleoporins are located symmetrically on both sides of the NPC, and only a few are
asymmetrically located either on the cytoplasmic or nuclear
face. NPCs are highly dynamic in configuration and composition, and are involved in diverse cellular processes that
range from organization of the cytoskeleton to gene expression, including the selective export of mRNA and establishment of higher-order levels of organization of the nuclear
genome (Tran and Wente, 2006; D’Angelo and Hetzer, 2008).
Molecules with a mass greater than 40 kDa need to be
actively transported into the nucleus. Such active transport
259
260 Qing Lu et al.
is often dependent on association with receptors and
facilitated by NPC-associated complexes. The TREX-2 (transcription-coupled export 2) complex, formerly called Sac3–
Thp1–Sus1–Cdc31, is an mRNA export complex in yeast
(Fischer et al., 2002, 2004; Gallardo et al., 2003; Rodrı́guezNavarro et al., 2004; Kohler and Hurt, 2007). The TREX-2
complex is tethered to the inner side of the NPC via the
nucleoporin Nup1 (Fischer et al., 2002). The Sus1 component interacts with the SAGA transcriptional co-activator
complex (Köhler et al., 2006; Shukla et al., 2006), and thus
the TREX-2 complex has been proposed to functionally
couple SAGA-dependent transcription to mRNA export at
the inner side of the NPC (Rodrı́guez-Navarro et al., 2004;
Kohler and Hurt, 2007). Mutations of any components of the
TREX-2 complex and Nup1 cause accumulation of polyadenylated RNA in the nucleus. Components of the TREX-2
complex have also been identified recently in Drosophila
and humans (Kurshakova et al., 2007; Jani et al., 2009).
Here we show that an Arabidopsis gene identified in an
unrelated screen (Tang et al., 2008) is the ortholog of yeast
Thp1. Further protein interaction studies and functional
analyses identified Arabidopsis homologs of another key
component of the yeast TREX-2 mRNA export complex,
Sac3, and its anchoring nucleoporin Nup1. Physical interactions of the previously identified putative Arabidopsis
homologs of yeast Cdc31 (AtCEN1 and 2; Molinier et al.,
2004) and Sus1 (AtSUS1; Rodrı́guez-Navarro et al., 2004)
with the newly identified TREX-2 components were tested.
Moreover, the Arabidopsis homolog of yeast Dss1/Sem1
was identified as an AtTHP1 interaction partner and thus a
TREX-2 component.
RESULTS
Identification of the Arabidopsis homolog of yeast Thp1
We identified the Arabidopsis homolog of yeast Thp1 fortuitously in a genetic screen to identify mutants exhibiting
ectopic expression of a soybean conglycinin (7S storage
protein) gene promoter–GUS transgene (bCGpro:GUS) (Tang
et al., 2008). Mutant plants (initially named essp1) had diverse developmental defects, such as curly leaves, shorter
siliques, shorter primary roots and fewer lateral roots (Figure 1a–d). GUS staining was observed only along the leaf
margins, and we detected mRNA from the Arabidopsis gene
encodes a putative 7S storage protein (At7S1/At4g36700;
Tang et al., 2008) (Figure 1e).
The mutation segregated 3:1 with respect to GUS
phenotype, and is thus a single recessive mutation. We
mapped the essp1 locus to a genomic interval of approximately 37 kb on chromosome 2 (8 447 799–8 484 961 bp;
Figure 1f), based on the curly leaf and GUS phenotype,
using standard procedures (Lukowitz et al., 2000; Tang et al.,
2008). To identify the molecular lesion in essp1, the genomic
region was amplified by PCR and sequenced. A single
C fi T point mutation was identified in At2g19560, a gene
that is predicted to encode a PCI-domain protein (Kim et al.,
2001). The mutation is predicted to result in a premature stop
codon, leading to truncation of the PCI domain (Figure 1g).
The gene was also recently identified as EER5, acting in the
ethylene signaling pathway (Christians et al., 2008).
To confirm that ESSP1 is At2g19560, we first introduced
the cDNA, under the control of the 35S promoter, into the
essp1 plants. In total, more than 100 transgenic lines were
obtained, all of which showed wild-type morphology and no
GUS activity in leaves (data not shown). Further, two T-DNA
insertion lines, SK6095 and SAIL_82A02, were obtained, and
plants homozygous for the T-DNA insertions were crossed
with bCGpro:GUS. In the F2 generation, about a quarter of the
plants showed the curly leaf phenotype, and all these plants
showed GUS staining at the leaf margin (data not shown).
Together, these data strongly suggest that ESSP1 is
At2g19560. We also used RT-PCR to show that At7S1 is
expressed in the two T-DNA lines (Figure 1e), further
confirming that ESSP1 is At2g19560. In addition, we generated promoter–GUS gene transgenic lines, and observed
GUS expression in seedlings, roots, leaves and anthers
(Figure S1). The expression patterns observed for ESSP1
are consistent with the results of several DNA microarray
experiments (http://www.arabidopsis.org) and those
obtained by Christians et al. (2008).
It was reported by Ciccarelli et al. (2003) that ESSP1
contains both the PCI domain and the PCI-associated
module (PAM), and also identified ESSP1 as a potential
ortholog of yeast Thp1 using refined search programs. We
localized ESSP1 by expressing a gene encoding an ESSP1–
YFP fusion protein under the control of the 35S promoter in
the essp1 mutant. The transgene rescued the essp1 mutant
phenotype (data not shown). As shown in Figure 1(h,i), the
YFP signal is localized in the nucleus, consistent with its
putative identity as Arabidopsis THP1, which is part of the
TREX-2 complex. Therefore, we refer to ESSP1 as AtTHP1,
and describe below experiments investigating whether
AtTHP1 is a component of the Arabidopsis TREX-2 complex.
Identification of AtTHP1-interacting proteins by a yeast
two-hybrid screen
Based on the bioinformatic prediction (Ciccarelli et al., 2003)
and protein localization (Figure 1h,i), we hypothesized that
AtTHP1 is the Arabidopsis homolog of yeast Thp1, and
predicted that it would interact with other Arabidopsis
TREX-2 components. In addition, AtTHP1 is one of three PCIdomain proteins whose function remains uncharacterized,
out of 20 such proteins in Arabidopsis. The others are all
subunits of large protein complexes: the proteasome, COP9
singnalosome or the translational initiation factor elF3 (Kim
et al., 2001). To test the hypothesis that it would interact with
other Arabidopsis TREX-2 components, we performed a
yeast two-hybrid (Y2H) screen to search for its interacting
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TREX-2 mRNA export complex 261
(a)
(f)
(c)
(g)
(b)
(h)
(i)
(j)
(k)
(d)
(e)
Figure 1. Identification and characterization of AtTHP1.
(a) Histochemical staining for GUS activity of a typical 14-day-old essp1 mutant seedling grown on an MS agar plate; the inset shows a close-up view of a single leaf.
(b) 35-day-old wild-type (Col) and essp1 plants grown under long-days condition (18 h light/6 h dark).
(c) Two-week-old seedlings grown on MS plates, showing comparison of roots of wild-type (Col) and essp1.
(d) Comparison of mature siliques between essp1 and wild-type (Col).
(e) RT-PCR analysis of At7S1 expression in 14-day-old wild-type (Col) and essp1 mutants grown on MS agar plates. Elongation factor 1a was used as a control.
(f) Map-based cloning of essp1. Fine mapping locates the essp1 locus to the middle of chromosome 2. The numbers of recombination events out of the total number
of chromosomes examined (2758) are indicated.
(g) Structure of ESSP1/AtTHP1 and the locations of mutations/T-DNA insertion sites in mutant alleles. Boxes and lines represent exons and introns, respectively. The
red and blue boxes indicate the coding regions for the PCI and PAM domains, respectively.
(h, i) Subcellular localization of AtTHP1. Shown are the root tip and elongation zone from transgenic Arabidopsis expressing 35Spro:AtTHP1-YFP in the atthp1-3
background. Cell walls are stained by propidium iodide. Scale bars = 40 lm.
(j, k) mRNA export assay by whole-mount in situ localization of poly(A) RNA for wild-type (Col) and atthp1-3. Leaves from 10-day-old seedlings were used. The bright
green signal in the atthp1-3 nuclei represents the accumulation of mRNA. Scale bars = 40 lm.
partners. Using full-length AtTHP1 as bait, six genes were
identified, including genes predicted to encode a SAC3
family protein, a nucleoporin-like protein, and one of the two
Table 1 Arabidopsis proteins identified
from a Y2H library screening using AtTHP1
as bait
Arabidopsis homologs of the 26S proteasome subunit DSS1
[AtDSS1(V)] (Table 1). Because Thp1 and Sac3 are components of the yeast TREX-2 complex that interacts with a
At code
Protein products
Peptides identifieda Yeast homolog Key references
At3g10650
At2g39340
At5g45010
At5g16310
At5g61290
At2g38280
Nucleoporin-like
SAC3 domain protein
AtDSS1(V)
UCH1
FMO family mamber
AMP deaminase
850–1309
134–1006
1–73
123–334
304–461
1–839
Nup1
–
Sem1/Dss1
UCH2/UCH37
–
AMD1
a
Numbers refer to the number of amino acids.
ª 2009 Her Majesty the Queen in Right of Canada
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 259–270
Neumann et al. (2006)
–
Dray et al. (2006)
Yang et al. (2007)
–
Xu et al. (2005)
262 Qing Lu et al.
Figure 2. Characterization of the three Arabidopsis homologs of the yeast
Sac3 protein.
(a) Unrooted dendrogram of SAC3 domain family proteins from Arabidopsis
(At), yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe, Sc
and Sp), Caenorhabditis elegans (Ce), human (Hs) and rice (Oryza sativa, Os).
Clades of functionally distinct subgroups are shaded. The three Arabidopsis
SAC3s are indicated in bold.
(b) Yeast two-hybrid (Y2H) assay showing interaction between the Arabidopsis SAC3s, AtTHP1 and AtCEN1/2, which are the orthologs of the yeast Cdc31
protein. SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade correspond to dropout
medium lacking Trp and Leu and Trp, Leu, His and Ade, respectively. Series
of 10-fold dilutions of the co-transformed yeast cultures were spotted on SD/Trp/-Leu and SD/-Trp/-Leu/-His/-Ade plate and incubated at 30C for 2-4 days.
Positive interactions result in yeast growth on the SD/-Trp/-Leu/-His/-Ade
plate. BD and AD, genes fused with Gal4 binding and activation domains,
respectively. pGBKT7 and pGADT7-Rec, empty vectors for bait and prey,
respectively.
(c–h) BiFC images confirming the positive interactions shown by Y2H in (b).
N. benthamiana leaves co-transformed with constructs of genes fused N- and
C-terminally to YFP, respectively (as indicated) were imaged 48-72 h after
infiltration, using a Leica TCS SP2 confocol microscope. Images are shown as
merged confocal YFP and bright-field images of epidermal N. benthamiana
leaf cells. Scale bars = 40 lm.
(i, j) Subcellular localization of AtSAC3A and B. Confocal image of transgenic
roots (35Spro:AtSAC3A-YFP and 35Spro:AtSAC3B-YFP). Cell walls are stained
by propidium iodide. Scale bars = 40 lm.
(a)
(b)
nucleoporin, we tested the possibility that we have identified
key components of the Arabidopsis TREX-2 mRNA export
complex.
Identification of the Arabidopsis homologs of yeast Sac3
Yeast Sac3, a 1293 amino acid protein, is evolutionarily
conserved from yeast to human (Jones et al., 2000),
although the Arabidopsis ortholog(s) had not yet been
identified. Using the conserved approximately 400 amino
acid central region of Sac3 (Jones et al., 2000; Fischer et al.,
2002), we identified three potential Arabidopsis SAC3 family
genes, At2g39340, At3g06290 and At3g54380, which we
designated AtSAC3A, B and C, respectively. Of the three
AtSAC3s, AtSAC3B and AtSAC3C are grouped together in
the dendrogram shown in Figure 2(a), and are closest to the
Sac3 proteins from yeast and animals, whereas AtSAC3A is
divergent and does not appear to be closely related to any
Sac3 proteins.
The Y2H screen identified a peptide containing the
conserved central domain of AtSAC3A (AtSAC3A134-1006)
(Table 1) as the interacting partner of AtTHP1. The interaction was verified by directed Y2H assay, as determined by
colony growth on selective media (Figure 2b). We further
examined interactions of the AtSAC3s with other putative
Arabidopsis TREX-2 components, and showed that AtSAC3A interacted with AtSAC3B and AtCEN1, and that
AtSAC3B interacted with AtTHP1 and AtCEN1 and 2 (Figure 2b). No interactions of AtSAC3C and AtSUS1 with any of
the putative TREX-2 components were observed (Table S1).
The interactions observed between AtSACs and AtTHP1/
AtCENs by Y2H were validated using a bi-molecular
fluorescence complementation (BiFC) assay in a tobacco
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
ª 2009 Her Majesty the Queen in Right of Canada
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 259–270
TREX-2 mRNA export complex 263
transient expression system (Kerppola, 2006; Figure 2c–h).
Thus, putative components of the Arabidopsis TREX-2
complex interact in yeast and in planta.
To obtain clues about whether the AtSAC3s are functional
orthologs of the yeast Sac3 protein, we fused AtSAC3s with
YFP and localized the fusion proteins in stably transformed
plants. The AtSAC3A–YFP fusion protein was highly abundant and localized in the nucleus, whereas AtSAC3B–YFP
appeared to be concentrated at the nuclear periphery
(Figure 2i,j). We did not observe a fluorescence signal from
AtSAC3C–YFP. These localization experiments indicate that
the nucleus is the primary location of AtSAC3A and B, as
suggested by the BiFC experiments (Figure 2c–h). The
nuclear localization of AtSAC3s is consistent with the
hypothesis that they are subunits of the Arabidopsis
TREX-2 complex.
The Arabidopsis ortholog of yeast Nup1
The Y2H screen with AtTHP1 identified a protein (At3g10650,
Table 1) that was identified by the advanced bioinformatics
strategy described by Neumann et al. (2006) as a putative
ortholog of the yeast nucleoporin Nup1 and vertebrate nucleoporin Nup153. Thus, we designated this gene AtNUP1.
We verified the interaction of AtTHP1 with AtNUP1 by
independent Y2H assays (Figure 3a) and BiFC analysis (Figure 3b). Similar to the situation in yeast, the putative TREX-2
component AtTHP1 interacts with the putative Nup1/Nup153
nucleoporin.
To further validate the identity of AtNUP1 as a nucleoporin, we determined its subcellular localization by
expressing an AtNUP1 reporter protein in an atnup1
mutant, atnup1-1 (Figure 3c,d). The morphological phenotype of atnup1-1 is shown in Figure 3(e). A gene encoding
a translational fusion between AtNUP1 and YFP under the
control of its native promoter was introduced into the
atnup1-1 background. The fusion gene rescued the morphological mutant phenotype (data not shown), suggesting that the YFP fusion gene is functional in vivo. As
shown in Figure 3(f), the YFP signal is clearly found only
along the nuclear periphery, as expected for a nucleoporin.
The subcellular location of AtNUP1 is consistent with its
potential role as a nuceloporin.
Functional evidence that AtTHP1 and AtSAC3B are TREX-2
components and AtNUP1 is an anchoring nucleoporin
We have shown that AtTHP1, AtSAC3s and AtNUP1 interact
physically and are localized to the periphery of the nucleus,
suggesting that they are components of the TREX-2 mRNA
export complex. To test this hypothesis, we determined
whether mRNA export was compromised in plants with
mutations in putative TREX-2 components by localizing
poly(A) mRNA in young leaves using in situ hybridization.
The poly(A) mRNA localization experiments suggested
that both AtTHP1 and AtNUP1 are required for nuclear
RNA export. As shown in Figure 1(k), poly(A) mRNA
preferentially accumulated in the nuclei of the atthp1-3
mutant compared with wild-type (Figure 1j), suggesting a
defect in export. Similarly, poly(A) mRNA localized in
atnup1-1 mutant nuclei but not wild-type nuclei (Figure 3g,h). The findings that AtTHP1 and AtNUP1 are
necessary for nuclear mRNA export provide support for
the hypothesis that they are components of the putative
Arabidopsis TREX-2 complex. Consistent with its mRNA
export defect, atnup1-1, a knockdown line (Figure 3d),
exhibited diverse developmental defects, including fewer
rosette leaves, longer secondary branches and reduced
fertility (Figure 3e). atnup1-2 is apparently a gametophytic-lethal or a homozygous sporophytic-lethal mutation, as over 300 progeny from heterozygous parent were
genotyped and no homozygous insertion plants were
found.
By contrast, experiments with AtSAC3s suggest that they
are not required for nuclear mRNA export. We identified TDNA insertion mutations in each of the Arabidopsis SAC3
genes (Figure 4a) that all appeared to be null mutations
(Figure 4b), and created all possible atsac3a, atsac3b and
atsac3c double mutant combinations and a triple mutant.
However, none of the single, double or triple atsac3 mutations showed defects in nuclear mRNA export (data not
shown). Consistent with these findings, we did not observe
any morphological mutant phenotype in the single, double
or triple mutants.
Although the AtSAC3 subunits do not appear to be
required for nuclear mRNA export, we obtained evidence
of their functional interaction with Arabidopsis TREX-2
subunits. As shown in Figure 4(c), an atthp1 atsac3b
double mutant was smaller in stature and exhibited a
reduction in fertility compared with the single mutants.
atthp1-3 atsac3b-1 is sterile, while atthp1-3 atsac3b-2 set a
few seeds. We also investigated the effects of AtTHP1 and
AtSAC3B on ethylene responses, because both have been
implicated recently as playing similar roles to each other
in the ethylene-signaling pathway (Christians et al., 2008;
McClellan et al., 2008, see http://www.arabidopsis.org –
TAIR accession/publication number 501727765). We found
that atthp1-3 and atsac3b-1 but not atsac3a and atsac3c
single mutants exhibited enhanced sensitivity to ethylene
(Figure 4d). However, the atthp1 atsac3b double mutant
showed even greater sensitivity to ethylene (Figure 4d).
The morphological analysis and ethylene response experiments suggest a synergistic interaction between AtTHP1
and AtSAC3B.
Arabidopsis DSS1 is associated with both the TREX-2
complex and the proteasome
An Arabidopsis homolog of DSS1, AtDSS1(V), was identified in the Y2H screen using AtTHP1 (Table 1). Consistent
with this finding, the yeast homolog of DSS1, Sem1, has
ª 2009 Her Majesty the Queen in Right of Canada
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 259–270
264 Qing Lu et al.
(a)
(b)
(e)
(d)
(c)
(f)
(g)
(h)
Figure 3. Characterization of the putative nucleoporin NUP1 encoded by At3g10650.
(a) Y2H assay showing interaction between AtTHP1 and the putative AtNUP1. A truncated version (the one uncovered from the original Y2H library, see Table 1) was
used as the full-length version shows self-activation.
(b) BiFC assay showing the in vivo interaction between AtTHP1 and AtNUP1. The reconstituted fluorescent signals are indicated by arrows. Scale bar = 40 lm.
(c) Structure of the AtNUP1 gene and location of T-DNA insertion sites. The red vertical lines indicate the coding for FG repeats, the only recognizable structural
motif.
(d) RT-PCR analysis of the expression of AtNUP1 in wild-type and mutant atnup1-1. The primers used are indicated in (c). Genomic DNA (gDNA) was included as size
control for RT-PCR products, and the actin2 gene was used as an internal control.
(e) Phenotypes of 5-week-old wild-type (Col) and atnup1-1 plants.
(f) Subcellular localization of AtNUP1. Confocal image of transgenic root (NUP1pro:NUP1-YFP). The same pattern was also observed for 35Spro:NUP1-YFP
transgenics. Cell walls are stained by propidium iodide. Scale bars: 40lm.
(g, h) mRNA export assay by whole-mount in situ localization of poly(A) RNA for wild-type (Col) and atnup1-1, respectively. Leaves from 10-day-old seedlings were
used. The bright green signal in the atthp1 nuclei represents the accumulation of mRNA. Scale bars = 40 lm.
also been shown recently to be a functional component of
the TREX-2 complex (Wilmes et al., 2008; Faza et al., 2009)
and is required for nuclear RNA export (Thakurta et al.,
2005; Mannen et al., 2008). As shown in Figure 5(a), the
interaction between AtDSS1(V) and AtTHP1 was confirmed
in a Y2H assay. In addition, interaction of AtDSS1(V) with
AtCEN2 was also observed (Figure 5a). We validated these
interactions using the BiFC assay (Figure 5b,c). We did
not detect interaction between AtTHP1 and a second
Arabidopsis homolog, AtDSS1(I), in the Y2H assay
(Table S1).
DSS1 is also a subunit of the 26S proteasome regulatory
particle (Funakoshi et al., 2004; Krogan et al., 2004; Sone
et al., 2004; Baillat et al., 2005; Schmidt et al., 2005; Josse
et al., 2006). We used Y2H experiments to show that
AtDSS1(V) and its paralog, AtDSS1(I), both interact with
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Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 259–270
TREX-2 mRNA export complex 265
(a)
(b)
(c)
(d)
Figure 4. Phenotypes of the atthp1 atsac3b double mutants.
(a) Structure and T-DNA insertion sites of the AtSAC3A, B and C genes. Arrows represent the location of primers used for RT-PCR analysis. Blue boxes indicate the
SAC3 domain.
(b) RT-PCR analyses of the AtSAC3A, B and C genes in the T-DNA insertion lines shown in (a) and in wild-type (Col). Genomic DNA (gDNA) was included as a size
control for RT-PCR products, and the actin2 gene was used as an internal control.
(c) Phenotypes of 6-week-old plants. Genotypes are indicated.
(d) Ethylene response phenotype of atthp1-3, atsac3b-2 and atthp1-3 atsac3b-2 double mutants at the stated ACC concentration. Scale bar = 2 mm.
the 26S proteasome subunits RPN3A and RPN3B (Figure 5a), and confirmed these interactions using BiFC assays
(Figure 5c–g). This result is consistent with those of Wei
et al. (2008), who demonstrated direct binding of DSS1s
from diverse species, including AtDSS1(I), to the protea-
some subunit RPN3. The BiFC signal patterns in Figure 5c–g
indicating that the AtDSS1–RPN3 complexes are localized to
the nucleus and cytoplasm are consistent with the subcellular localization of the Arabidopsis DSS1s (Figure S2) and
proteasome subunits. In summary, our data show that
ª 2009 Her Majesty the Queen in Right of Canada
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266 Qing Lu et al.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Figure 5. Physical association of AtDSS1(I)/(V) with the TREX-2 complex and
the proteasome.
(a) Y2H assay testing the interactions of AtDSS1 proteins with TREX-2 and 26S
proteasome subunits.
(b–f) BiFC images confirming the interactions shown in (a). (b) Merged YFP
signal and bright-field image. (c–g) Non-merged YFP signal. Scale
bars = 40 lm.
Arabidopsis DSS1s are associated with both the TREX-2
complex and the proteasome.
DISCUSSION
The Arabidopsis TREX-2 complex: composition and
anchoring nucleoporin
The results of our Y2H screen and bioinformatic predictions
indicating that several of the identified proteins are homologous to the yeast TREX-2 components prompted us to look
into the possibility that key components of the Arabidopsis
TREX-2 complex had been identified. Bioinformatic predictions were key in the initial identification of AtTHP1 and AtNUP1, both of which are poorly conserved compared with
their yeast counterparts at the amino acid sequence level,
and the identifications were made possible only by advanced bioinformatic programs (Ciccarelli et al., 2003; Neumann et al., 2006). Our subcellular localization and mRNA
export data provide support for the bioinformatic prediction
results, and suggest that AtNUP1 is an ortholog of yeast
Nup1 and an anchor of the Arabidopsis TREX-2 complex.
Our data suggests that we have identified components of
the Arabidopsis TREX-2 complex. The subcellular localization and mRNA export studies with AtTHP1, and its physical
interaction with AtNUP1, demonstrate that AtTHP1 is the
ortholog of yeast Thp1. Three homologs of yeast Sac3 were
characterized, but the results were inconclusive regarding
which is the functional ortholog. It was unexpected that the
double mutants and even the triple mutant did not show
mRNA export or morphological defects. The possibility that
the current mRNA export assay is not sensitive enough
cannot be excluded, nor can the possibility that the young
leaves used are not the best tissue in which to observe a
defect. Nevertheless, the data strongly suggest that AtSAC3B and AtSAC3A are part of the TREX-2 complex. First,
the Y2H and BiFC data demonstrate that the two AtSAC3s
interact with each other and both interact with AtTHP1.
Second, both AtSAC3A and AtSAC3B are localized in the
nucleus, and AtSAC3B shows an apparent concentration to
the nuclear periphery. Third, we observed a synergistic
genetic interaction between AtSAC3B and AtTHP1. The
ethylene response phenotype of the double mutants is
consistent with their growth defects (Figure 4c,d). Similar
phenotypes have been observed for constitutive/hypersensitive ethylene-response mutants (Hua and Meyerowitz,
1998; Qu et al., 2007). These observations clearly suggest
that AtTHP1 and AtSAC3B function in the same cellular
process. One possibility is that they are present in the same
TREX-2 complex and are mutually required for optimal
stability and function.
The putative Arabidopsis homologs of yeast Cdc31 and
Sus1 had been identified previously (Molinier et al., 2004;
Rodrı́guez-Navarro et al., 2004), but it had not been shown
that they are components of the Arabidopsis TREX-2 complex. Our observation that AtCEN1/2 interact with AtSAC3A/
B suggests that the two Cdc31 homologs may well be
components of the Arabidopsis TREX-2 complex. No interaction of AtSUS1 with any of the putative TREX-2 components described here was detected. Thus it remains to be
determined whether it is an ortholog of yeast Sus1. It is also
possible that AtSUS1 interacts with the complex through
an adaptor protein, or that its interactions are too weak to
be detected under the assay conditions. Lastly, the data
strongly suggests that AtDSS1(V) is also part of the
ª 2009 Her Majesty the Queen in Right of Canada
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 259–270
TREX-2 mRNA export complex 267
Arabidopsis TREX-2 complex. It remains to be investigated
whether these three small TREX-2 components are required
for mRNA export, and whether the two AtCENs play
redundant roles. In addition, the observation that AtDSS1(V)
is a component of both the TREX-2 complex and proteasome
is reminiscent of the physical and functional roles of the
yeast Sus1 in coupling TREX-2 and SAGA complexes
(Rodrı́guez-Navarro et al., 2004). It is tempting to speculate
that AtDSS1(V) may play a similar role as a link between the
TREX-2 complex and the proteasome when it is necessary to
bring the proteasome into close physical proximity with the
TREX-2 complex and NPC (Figure 6). Indeed, it has been
proposed that the 19S proteasome lid complex may play a
non-proteolytic role in the remodeling of mRNP complexes,
thereby facilitating mRNA export (Iglesias and Stutz, 2008).
The interaction network uncovered in this work is summarized in Figure 6. The AtTHP1–AtNUP1 interaction links
the Arabidopsis TREX-2 complex to NPC. AtSAC3A and
AtSAC3B interact with each other, and both of them interact
with AtTHP1. In yeast, it is believed that Sac3 links the TREX2 complex to Nup1 (Lei et al., 2003). In our Y2H study, we did
not find an interaction between AtSAC3B and AtNUP1 and
were unable to test the interaction between AtSAC3A and
Figure 6. Schematic representation of the interactions uncovered in this
study.
At the top is a schematic representation of a nuclear pore embedded in the
nuclear membrane. The cytoplasmic filaments and nuclear basket are also
shown. The AtTHP1–AtNUP1 interaction links the Arabidopsis TREX-2 complex and the nuclear basket. AtDSS1(I)/(V) are stably associated with the lid
complex of the proteasome. AtDSS1(V) is likely to be associated with the
TREX-2 complex and the proteasome independently, but may serve as a link
to recruit the proteasome, or just the lid complex, to TREX-2/NPC when
required. The interacting partner of AtSUS1 remains to be identified. RP,
regulatory particle; CP, core particle.
AtNUP1 due to self-activation of the constructs. However,
we did observe interactions in a BiFC assay between
AtSAC3A/B and AtNUP1 (data not shown), suggesting that
these proteins are in close physical proximity. Future work is
required to elucidate the complete composition of the
Arabidopsis TREX-2 complex and its roles in diverse developmental processes.
Roles of NPC and the TREX-2 complex in gene expression
The findings presented here suggest a physical link between
NPC and the TREX-2 complex via AtNUP1 (Figure 6). This
raises the possibility that there are other similar association
networks between NPC and other complexes present in
Arabidopsis cells, as there are in yeast. In yeast, there are
numerous recent observations demonstrating that NPC, the
TREX-2 complex and other associated complexes play a role
in gene regulation/chromatin remodeling (for reviews, see
Ahmed and Brickner, 2007; Akhtar and Gasser, 2007; Taddei,
2007; Luna et al., 2008; Iglesias and Stutz, 2008).
The link between NPC and gene expression was first
proposed by Blobel (1985) in his ‘gene gating’ hypothesis,
which states that every gene in the nucleus is physically
linked (gated) to a particular NPC. Recent findings support
the hypothesis that gene association with NPC can play an
active role in regulating transcription, possibly by creating
a docking surface on which all the nuclear steps of gene
expression are coordinated (Taddei, 2007). The best known
example is the functional and physical coupling of the
SAGA transcription initiation complex and the TREX-2
complex via Sus1 in the relocation and subsequent
confinement of GAL genes to NPCs during transcriptional
activation in S. cerevisiae (Rodrı́guez-Navarro et al., 2004;
Cabal et al., 2006).
The initial observation described here was identification
of the AtTHP1 gene based on a mutant allele that altered
expression of a seed storage protein promoter–GUS transgene. As AtTHP1 is part of the TREX-2 complex, this finding
supports the hypothesis that the complex can affect specific
gene expression. The hypothesis is further supported by
the ethylene response phenotypes of atthp1 and atsac3b
mutations. However, the functional significance of such
physical associations for specific gene expression requires
additional delineation. Similarly, in other higher eukaryotes,
little is known about the connection between the NPC and
associated complexes and the regulation of gene expression
(Ruault et al., 2008), and this will undoubtedly be an
important area of investigation.
EXPERIMENTAL PROCEDURES
Plant materials, growth conditions and genotype analysis
Arabidopsis thaliana T-DNA insertion lines for AtSAC3A, B, C, AtNUP1 and AtTHP1 were obtained from the Arabidopsis Biological
Resource Center, and SK6095 was obtained from Agriculture and
Agri-Food Canada (http://www.agr.gc.ca). For propagation, seeds
ª 2009 Her Majesty the Queen in Right of Canada
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 259–270
268 Qing Lu et al.
were treated at 4C in the dark for 3 days before moving them into
the growth room. Plants were grown for 16 h in the light at 22C/8 h
in the dark at 18C. For ethylene response tests, seeds were plated
on MS agar plates with or without the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), incubated in the dark and
photographed 4 days after germination. Genotypes of mutants
were identified by PCR. For RT-PCR analysis, homozygous seeds
were spotted on MS agar plates, vernalized, and grown at 22C for
2 weeks. An appropriate amount (100 mg) of leaves was collected,
and RNA was extracted using an RNeasy Plant Mini kit (Qiagen,
http://www.qiagen.com/) according to the manufacturer’s
instructions.
Subcellular localization
Map-based cloning of essp1
New BiFC vectors pEarleyagte201-YN and pEarleygate202-YC were
created based on the Gateway-compatible vectors pEarleygate201
and 202 (Earley et al., 2006). The N-terminus (amino acids 1–174)
and C-terminus (amino acids 175–239) of YFP from vector pEarleygate101 were PCR-amplified using the primers listed in Table S2,
and cloned into the TA cloning vector pGEM-T Easy. The N- and
C-terminal YFP fragments were then cut out and subcloned into
pEarleyhgate201 and pEarleygate202, respectively, at the AvrII site.
For infiltration of epidermal N. benthamiana cells, the OD600 of an
Agrobacterium strain GV3101 overnight culture containing gene
fused N- or C-terminally with YFP was measured, and brought to an
optical density of 0.6–1.0. Equal volumes of each culture were mixed
and used for infiltrating the epidermal N. benthamiana leaves as
previously described (Sparkes et al., 2006). The YFP signal was
imaged 2–3 days after infiltration. For protein pairs showing a
reconstituted YFP signal in the nuclei, the YFP signal and bright-field
images were overlaid. For those showing YFP signal in both the
nucleus and cytoplasm, only the YFP signal was imaged.
For genetic mapping of the essp1 mutation, mutant plants from
the Col background were crossed with wild-type plants of the Ler
ecotype. Homozygous essp1 mutants were selected from the F2
segregating population. Genomic DNA extracted from these
seedlings was used for PCR-based mapping with simple sequence
polymorphism markers, and the essp1 locus was mapped to
approximately 37 kb on BAC F3P11. Sequencing of the genomic
region revealed a mutation in At2g19560. For the complementation test, a cDNA clone was obtained from RIKEN (http://www.
riken.go.jp), and inserted into the pCAMBIA 3300 vector. The
resulting construct was introduced into essp1 plants. Over 100 T1
transgenic plants were obtained, and all showed complete rescue
of the essp1 phenotype, i.e. wild-type morphology and no GUS
activity in leaves. The histochemical GUS assay was performed as
described previously (Tang et al., 2008).
Y2H library screening and directed assay
Two-week-old wild-type Columbia leaves from plants growing on
agar plates were used for cDNA library construction. The AtTHP1
coding sequence was amplified and cloned into the yeast twohybrid bait vector pGBKT7 (Clontech, http://www.clontech.com/)
and confirmed by sequencing. The self-activation test for AtTHP1,
leaf cDNA library construction and screening were performed
according to Clontech protocols PT3024-1 and PT3529-1. For
co-transformation of the bait construct and prey vector harboring
the cDNA library, yeast strain AH109 was used, with the LiAc
transformation method. After transformation, cells were spread
on SD/-Trp/-Leu/-His agar plates, and incubated at 30C for 5 days
and then room temperature for another 3 days. Colonies growing
on the SD/-Trp/-Leu/-His plates were analyzed following the
manufacturer’s protocol (Clontech).
For the directed Y2H test, we constructed two Gateway-compatible vectors pGBKT7-DEST (bait) and pGADT7-DEST (prey) by
modifying the pGKBT7 and pGADT7-Rec vectors. The attR1–attR2
cassette from pEarleygate101 (Earley et al., 2006) was PCR-amplified and cloned into the pGEM-T Easy vector (Promega, http://
www.promega.com/) followed by sequence confirmation. The
plasmid was digested using NdeI and XhoI, and the attR1–attR2
cassette was ligated into pGBKT7 digested with NdeI and SalI, and
pGADT7-Rec digested with NdeI and XhoI. A series of 5 ll aliquots
of diluted AH109 culture co-transformed with bait and prey
constructs was spotted onto /-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade.
Plates were incubated at either 23 or 30C for up to 5 days. As
negative controls, the intact pGBKT7, pGADT7-Rec and newly
developed pGBKT7-DEST and pGADT7-DEST vectors were used,
but only the pGBKT7 and pGADT7 results are shown as there was no
difference between them. All constructs were produced using
full-length cDNAs, except AtNUP1, as shown in Figure 3, as the
full-length version showed self-activation.
Constructs were produced by cloning the target genes into pEarleygate 101 (Earley et al., 2006). The primers used are the same as
those used for Y2H and BiFC (Table S2). The constructs were
introduced into Arabidopsis for stable transformation or epidermal
Nicotiana benthamiana cell for transient assays. For AtNUP1, a
native promoter-driven gene was cloned into pEarleygate301-YFP,
which was made by transferring the NcoI–PacI fragment from
pEarleygate101 to pEarleygate301. The primers used to amplify the
AtNUP1 genomic region, npNupF and NupR, are listed in Table S2.
BiFC assay
mRNA export assay
The accumulation of mRNA in the wild-type (Col) and mutant nuclei
were assayed by in situ hybridization using oligo(dT)50 as a probe as
described by Gong et al. (2005) with minor modifications. Leaves
from 10-day-old seedlings grown on MS agar plates were fixed in
5 ml fixation buffer (120 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4,
2.7 mM KCl, 80 mM EGTA, 0.1% Tween-20, 10% DMSO and 5%
formaldehyde) under vacuum for 10 min, followed by adding an
equal volume of heptane and gently shaking for 20 min. After 5 min
washes, twice in methanol and three times in absolute ethanol,
samples were incubated in 1:1 ethanol:xylene for 30 min. Leaves
were then sequentially washed for 5 min twice with ethanol, once
with methanol and once with 1:1 methanol:fixation buffer without
formaldehyde, before being post-fixed using fixation buffer for an
additional 30 min. Samples were then incubated for 5 min once
with fixation buffer and twice with 1 · blocking buffer (Roche, http://
www.roche.com) before being subjected to blocking for 1–2 h at
50C. After blocking, oligo(dT)50 with a 5¢ 6-fluorescein tag was added to a final concentration of 0.5 lg ml)1, and incubated for more
than 8 h at 50C in the dark. After hybridization, leaves were washed
once for 30 min with 2 · SSC, 0.1% SDS, and once for 20 min with
0.2 · SSC, 0.1% SDS at 50C. The fluorescein signal was assayed
using a Leica TCS SP2 confocal microscope (http://www.leica.com/)
with an argon excitation laser of 514 nm and fluorescein isothiocyanate filter. Each experiment was repeated at least twice.
Phylogenetic analysis
The amino acid sequence alignment and the subsequent unrooted
dendrogram construction of the three Arabidopsis SCA3 family
proteins identified in this study and other SAC3 proteins from
Arabidopsis, yeast, human and rice were performed following the
ª 2009 Her Majesty the Queen in Right of Canada
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 259–270
TREX-2 mRNA export complex 269
MEGA4.0.2 program package (http://www.megasoftware.net/
index.html). The alignment was performed by ClustalW using a
BLOSUM matrix with a gap opening penalty of 10 for both pairwise
and multiple alignment, and gap extension penalties of 0.1 and 0.2
for pairwise and multiple alignment, respectively. The resulting
alignment was then used to generate the unrooted dendrogram
using the neighbor-joining Poisson correction with 1000 bootstrap
replicates. The input order of the sequences was randomized.
Accession numbers
Sequence data from this article can be found in the GenBank/EMBL
data libraries under accession numbers At2g19560 (THP1),
At2g39340 (SAC3A), At3g06290(SAC3B), At3g54380 (SAC3C),
At3g10650 (NUP1), At1g64750 [DSS1(I)], At5g45010 [DSS1(V)],
At3g50360 (CEN1), At4g37010 (CEN2), At3g27100 (SUS1),
At1g20200 (RPN3A), At1g75990 (RPN3B), At1g64520 (RPN12A),
At5g42040 (RPN12B), NP_588432 (Sp Sac3), Q12049 (Sc Ypr045c),
EDN60497 (Sc Sac3), NP_501328 (Ce Sac3), AAI04959 (Hs Sac3),
NP_195051 (At eIF3K), NP_116710 (Sc RPN12), NP_037366 (Hs
eIF3K), NP_001049175 (Os eIF3K), NP_002803 (Hs RPN12),
NP_596750 (Sp RPN12) and NP_496489 (Ce RPN12).
ACKNOWLEDGEMENTS
We thank Carol Hannam for help with the Y2H assay, Alex Molnar for
help with preparing the figures, Ida van Grinsven for the sequencing
service, Isabelle Parkin (Agriculture and Agri-Food Canada, Saskatoon Research Centre) for seeds of SK6095, the Arabidopsis Biological Resource Center for seeds of T-DNA insertion lines and
transformation vectors, and RIKEN for the AtTHP1 cDNA clone.
Funding was provided by the Agriculture and Agri-Food Canada Abase and Canadian Crop Genomics Initiative (to Y.C.), Genome Prairie/Genome Canada (to Y.C., E.W.T.T. and W.A.K.) and the Natural
Sciences and Engineering Research Council of Canada (to S.J.R.).
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Expression pattern of GUS driven by the ESSP1
promoter.
Figure S2. Subcellular localization of AtDSS1(I)/(V).
Table S1. Interactions tested by Y2H and BiFC.
Table S2. PCR primers used in this study.
Please note: Wiley-Blackwell are not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
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Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2010), 61, 259–270