Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export

Abstract

RNA undergoing nuclear export first encounters the basket of the nuclear pore. Two basket proteins, Nup98 and Nup153, are essential for mRNA export, but their molecular partners within the pore are largely unknown. Because the mechanism of RNA export will be in question as long as significant vertebrate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and Nup153 were used for pulldown experiments from Xenopus egg extracts, which contain abundant disassembled nuclear pores. Strikingly, Nup98 and Nup153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced. Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg extracts and in assembled pores, now termed the Nup160 complex. Sec13 is prominent in Nup98 and Nup153 pulldowns, and we find it to be a member of the Nup160 complex. We have mapped the sites that are required for binding the Nup160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153 to the nuclear pore. With transfection and in vivo transport assays, we find that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not protein import or export. These results demonstrate that two novel vertebrate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup153, but themselves play a role in mRNA export.

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The Journal of Cell Biology, Volume 155, Number 3, October 29, 2001 339–353
http://www.jcb.org/cgi/doi/10.1083/jcb.200108007
JCB
Article
339
Novel vertebrate nucleoporins Nup133 and Nup160
play a role in mRNA export
Sanjay Vasu,
1
Sundeep Shah,
1
Arturo Orjalo,
1
Minkyu Park,
2
Wolfgang H. Fischer,
2
and Douglass J. Forbes
1
1
Section of Cell and Developmental Biology, Division of Biology 0347, University of California at San Diego,
La Jolla, CA 92093
2
Clayton Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, CA 92037
NA undergoing nuclear export first encounters the
basket of the nuclear pore. Two basket proteins,
Nup98 and Nup153, are essential for mRNA export,
but their molecular partners within the pore are largely
unknown. Because the mechanism of RNA export will be in
question as long as significant vertebrate pore proteins remain
undiscovered, we set out to find their partners. Fragments of
Nup98 and Nup153 were used for pulldown experiments
from
Xenopus
egg extracts, which contain abundant disas-
sembled nuclear pores. Strikingly, Nup98 and Nup153
each bound the same four large proteins. Purification
and sequence analysis revealed that two are the known
vertebrate nucleoporins, Nup96 and Nup107, whereas two
mapped to ORFs of unknown function. The genes encoding
the novel proteins were cloned, and antibodies were
produced. Immunofluorescence reveals them to be new
R
nucleoporins, designated Nup160 and Nup133, which are
accessible on the basket side of the pore. Nucleoporins
Nup160, Nup133, Nup107, and Nup96 exist as a complex
in
Xenopus
egg extracts and in assembled pores, now termed
the Nup160 complex. Sec13 is prominent in Nup98 and
Nup153 pulldowns, and we find it to be a member of
the Nup160 complex. We have mapped the sites that are
required for binding the Nup160 subcomplex, and have
found that in Nup98, the binding site is used to tether Nup98
to the nucleus; in Nup153, the binding site targets Nup153
to the nuclear pore. With transfection and in vivo transport
assays, we find that specific Nup160 and Nup133 fragments
block poly[A]

RNA export, but not protein import or export.
These results demonstrate that two novel vertebrate nucle-
oporins, Nup160 and Nup133, not only interact with Nup98
and Nup153, but themselves play a role in mRNA export.
Introduction
The nuclear pore mediates export from the nucleus. For
proteins, a soluble nuclear export receptor binds Ran-GTP
and a protein cargo bearing a nuclear export sequence
(NES)* to form a trimeric export complex (Mattaj and
Englmeier, 1998; Gorlich and Kutay, 1999; Damelin and
Silver, 2000; Ryan and Wente, 2000; Conti and Izaurralde,
2001; Vasu and Forbes, 2001). The complex translocates
through the pore, pausing on the cytoplasmic filaments of
the pore. There, Ran-GTP hydrolysis disassembles the com-
plex and completes export. Individual receptors have been
tailored for specific cargo, such that Crm1/exportin1 carries
proteins bearing leucine-rich NESs, whereas exportin-t car-
ries newly transcribed tRNAs. Multiple different proteins
have been implicated in mRNA export (for review see Conti
and Izaurralde, 2001).
The vertebrate pore at 120 million daltons is estimated
to contain

30–60 different proteins. Each is present in

8–32 copies, giving perhaps 1,000 proteins per pore. Only
a subset of vertebrate pore proteins is known (Vasu and
Forbes, 2001). Structurally, the pore consists of three
stacked rings of

1,200 Å. The middle ring contains eight
thick spokes surrounding a central transporter (Stoffler et
al., 1999; Allen et al., 2000; Ryan and Wente, 2000; Vasu
and Forbes, 2001). At one face of the pore, cytoplasmic
filaments extend to interact with incoming or outgoing
receptor complexes. On the opposite or nuclear face of the
pore, eight long filaments connect to a 500-Å ring to form
the nuclear basket of the pore. A large mRNA/protein cargo,
the Balbiani transcript, has been seen to thread through the
basket during export (Kiseleva et al., 1996). It is hypothe-
sized that other export cargos follow a similar pathway.
To date, only two basket nucleoporins have been shown
to play a critical role in vertebrate RNA export, Nup98 and
The online version of this article contains supplemental material.
Address correspondence to Douglass J. Forbes, Section of Cell and De-
velopmental Biology, Division of Biology 0347, University of California
at San Diego, La Jolla, CA 92093-0347. Tel.: (858) 534-3398. Fax:
(858) 534-0555. E-mail: dforbes@ucsd.edu
*Abbreviations used in this paper: aa, amino acid(s); AL, annulate lamel-
lae; GFP, green fluorescent protein; IB, immunoblotting; IF, immuno-
fluorescence; LMB, leptomycin B; NES, nuclear export sequence.
Key words: Nup133; Nup160; mRNA export; Nup98; Nup153

340 The Journal of Cell Biology
|
Volume 155, Number 3, 2001
Nup153 (Bastos et al., 1996; Powers et al., 1997; Ullman et
al., 1999). Antibodies to Nup98 block mRNA, snRNA, 5S
RNA, and preribosome particles export, whereas these anti-
bodies have no effect on tRNA export or NLS-mediated im-
port (Powers et al., 1997). Vertebrate Nup98 resembles
three yeast nucleoporins involved in RNA export, Nup100p,
Nup116p, and Nup145p, having features in common with
each and identity with none (Powers et al., 1995; Radu et
al., 1995; Stutz et al., 1996; Iovine and Wente, 1997; Bailer
et al., 1998; Pritchard et al., 1999; Zolotukhin and Felber,
1999; Bachi et al., 2000; Fontoura et al., 2000; Strasser et
al., 2000). Although all have GLFG repeats capable of bind-
ing different transport receptors in vitro, only Nup116p and
Nup98 contain a sequence that binds the small transport
factor Gle2 (Murphy et al., 1996; Bharathi et al., 1997;
Bailer et al., 1998; Zolotukhin and Felber, 1999). Nup98
also resembles yeast Nup145 in that both are synthesized as
precursors capable of self-cleavage into two nucleoporins:
Nup98 and Nup96 in vertebrates, and Nup145N and
Nup145C in yeast (Emtage et al., 1997; Teixeira et al.,
1997, 1999; Fontoura et al., 1999; Rosenblum and Blobel,
1999). The yeast GLFG proteins are found on both sides of
the yeast pore (Rout et al., 2000), whereas Nup98 is concen-
trated on the nuclear face of the pore and in the nuclear inte-
rior (Powers et al., 1995; Radu et al., 1995; Zolotukhin and
Felber, 1999). Clearly, evolution has responded to chal-
lenges yet to be elucidated.
Nup153 is the only vertebrate nucleoporin reported to
map to the distal ring of the basket (Panté et al., 1994).
Functionally, Nup153 is critical for export (Bastos et al.,
1996; Ullman et al., 1999). Overexpression of certain do-
mains of Nup153 causes poly[A]

RNA accumulation in the
nucleus (Bastos et al., 1996). Moreover, antibodies to
Nup153 block mRNA, snRNA, and 5S RNA export, as well
as NES protein export and Rev-dependent HIV RNA export
(Ullman et al., 1999). The antibodies do not block tRNA ex-
port, recycling of importin

to the cytoplasm, or NLS-
mediated import. Nup153 also functions in the terminal steps
of nuclear import, binding importin

–

–NLS complexes
and transportin (Shah et al., 1998; Shah and Forbes, 1998;
Nakielny et al., 1999). Interestingly, the presence of Nup153
on the nuclear pore depends on an intact nuclear lamina
(Smythe et al., 2000), whereas photobleaching shows that ver-
tebrate Nup153 can exchange on and off the pore with fast ki-
netics (Daigle et al., 2001; Lyman and Gerace, 2001), raising
further interest in the role of Nup153 on the pore.
In the rat, Nup153 is comprised of a unique NH
2
termi-
nus, four central zinc fingers, and 32 FXFG and FG repeats
at the COOH terminus (see Fig. 2 a) (Sukegawa and Blobel,
1993; Bastos et al., 1996; Shah et al., 1998). Yeast has no se-
quence homologue of Nup153, and no yeast nucleoporins
contain Zn fingers. It is possible that yeast Nup1 may fulfill
certain of the functions of Nup153, as it is localized to the
distal basket and can bind importin

(Davis and Fink,
1990; Rout et al., 2000). However, beyond possessing FG
repeats, yeast Nup1 bears no sequence resemblance to
Nup153.
Yeast and vertebrate nuclear pores are separated by a bil-
lion years of evolution (Gouy and Li, 1989). The vertebrate
pore is reported to be five times the volume and twice the
longitudinal axis of the yeast pore, containing a number of
different structural elements (Yang et al., 1998; Rout et al.,
2000). Interestingly, whereas the soluble receptors and fac-
tors used in nuclear transport have been relatively well con-
served, the nuclear pore proteins themselves have diverged
dramatically (Mattaj and Englmeier, 1998; Stoffler et al.,
1999; Ryan and Wente, 2000; Conti and Izaurralde, 2001;
Vasu and Forbes, 2001). Four different protein scenarios
have been observed: (A) A small subset of nucleoporins are
fairly similar in sequence in vertebrates and yeast (vNup155/
ScNup157/ScNup170 and vNup93/ScNic96; 21 and 24%
identity, respectively); (B) Other vertebrate pore proteins
such as Nup98 are related to multiple different yeast nucle-
oporins; (C) Others have no yeast homologues and vice
versa (gp210, POM121, POM152); and (D) Yet others,
such as Nup153 and Nup214, have no sequence homo-
logues in yeast but are suspected to have analogues. One last
difference is that the majority of yeast nucleoporins are sym-
metrically localized to both sides of the pore (Rout et al.,
2000), whereas many vertebrate nucleoporins are found on a
specific face of the pore (Vasu and Forbes, 2001). Given the
evolutionary divergence in size, architecture, composition,
and protein sequence between yeast and vertebrate pores,
identifying the proteins of and providing a structure for the
120 million dalton vertebrate pore remains a daunting task.
The importance of Nup98 and Nup153 is clear from the
findings that they function in RNA export, protein import,
and most recently, viral infection (Petersen et al., 2000; von
Kobbe et al., 2000; Gustin and Sarnow, 2001). In the five
years since their discovery, little evidence has been found to
connect them to one another or to other nucleoporins. A re-
cent exception is Nup50, required for protein export (Guan
et al., 2000). Here we report four large proteins that interact
with Nup98. The same four proteins also bind Nup153. We
demonstrate that all four are nucleoporins, two known and
two hitherto unknown, which we now term vertebrate
Nup160 and Nup133. All are present in a large subcomplex
of the nuclear pore. The complex appears to play a role not
only in tethering Nup98 and Nup153 to the nucleus and
the pore, but also in vertebrate mRNA export.
Results
Novel molecular partners for the RNA export
nucleoporin, Nup98
To more clearly define those components of the nuclear
pore required for RNA export, the protein partners of
Nup98 were sought. The amino half of Nup98, containing
GLFG repeats and a Gle2-binding site, is thought to inter-
act primarily with transport factors. Thus, we focused on
the carboxyl half of Nup98 as a likely site of interaction
with putative nucleoporins. Recombinant Nup98 frag-
ments complexed to Sepharose beads were used for pull-
downs from extracts of
Xenopus
eggs containing the disas-
sembled proteins of

2.5

10
8
nuclear pores (Cordes et
al., 1995). The Nup98 COOH terminus was found to
bind a distinct set of silver staining proteins from
Xenopus
egg extracts, (Fig. 1 b, lanes 3–5). Those

60 kd were de-
termined to be either nonspecifically bound, as they were
also pulled down by protein A-Sepharose (Fig. 1 b, lane 2),

Nup160 and Nup133 play a role in mRNA export |
Vasu et al. 341
or were obscured by BSA. An

97-kd protein was also ob-
served to bind (*, Fig. 1 b, lanes 3–5), but because this pro-
tein was greatly decreased after Ran-GTP addition (*, Fig. 2
c, compare lanes 3 and 4), it resembled a Ran-sensitive trans-
port receptor and was not analyzed further. Bands present in
lesser amounts or variably present from experiment to experi-
ment (dot, Fig. 1 b, lanes 3–5) also were not analyzed further.
Our attention focused on four large proteins, designated
A–D, consistently pulled down by the COOH terminus of
Nup98 (Fig. 1 b, lanes 3–5). Proteins A–D bound to
Nup98 fragments I, II, and III. We found that fragments I
and II become self-cleaved in vitro, as occurs with endoge-
nous Nup98, to produce a COOH terminus at amino acid
(aa) 876 (unpublished data; Fontoura et al., 1999; Rosen-
blum and Blobel, 1999). We concluded that the Nup98
binding site(s) for the A–D proteins must be upstream of the
HF
876
SKY cleavage site (Fig. 1 a, dotted line). When Nup98
aa 470–824 was used, proteins A–D no longer bound (Fig. 1
b, fragment IV). Thus, Nup98 aa 825–876 are critical for
binding the A–D proteins in that removal of these terminal
52 aa abolishes binding. The fact that all four are lost with
the same small deletion first suggested that they might bind
to Nup98 as a complex.
The A–D proteins migrate on gels at

145, 130, 112,
and 103 kd, respectively (Fig. 1 b). Their binding to Nup98
fragments is Ran insensitive (Fig. 2 c, lanes 3–4), as well as
stable to 500 mM NaCl (unpublished data), indicative of
strong protein–protein interactions. Because the Nup98
fragments used above all contain a potential, if abbreviated,
RNA-binding motif (Radu et al., 1995), and because
Nup98 binds to certain homoribopolymers in vitro (Ullman
et al., 1999), we tested for an effect of RNase on the Nup98/
A–D interaction; we found none (unpublished data). This
suggests that no RNA moiety is required for the formation
or the maintenance of the Nup98/A–D interactions.
The basket nucleoporin Nup153 interacts with
proteins A–D
Nup153 is localized to the most distal ring of the nuclear
pore basket (Panté et al., 1994). The NH
2
terminus of hu-
man Nup153 (aa 1–339) is sufficient to target to nuclear
pores in transfected cells (Enarson, et al., 1998). To search
for nucleoporin partners of Nup153, fragments of the NH
2
terminus (aa 1–339) (Fig. 2 a) were coupled to beads and
used in pulldowns from
Xenopus
egg cytosol. Proteins

60
kd were either nonspecifically bound or were obscured by
BSA (Fig. 2 b). Strikingly, four large proteins with a mobil-
ity identical to proteins A–D bound to aa 1–339 of human
Nup153 (Fig. 2 b, lane 4); these were designated A

–D

. A
very abundant protein of

97 kd also bound (*, Fig. 2 b,
lanes 2–4), but was not studied further as it was Ran sensi-
tive (*, Fig. 2 c, compare lane 5 with lane 6) and bound even
in absence of A

–D

binding (*, Fig. 2 b, lanes 2 and 3). The
A

–D

proteins did not bind to Nup153 aa 1–245 (Fig. 2 b,
lane 3), to a
Xenopus
Nup153 fragment equivalent to aa
431–723 of human Nup153 (Nup153-N

, Fig. 2 b, lane 2),
or to Sepharose beads coupled to control proteins (Fig. 2 b,
lane 1, and unpublished data). To test whether A

–D

were
identical to A–D, the A–D proteins from a Nup98 column
were biotinylated, and added to beads containing either
green fluorescent protein (GFP), Nup153 aa 1–339, or
Nup153-N

. The A–D proteins bound only to the Nup153
1–339 beads (unpublished data), indicating the A

–D

and
A–D proteins are identical. For Nup153, aa 246–339 are
most critical for interaction with the A–D proteins.
Protein purification reveals nucleoporins Nup96
and Nup107
To identify the A–D proteins, small scale pulldown reactions
were probed for known vertebrate nucleoporins and transport
factors by immunoblotting (IB). The pulldowns did not
contain substantial amounts of Nup62, Nup93, Nup98,
Nup155, Nup205, Nup214, Nup358, the pore-associated
protein Tpr, importin

, importin

, Gle2, Crm1, or the nu-
clear protein RCC1, although traces of importin

and

, and
Nup93 and Nup205 were observed (Table SI, available at
http://www.jcb.org/cgi/content/full/jcb.200108007/DC1).
Overall, the finding that these other proteins did not bind to
the Nup98 and Nup153 fragments in question underlined
the specificity of association with proteins A–D.
The A–D proteins were purified and examined by pro-
teolytic cleavage and peptide sequence analysis. The A

–D

proteins gave the same peptide sequences as the A–D proteins
Figure 1. Proteins A–D bind to the COOH terminus of Nup98.
(a) Subcloned fragments I–IV of rat Nup98. The dotted line shows the
cleavage site for Nup98 endoproteolytic activity (Fontoura et al.,
1999; Rosenblum and Blobel, 1999). Gle2 binds to Nup98 aa
150–224, whereas GLFG repeats occupy much of the remainder of aa
1–470. (b) Nup98 fragments I–IV coupled to beads were mixed with
Xenopus egg cytosol in pulldown reactions. The * indicates a 97-kd
Ran-sensitive protein. The filled circle indicates a band variably seen.
Proteins A–D bound to fragments I–III (lanes 3–5), but not to fragment
IV (lane 6) or S. aureus protein A beads (Ctl, lane 2). Xenopus egg
extract (Ext; 0.02 l) is shown for comparison (lane 1). Size markers
are 205, 116, 97, and 66 kd, respectively (left hatchmarks).

342 The Journal of Cell Biology
|
Volume 155, Number 3, 2001
(unpublished data).
Xenopus
band C produced peptides with
high homology to human Nup96 (Fig. 3 a), whereas band D
revealed peptides with almost complete identity to rat
Nup107 (Fig. 3 b) (Radu et al., 1994; Fontoura et al., 1999;
Rosenblum and Blobel, 1999). Human Nup96 migrates at
115 kd, a molecular mass essentially identical to that of
Xeno-
pus
protein C at

112 kd. Rat Nup107 migrates at

107 kd,
almost identically to
Xenopus
protein D at

103 kd. We con-
cluded from the size identity, sequence homology, and bio-
chemical behavior of the proteins (see below) that C and D
are the
Xenopus
homologues of Nup96 and Nup107, respec-
tively.
A new vertebrate nucleoporin, Nup133
Peptide analysis of
Xenopus
protein B yielded compelling
matches to a predicted human protein of 1156 aa with un-
known function (AK001676), as well as to mouse ESTs.
When a sensitive Psi-BLAST search was done with the hu-
man sequence, it revealed relatedness to proteins of identical
length in
Drosophila
(1,154 aa; AAF56042; 24% iden-
tity, 44% similarity) and
Schizosaccharomyces pombe
(1162
aa; CAB55845.1). Strikingly, the
S. pombe
protein had
22% identity with a
Saccharomyces cerevisiae
nucleoporin,
Nup133 (1157 aa) (Doye et al., 1994; Li et al., 1995; Pem-
berton et al., 1995). All are predicted to migrate at

130
kd, identical in size to
Xenopus
protein B. The human se-
quence shows very distant, but discernable sequence related-
ness to ScNup133 (18% identity for aa 536–903).
An antibody was raised to aa 777–1105 of the human
protein. This recognized a single band of

130 kd in HeLa
cells and in rat liver nuclei (Fig. 4 a). When used to probe
Nup98 aa 470–876 pulldowns from
Xenopus
extracts, the
antibody recognized a single

130-kd protein (Fig. 4 b, lane
6) identical in size to
Xenopus
protein B as visualized by sil-
ver stain (lane 3). The band was not seen in Nup98 aa 470–
824 pulldowns (Fig. 4 b, lanes 2 and 5). Immunofluores-
cence (IF) of HeLa cells gave a punctate nuclear rim stain
(Figs. 4 c and 5 b). We conclude that the 130-kd human
Figure 3. Proteins C and D are the known vertebrate nucleoporins
Nup96 and Nup107. (a) Two of the peptides obtained from Band C
are shown. (b) Three of the peptides obtained from Band B and D
are shown and match with near identity to rat nucleoporin Nup107.
Identity is boxed and homology is indicated in gray, as defined by
Kyte-Doolittle algorithms.
Figure 2. Nucleoporin Nup153 binds the same
four proteins. (a) A map of Nup153 and the
fragments used. (b) Nup153 fragments coupled to
beads were used in pulldowns. Nup153 aa 1–339
bound four proteins similar in size to A–D, termed
A’–D’ (lane 4). A zz tag control fragment and two
Nup153 fragments, xNup153-N’ and human
Nup153 aa 1–245, did not (lanes 1–3). Size
markers are 205, 116, 97, and 66 kd, respectively.
(c) Nup98 fragment II and Nup153 aa 1–339 bind
proteins identical in size (upper panel). Each also
bound a protein of 97 kd (*; lanes 3 and 5) that
was largely removed by the addition of RanQ69L
(lanes 4 and 6). A–D and A’–D’ binding were
not sensitive to RanQ69L (lanes 3–6). Control
S. aureus protein A-Sepharose beads did not bind
A–D (lanes 1 and 2). RanQ69L was functional
(lower panel), as it dissociated transportin from
a Nup153 fragment (Shah and Forbes, 1998).
The lower panel is an immunoblot with anti-
transportin antibody; compare lane 5 (no Ran)
with lane 6 (Ran).

Nup160 and Nup133 play a role in mRNA export |
Vasu et al. 343
and the
Xenopus
B protein are new vertebrate nucleoporins,
now termed vertebrate Nup133. A comparison to the
Dro-
sophila
Nup133 homologue is shown in Fig. 4 d. Addition-
ally, we have raised an antibody to a
Xenopus
sequence
homologue of human Nup133. This recognizes
Xenopus
protein B (see Fig. 7 c, lane 2), closing the circle and demon-
strating that
Xenopus
protein B is xNup133.
Antibodies to vertebrate Nup133 bind to the
nucleoplasmic face of the pore
To localize Nup133, IF was performed using digitonin per-
meabilization of HeLa cells, where only the cytoplasmic side
of the pore is accessible to antibody (Fig. 5, exterior), and
Triton permeabilization, which renders both sides of the
pore accessible (Fig. 5, interior

exterior). Anti-Nup133
stained the nuclear pore only when the nuclear envelope was
permeabilized by Triton X-100 (Fig. 5 b) or by exception-
ally long digitonin permeabilization (unpublished data). An-
tibodies to Nup153 (Fig. 5, i–j), Nup98 (Fig. 5, m–n), and
lamin B (Fig. 5, c–h, k–p) also stained their antigen only
when the nuclear envelope was permeabilized. Monoclonal
mAb414, which can recognize Nup214 and Nup358 on the
cytoplasmic filaments of pores, gave a punctate nuclear rim
in both digitonin- and Triton-permeabilized cells (Fig. 5,
e–f). We conclude that Nup133 is primarily accessible on
the basket side of the pore.
Identification of a large novel vertebrate
nucleoporin, Nup160
Multiple peptides were obtained from
Xenopus
band A (Fig.
6 a). All showed high homology to a putative 160-kd mouse
protein (1402 aa; AAD17922) (Fig. 6, a and d), a hu-
man protein of unknown function (1314 aa; KIAA0197;
91% identity), a 160-kd
Drosophila
protein (1411 aa;
AAF53075.1; 28% identity; 47% homology), and a more
distantly related 176-kd
Caenorhabditis elegans
protein
(AAB37803.1). A search of
Xenopus
cDNAs revealed a
Xeno-
pus
EST. This encoded a highly homologous
Xenopus 160-
kd protein. Antibody raised to the Xenopus 160-kd protein
cross reacted with a single protein in Xenopus egg cytosol
identical in size to Band A (Fig. 7 c, lane 1). IF with both
this antibody (unpublished data) and an antibody raised to
the human 160-kd protein gave a punctate nuclear rim stain
(Fig. 6 c). Thus, we have designated Xenopus band A and its
Figure 4. A novel vertebrate
nucleoporin, Nup133. (a) Antibody
to aa 777–1105 of the human protein
AK001676, homologous to Xenopus
protein B, recognized a single 130-kd
protein in HeLa cell extracts (lane 1) and
in rat liver nuclei (lane 2). Markers are
205, 116, and 97 kd. (b) Pulldowns with
Nup98 fragments were split and
electrophoresed on two gels. One was
silver stained (lanes 1–3) and one
immunoblotted with anti-Nup133
antibody (lanes 4–6). An antibody-
reactive band of 130 kd was observed
only in the Nup98 aa 470–876 pull-
down (lane 6) and ran coincident with
silver-stained protein B (lane 3). Ctl
represents a pulldown with S. aureus
protein A beads. (c) IF on HeLa cells
using affinity-purified anti-Nup133
antibody; the inset shows a portion of
the same nucleus magnified to reveal
the punctate nuclear rim stain. (d) The
sequence of the novel human nucle-
oporin Nup133 (AK001676) is
compared with a highly related
Drosophila homologue (AAF56042).
Identity is boxed and homology is
indicated in gray, as defined by
Kyte-Doolittle algorithms.

344 The Journal of Cell Biology | Volume 155, Number 3, 2001
relatives in mice, humans, and Drosophila as nucleoporin
Nup160. A comparison of mouse and Drosophila Nup160 is
shown in Fig. 6 d. Like Nup133, Nup160 is primarily acces-
sible to antibody only on the nuclear side of the pore (Fig. 6
c, right panel).
We searched for homology to any known yeast nucle-
oporins. Yeast homologues of Nup96 and Nup107 are nor-
mally in complex with four other nucleoporins, Nup120p
(120 kd), Nup85p (85 kd), sec13p (33 kd), and seh1p (39
kd) (Siniossoglou et al., 1996, 2000), all seemingly too
small to be a homologue of metazoan Nup160. Mouse
Nup160 showed no sequence homology to yeast Nup85p,
sec13p, or seh1p. A highly sensitive Psi-BLAST search us-
ing S. cerevisiae Nup120 (1037 aa) (Aitchison et al., 1995;
Heath et al., 1995; Altschul et al., 1997) brought up no
Drosophila or vertebrate homologues, but did show NH2-
terminal homology with aa 100–445 of an S. pombe se-
quence of 1136 aa (PIR T40355) (Fig. 6 b). When a stan-
dard BLAST was done using this putative S. pombe
Nup120p, it identified S. cerevisiae Nup120 and the same
Drosophila and C. elegans sequences we had observed with
mouse Nup160. When a more sensitive Psi-BLAST search
was done starting with the putative SpNup120p (rather
than ScNup120p), only five sequences were observed as re-
lated: the mouse 160-kd protein, the related human pro-
tein, the Drosophila and C. elegans sequences, and Sc-
Nup120. The metazoan Nup160 proteins and ScNup120p
contain no FG or other repeat sequences.
Direct comparison of the metazoan Nup160 proteins and
the shorter yeast Nup120 proteins shows that they differ
greatly in sequence. The highest homology between the puta-
tive S. pombe Nup120p and the Nup160 proteins is at the S.
pombe COOH terminus (gray box, Fig. 6 b). S. cerevisiae
Nup120p (aa 756–1033) has quite good homology to Dro-
sophila Nup160 in this region (aa 861–1130) (black rimmed
boxes; Fig. 6 b), but not to human or mouse Nup160. The
NH2 termini of S. cerevisiae and putative S. pombe Nup120
show the most resemblance to one another, but show little
similarity to the same region in the Nup160 proteins. The
central domain of ScNup120p (aa 426–755) has diverged the
most, both from the Nup160 proteins and from the S. pombe
protein. Most strikingly, the metazoan Nup160 proteins con-
tain an additional 33 kd at their COOH termini (Fig. 6 b).
Clearly, of the four A–D nucleoporins, Nup160 has under-
gone the most changes through evolution.
A Nup160 complex of nucleoporins in vertebrates
To determine whether the A–D proteins interact with
Nup98 and Nup153 individually or as a complex, egg ex-
tract was subjected to gel filtration, and 54 fractions were
collected. The odd fractions were subjected to pulldown
with Nup153 aa 1–339 beads and the bound proteins were
analyzed by SDS-PAGE and silver staining. All four nucle-
oporins A–D were pulled down from the same fractions
(33–41, Fig. 7 a) and peaked in fraction 37 (dots). An im-
munoblot using anti-hNup133 antibody on the even frac-
tions (i.e., total cytosol with no pulldown) gave essentially
the same pattern of migration for Nup133 (Fig. 7 b).
Immunoprecipitation was performed from unfractionated
egg extract under native conditions (Fig. 7 c). Anti-Nup160
antibody coimmunoprecipitated Nup133 (Fig. 7 c, lane
5), whereas anti-Nup133 antibody coimmunoprecipitated
Nup160 (Fig. 7 c, lane 6). A control anti-Nup62 antibody
did not immunoprecipitate either Nup160 or Nup133 (lane
7). Thus, Nup160 and Nup133 exist in the same complex.
From this and the cofractionation of proteins A–D in pull-
downs (Figs. 1, 2, 4, and 7 a), we designate Xenopus Nup160,
Nup133, Nup107, and Nup96 proteins as members of a
Nup160 complex. The Xenopus Nup160 complex migrates
at 700–800 kd.
As stated above, the yeast homologues of Nup96 and
Nup107 are extracted from yeast pores in complex with yeast
Nup120p, Nup85p, sec13p, and seh1p (Pryer et al., 1993;
Siniossoglou et al., 1996, 2000). By silver stain, we never ob-
Figure 5. Antibody to Nup133 localizes
to the nuclear side of the pore. HeLa
cells were permeabilized with digitonin
to allow antibodies access only to the
exterior face of the nuclear envelope
(Exterior). The monoclonal antibody
mAb414 detects the presence of Nup214
and Nup358 on the cytoplasmic face of
the nuclear pores (e). Nup133, Nup153,
or Nup98 were not detected on this side
of the pores (a, i, and m, respectively). As
a control for nuclear envelope integrity,
the cells were co-stained with the anti–
lamin B antibody, LS-1 (c, g, k, and o). To
probe the nuclear interior, Triton X-100
(Interior  Exterior) or longer digitonin
(unpublished data) permeabilization was
used. Anti–lamin B stain confirms nuclear
envelope penetration of the antibody
(d, h, l, and p) after TX-100. Nup153 and
Nup98 are detected on the intranuclear
face of pores, as expected (j and n).
Nup133 is detected on the interior (b),
but not the exterior (a) of the nuclear
envelope.

Nup160 and Nup133 play a role in mRNA export | Vasu et al. 345
serve a vertebrate 85-kd protein in our pulldowns. However,
we have recently identified a sequence with homology to
ScNup85, implying the Nup160 complex may also contain
vertebrate Nup85 protein.
Putative proteins of the size of sec13 and seh1 would have
been obscured by small nonspecifically bound proteins in our
silver-stained pulldowns (Figs. 1 and 2). Although the existence
of a vertebrate seh1 is controversial, human sec13 has been
cloned (Shaywitz et al., 1995). Using anti–human sec13 anti-
body (unpublished data), we probed the gel filtration fractions
of total Xenopus egg cytosol and found that human sec13 mi-
grates in a region identical to that of Nup133 (Fig. 7 b) and to
A–D (Fig. 7 a). Sec13 protein is present in Nup98 aa 470–876
pulldowns (Fig. 7 d, lane 3), but not in Nup98 aa 470–824
pulldowns (Fig. 7 d, lane 4). Similarly, sec13 is present in
Nup153 aa 1–339 pulldowns (Fig. 7 d, lane 5), but not in
Nup153 aa 1–245 pulldowns (Fig. 7 d, lane 6). Most convinc-
ingly, sec13 is coimmunoprecipitated by both anti-Nup160 and
Nup133 antibodies (Fig. 7 c, lanes 5 and 6). Thus, sec13 is a
member of the vertebrate Nup160 complex, which now mini-
mally contains vertebrate Nup160, Nup133, Nup107, Nup96,
sec13, and likely a vertebrate Nup85 (unpublished data).
Neither Nup98 nor Nup153 were found in anti-Nup160
or anti-Nup133 immunoprecipitates (Fig. 7 c, lanes 9 and
10; see also Table I in the online supplement), as determined
by IB. The absence of Nup98 and Nup153 from the
Nup160 complex is entirely consistent with previous find-
ings that, when nuclear pores disassemble at mitosis, Nup98
is found primarily in a pore subcomplex containing the
transport factor Gle2, both in egg extract (unpublished data)
and in human mitotic extracts (Matsuoka et al., 1999). Sim-
ilarly, Xenopus Nup153 is found primarily in complex with
the transport receptors importin , , and transportin in egg
extract (Shah et al., 1998; Shah and Forbes, 1998).
Assembled pores contain the Nup160 complex
We examined assembled pores for the Nup160 complex.
Annulate lamellae (AL), cytoplasmic stacks of membranes
Figure 6. A new vertebrate nucleoporin,
Nup160. (a) Peptides from Xenopus
band A match sequences of a 160- kd
unknown mouse protein (AAD17922),
a human clone (KIAA0197), and an
overlapping human EST (N53299).
(b) A schematic comparison of the
human (Hu), mouse (Mo), and
Drosophila (Dm) Nup160 proteins and
distantly related S. cerevisiae ScNUP120
and putative S. pombe Nup120 proteins.
Thick lines indicate relatedness, hatched
boxes show relatedness between the two
yeast proteins, gray boxes indicate a
region of moderate homology between
S. pombe and metazoans, and the black
outlined box indicates a region of
homology between ScNup120 and
Drosophila Nup160. The hatched box
and thin lines of the yeast proteins show
little relatedness to the vertebrate
proteins. (c) Antibody raised to the
putative human Nup160 protein gives a
punctate nuclear rim stain on TX-100
permeabilized HeLa cells (right panel),
but not on digitonin-permeabilized cells
(left panel), indicating localization of
Nup160 on the basket face of the pore.
(d) A comparison of mouse Nup160 and
the highly related Drosophila homo-
logue, aligned using Clustal W. Identi-
ties are boxed, homologies are in gray.

346 The Journal of Cell Biology | Volume 155, Number 3, 2001
that contain abundant pores identical to nuclear pores, can
easily be assembled in Xenopus extracts in vitro (Dabauvalle
et al., 1991; Cordes et al., 1995; Meier et al., 1995; Miller et
al., 2000; Miller and Forbes, 2000). AL were formed in egg
extract, purified, and treated with 0.5 M NaCl to partially
solubilize the pores (Miller et al., 2000). When this mixture
was added to Nup98 beads (fragment II), all four A–D pro-
teins were pulled down (Fig. 8 a, lane 4), as was sec13 (Fig. 7
d, lane 1). Neither A–D (Fig. 8 a, lane 3) nor sec13 (Fig. 7
d, lane 2) were pulled down from AL reactions done in the
presence of the pore assembly inhibitor BAPTA (Macaulay
and Forbes, 1996; Goldberg et al., 1997). The A–D proteins
were pulled down from normal AL pores by Nup153 aa
1–339 beads (unpublished data). We conclude that proteins
A–D and sec13 are present together in the assembled pores
of AL. Also indicative of this, we found that the A–D pro-
teins, when purified and biotinylated, could be incorporated
into AL in vitro (unpublished results).
Rat liver nuclei were also gently solubilized with Triton
X-100, and the extracted proteins were added to Nup153 aa
1–339 beads. Two rat proteins very similar in size to Xeno-
pus Nup96 (C) and Nup107 (D) were observed by silver
stain to pulldown (Fig. 8 b, compare lane 3 with lane 4). A
dark band intermediate in size between Xenopus Nup160 (A)
and Nup133 (B) was also observed in the rat pulldowns
(Fig. 8 b, lane 3, upper band). This band reacted strongly
Figure 8. The Nup160 complex is in assembled pores. (a) AL
assembly reactions were done in the presence or absence of
BAPTA. AL were isolated and partially solubilized with 0.5 M NaCl
which does not destabilize the A–D complex. Solubilized AL was
clarified by centrifugation and used for pulldowns with Nup98
fragment II beads. Proteins A–D were pulled down from egg extract
(lane 2, Ext) and from AL (lane 4) by Nup98 beads, but not by
S. aureus protein A beads (lane 1, Ctl) or from AL reactions in which
pore assembly had been inhibited by BAPTA (lane 3). Mw markers
are 116 and 97 kd, respectively. (b) Rat liver nuclei were partially
disassembled with 2% Triton. The clarified supernatant was
incubated with Nup153 aa 1–339 beads. Three rat proteins in the
relevant size range were observed by silver staining to be pulled
down (lane 3), two similar to Xenopus C and D (lane 4), as well as
a thick band intermediate between Xenopus A and B. None bound
to zz-tag control beads (lanes 1 and 2). A portion of the Nup153 aa
1–339 pulldown from solubilized rat liver nuclei was probed with
anti–human Nup133 antibody and gave a single immunoreactive
band (lane 5) coincident with the thick band observed by silver
stain (lane 3), indicating that this is rat Nup133.
Figure 7. The Nup160 nuclear pore subcomplex. (a) After gel
filtration of Xenopus egg cytosol, odd fractions were subjected to
Nup153 aa 1–339 pulldown, SDS-PAGE, and silver staining. A–D
fractionated together and peaked at fraction 37 (dots). The left
hatchmark indicates a 116-kd mw marker protein. When size
controls were monitored for migration on the gel filtration column
(unpublished data), Nup214 was seen to fractionate in a complex
peaking at 1,000 kd (peak fraction  34) and Nup98 fraction-
ated in a complex peaking at 450 kd (peak fraction  44), as
expected (Macaulay et al., 1995). The dark band between C and D
was not seen in pulldowns of unfractionated extract (Figs. 1 and 2)
and is likely a breakdown product. (b) The even fractions (of total
egg extract) were probed with anti-Nup133 and anti-sec13
antibodies. Nup133 peaks coincident with the peak of the
silver-stained bands A–D indicated by dots in panel (a). The
majority of soluble sec13 in Xenopus extracts also peaks in this
region. (c) Immunoprecipitation from Xenopus cytosol using
anti-xNup160 (lanes 5 and 9), anti-xNup133 (lanes 6 and 10),
anti-xNup62 (lanes 3 and 7), and a mix of the preimmune sera
for xNup160 and xNup133 (lane 4). The top portion of the blot
corresponding to lanes 4–7 was probed with both anti-xNup160
and anti-xNup133 antibodies and the bottom with anti-sec13 anti-
body. The blot of lanes 8–10 was probed with both anti-rNup98
and anti-rNup153. Xenopus cytosol (0.2 l) is shown in lanes 1, 2,
and 8. Lane 1 was probed with anti-xNup160, lane 2 with anti-
xNup133, and lane 3 with anti-xNup62. The molecular markers
(left) were 200, 120, 90, 68, 53, 36, and 32 kd. Nup153 runs
aberrantly high at 180 kd (Sukegawa and Blobel, 1993) (d) To
ask whether sec13p is in Nup98 pulldowns, Nup98 aa 470–876
pulldowns were done from solubilized AL (lane 1), solubilized
mock AL made in the presence of the pore assembly inhibitor
BAPTA (lane 2), and egg extract (lane 3). A pulldown from egg
extract using Nup98 aa 470–824 beads was done in lane 4. Pull-
downs from Xenopus extract were also done with Nup153 aa
1–339 (lane 5) and Nup153 aa 1–245 (lane 6). All pulldowns were
probed with anti–human sec13p antibody.

Nup160 and Nup133 play a role in mRNA export | Vasu et al. 347
with our anti–human Nup133 antibody (Fig. 8 b, lane 5).
Thus, rat liver nuclei contain three proteins similar in size
and/or immunogenicity to those of the Xenopus Nup160
complex that are pulled down by Nup153 beads, consistent
with the existence of a Nup160 complex in assembled rat
nuclear pores.
Nup153 and Nup98 are tethered to nuclear sites by
their Nup160 complex binding domains
To determine the importance of its Nup160 complex bind-
ing site to Nup153, myc-tagged Nup153 constructs were
transfected into cells. A Nup153 fragment lacking the
Nup160 complex binding site, when transfected, was cyto-
plasmic (Fig. 9 e). However, Nup153 aa 1–339, which
binds the Nup160 complex, localized to the nuclear rim in a
punctate pattern typical of a nuclear pore stain (Fig. 9 f),
consistent with a previous localization study (Enarson et al.,
1998). In that study, Nup153 aa 1–245 localized to the nu-
clear rim, but not to the pore. Thus, the Nup153 aa that are
required for A–D binding, aa 246–339, are also essential for
Nup153’s targeting to the nuclear pore (Fig. 9 f).
When a Nup98 fragment that cannot bind the Nup160
complex was transfected into cells, it showed diffuse cyto-
plasmic localization (Nup98 aa 470–824; Fig. 9, a and c).
However, Nup98 aa 470–876, which contains the addi-
tional 52 aa that allow the Nup160 complex to bind, local-
ized to the nucleus (Fig. 9 b). This pattern mirrors that of
endogenous Nup98, i.e., a rim and intranuclear stain (Pow-
ers et al., 1995; Radu et al., 1995; Zolotukhin and Felber,
1999). We tested whether the shorter 470–824 fragment
might simply lack an NLS by testing localization of the
shorter fragment plus and minus leptomycin B (LMB), a
drug that inhibits NES-mediated export (Fornerod et al.,
1997a). When LMB was present, the shorter Nup98 aa
470–824 fragment localized to the nucleus (Fig. 9 d), indi-
cating that this shorter fragment can normally shuttle be-
tween the nucleus and cytoplasm. We conclude that Nup98
aa 470–876 localizes to the nucleus, not because it contains
an NLS that aa 470–824 lacks, but because it is actively
tethered to the nucleus by the 52 COOH-terminal aa crit-
ical for binding the Nup160 complex.
Smaller Nup98 constructs were made. Nup98 aa 611–
703 did not bind the Nup160 complex (unpublished data),
but aa 611–876 both bound the complex and localized to
the nucleus upon transfection (unpublished data). We con-
clude that aa 611–876 are sufficient for binding the Nup160
complex and for tethering the Nup98 fragment within the
nucleus. In particular, aa 824–876 are essential for both
complex binding and nuclear tethering.
Fragments of Nup160 and Nup133 inhibit nuclear
export of poly[A] RNA
The novel vertebrate nucleoporins Nup160 and Nup133
were discovered through their interaction with Nup98 and
Nup153, both critical for vertebrate mRNA export. To ask
whether Nup160 and Nup133 play a role in RNA export,
myc-tagged fragments of the genes were transfected into
HeLa cells and poly[A] RNA was monitored by hybridiza-
tion to Texas red oligo[dT]50. Cells were stained with FITC
anti-myc antibody to reveal successful transfection. Untrans-
fected cells (Fig. 10 for those not stained by anti-myc anti-
body), as well as cells transfected with the negative control
malate dehydrogenase gene (Fig. 10, e and h), showed dif-
fuse cytoplasmic poly[A] RNA staining with intranuclear
spots. This pattern is typical of poly[A] RNA localization
in normal cells (Heath et al., 1995; Bastos et al., 1996; Wat-
kins et al., 1998; Pritchard et al., 1999). A Nup98 fragment
containing the Gle2 binding site (aa 150–224), which is
known to cause nuclear poly[A] RNA accumulation (Prit-
chard et al., 1999), served as a positive control for inhibition
of mRNA export (Fig. 10, f and i). A fragment containing
residues 1–1149 of human Nup133 had little effect on
poly[A] RNA (Fig. 10, g and j). However, human Nup133
residues 587–936, when transfected into HeLa cells, caused
strong nuclear poly[A] accumulation (transfected cells, Fig.
10 d). Transfection of residues 317–697 of human Nup160
also caused nuclear poly[A] RNA accumulation (Fig. 10 b,
transfected cells). None of the constructs above affected the
hormone-inducible nuclear import or the export, upon hor-
mone withdrawal, of a shuttling reporter protein RGG
(Fig. S1, available at http://www.jcb.org/cgi/content/full/
jcb.200108007/DC1). In RGG, HIV Rev is fused to GFP
and to the hormone binding domain of the glucocorticoid
Figure 9. Nup98 and Nup153 tether in the nucleus and to the
pore, respectively, using their Nup160 complex binding domains.
After transfection, myc-tagged Nup98 aa 470–824 is cytoplasmic (a
and c), whereas Nup98 aa 470–876 is nuclear (b), like endogenous
Nup98 (Powers et al., 1995). Nup98 aa 470–824 has access to the
nucleus since it localizes there upon LMB addition (LMB; 100 nM, l
hr) (d). Fragment 1C3 from the COOH terminus of Xenopus Nup153
(Shah et al., 1998) localized throughout the cell with no nuclear rim
(e). Human Nup153 aa 1–339, which binds the Nup160 complex,
localized to nuclear pores (f), as in Enarson et al. (1998).

348 The Journal of Cell Biology | Volume 155, Number 3, 2001
receptor (Love et al., 1998; Gustin and Sarnow, 2001). Ex-
port of the RGG reporter protein could be inhibited by the
FG domain of Nup214, which is known to inhibit NES
protein export (Zolotukhin and Felber, 1999) (Fig.
S1, available at http://www.jcb.org/cgi/content/full/jcb.
200108007/DC1). We conclude that fragments of the novel
nucleoporins Nup133 and Nup160 elicit a dominant nega-
tive effect on poly[A] RNA export, while leaving RGG
protein import and export pathways intact.
Discussion
Only two proteins of the nuclear pore basket, Nup98 and
Nup153, have been identified previously as playing a role in
RNA export. We have now identified four large nucleopor-
ins that interact with Nup98 and Nup153. Two are Nup96
and Nup107, whereas two are nucleoporins previously un-
known in vertebrates, now designated vertebrate Nup160
and Nup133. Antibodies localize the novel proteins to the
basket side of the nuclear pore. Nup133 is distantly related
to S. cerevisiae Nup133, whereas Nup160 is very remotely
related to yeast Nup120. Pulldowns, gel filtration, and
coimmunoprecipitation all show that Xenopus Nup160,
Nup133, Nup107, Nup96, and the small secretory protein
sec13 form a complex, the Nup160 complex. The complex
is present in Xenopus egg cytosol, as well as in assembled
pores. We have mapped the sites that are required for bind-
ing the Nup160 subcomplex: in Nup98, the binding site
tethers Nup98 to the nucleus; in Nup153, the binding site
targets Nup153 to the nuclear pore. When fragments of the
Figure 10. Dominant negative
fragments of Nup133 and Nup160 cause
nuclear accumulation of poly[A] RNA.
HeLa cells were transfected with pCS2MT
vectors containing: human Nup160 aa
317–697 (a and b) and 133 aa 587–936
(c and d), malate dehydrogenase
(negative control; e and h), rat Nup98
aa 150–224 (positive inhibitory control;
f and i), or human Nup133 aa 1–1149
(g and j) in myc-tagged form. Successful
transfection was assayed by IF with
FITC–anti-myc antibody (a, c, and e–g),
whereas the localization of poly[A] RNA
was assessed by hybridization of the
coverslips with Texas red oligo[dT]50
(b, d, and h–j). Both human Nup160 aa
317–697 (b) and Nup133 aa 587–936 (d)
caused a striking accumulation of
poly[A] RNA in the nucleus, identical
to that caused by Nup98 aa 150–224 (i).

Nup160 and Nup133 play a role in mRNA export | Vasu et al. 349
novel nucleoporins Nup160 and Nup133 are overexpressed,
they cause strong intranuclear accumulation of poly[A]
RNA, indicating that the Nup160 complex plays a func-
tional role in mRNA export.
The existence of the Nup160 complex was suggested from
the short stretch of aa in Nup98 and Nup153 essential for
pulldown of the A–D proteins. Nup107, Nup96, and sec13
had been observed previously among 30 proteins extracted
from rat nuclear envelopes (Fontoura et al., 1999). However,
in that study it was not possible to determine an association,
as all 30 were present in only four protein-containing frac-
tions, and fifteen proteins were present with Nup107,
Nup96, and sec13p. Here, extensive gel filtration followed
by a Nup153 pulldown of each fraction showed that
Nup160, Nup133, Nup107, Nup96, and sec13 are all
pulled down from the same fractions with an apparent
complex mw of 700–800 kd. Definitive proof of a com-
plex came from the coimmunoprecipitation of Nup160,
Nup133, and sec13. We conclude that a Nup160 complex
exists and contains at a minimum Xenopus Nup160,
Nup133, Nup107, and Nup96, and sec13p. A vertebrate
Nup85 may also be present (unpublished data)
The related yeast Nup84 complex contains six members:
Nup107 and Nup96 homologues, as well as Nup120, sec13,
Nup85, and seh1 proteins (Siniossoglou et al., 1996, 2000).
Of these, we find Nup107, Nup96, Nup160 (a relative of
Nup120), and sec13. We cannot yet analyze whether a po-
tential vertebrate seh1 homologue is present. Instead, we
find an unexpected nucleoporin, the newly discovered verte-
brate Nup133. Vertebrate Nup133 is distantly related to S.
cerevisiae Nup133, which when mutant causes defects in
mRNA export (Doye et al., 1994; Li et al., 1995; Pemberton
et al., 1995). ScNup133p is not present in the yeast Nup84
complex. Either the pore basket differs in yeast and verte-
brates or, alternatively, the mitotic fracture of vertebrate
pores that produces the Nup160 complex occurs along dif-
ferent “fault lines” than those that create the yeast Nup84
complex.
In considering how the different subcomplexes of the ver-
tebrate pore basket connect one to another, we do not think
that Nup98 and Nup153 bind to the same Nup160 com-
plex protein. Excess soluble Nup153 fragment (aa 1–339)
has no effect on Nup98 pulldowns of A–D and visa versa
(unpublished data). Based on the affinity of recombinant
Nup96 and Nup98 (Rosenblum and Blobel, 1999), Nup96
may be the protein of the Nup160 complex that interacts
with Nup98. Careful examination of Nup153 pulldowns in-
dicates that Nup160 is more enriched than in Nup98 pull-
downs (Fig. 2 c, band A, lanes 3–6). This suggests that
Nup160 may be the complex protein that interacts with
Nup153.
We found no coimmunoprecipitation of the Nup160,
Nup98, and Nup153 subcomplexes with one another (Fig.
7). This is consistent with the finding that nuclear pore assem-
bly requires the presence of membranes (Dabauvalle et al.,
1991; Meier et al., 1995; Macaulay and Forbes, 1996). Pore
subcomplexes do not assemble into multisubcomplex struc-
tures unless membranes are present and subcomplex sizes are
identical in both interphase and mitotic extracts (Macaulay et
al., 1995; Matsuoka et al., 1999). Possibly the affinity of one
pore subcomplex for another is too low when they are present
as monomers dilute in interphase cytosol. However, when
subcomplexes are faced with the multiple adjacent copies of a
partner subcomplex in a forming pore, we predict that the af-
finity or avidity increases, allowing binding between subcom-
plexes. In the yeast pore, 8–52 copies of any given subcomplex
are present (Rout et al., 2000). In our experiments, numerous
closely apposed Nup98 or Nup153 molecules on beads may
mimic the multiple copies of these proteins found in the
forming pore, promoting the binding of the Nup160 com-
plex.
Functionally, the Nup160 complex plays at least three im-
portant roles. First, the ability of Nup98 to tether in the nu-
cleus coincides with its ability to bind the Nup160 complex.
Second, the ability of Nup153 to target to the pore requires
its Nup160 complex–binding site. Most importantly, the
Nup160 complex plays a role in RNA export: fragments of
Nup160 and Nup133 act as dominant negative inhibitors of
mRNA export. In yeast, the central portion of Nup133 is re-
quired for mRNA export (Doye et al., 1994; Li et al., 1995).
Here we find that a fragment from a similar region of verte-
brate Nup133 blocks mRNA export in mammalian cells.
Nup133 and Nup160 join a very small handful of verte-
brate nucleoporins involved in RNA export: Nup214,
Nup98, and Nup153 (Conti and Izaurralde, 2001; Vasu
and Forbes, 2001). Nup214 on the cytoplasmic filaments of
the pore acts as a late docking site for the mRNA export fac-
tor TAP (Katahira et al., 1999; Conti and Izaurralde, 2001).
Nup98 and Nup153 also bind TAP in vitro and may do so
in vivo (Bachi et al., 2000; Strasser et al., 2000; Tan et al.,
2000). They are also presumed to bind other export recep-
tors and proteins, such as Gle2. Nup133 and Nup160 may
function either directly by interacting with specific factors or
receptors involved in mRNA export, indirectly by tethering
Nup153 and Nup98 to the pore, or both.
It is worth reflecting on a recent mouse mutant engi-
neered to produce Nup98 that lacks the Gle2 binding site
(exon 3 of 31) but contains all other exons (Fontoura et al.,
2001; Wu et al., 2001). Yet unanswered in this mutant is
whether the bulk of the Nup98 protein is produced. Because
nucleoporin Nup96 is found to be present, the Nup 98 aa
715–920 that autocatalytically cleave their common precur-
sor must be also be present. Thus, at the very least, aa 715–
876 of the Nup160 binding site on Nup98 continue to be
present in the mutant mouse.
To form the nuclear pore basket, eight 1,000-Å filaments
connect to a 500-Å distal ring (Stoffler et al., 1999). Nup98
and Nup153 at 60 and 80 Å, if globular, are clearly much
smaller. ImmunoEM places Nup153 on the ring of the bas-
ket and Nup98 somewhere over the basket. In one model,
multiple copies of Nup98 could comprise the basket fila-
ments and use connectors to join to the 500-Å Nup153
ring. A molecular connector linking Nup98 and Nup153
has previously been lacking. The Nup160 complex, which
binds both Nup98 and Nup153, could fill the role of that
connector. The related Nup84 complex of yeast has a three-
legged or triskelion structure, perhaps suggesting a role for
the yeast complex at structural vertices (Siniossoglou et al.,
2000). In the future, the above proteins, together with the
new Nup50 and the finding that Nup153 cycles on and off

350 The Journal of Cell Biology | Volume 155, Number 3, 2001
the pore (Daigle et al., 2001), must be fit into the enigmatic
puzzle that forms the nuclear pore basket.
In a last consideration, the vertebrate Nup160 complex
may play a role in intranuclear architecture. This stems from
the finding that the yeast homologue of vertebrate Nup96,
SrNup145C, is instrumental in organizing multiple struc-
tures and functions within the yeast nucleus. Yeast Nup-
145C attaches intranuclear filaments to the yeast pore and
these in turn anchor telomeres, dsDNA repair enzymes, and
silenced genes to the yeast nuclear periphery (Galy et al.,
2000). In strains lacking Nup145C, yeast telomeres are re-
leased from the nuclear periphery, double strand break re-
pair is defective, and silenced genes become unsilenced.
Other intranuclear roles for vertebrate Nup96 have also
been suggested (Fontoura et al., 2001).
In summary, the novel nucleoporins Nup160 and Nup133
appear intimately involved in mRNA export and, as members
of a large complex, interact with the basket nucleoporins
Nup98 and Nup153. This is among the first evidence linking
subcomplexes of the vertebrate nuclear pore and, as such, allows
models of the vertebrate pore to be proposed and tested. Future
work may determine the Nup160 complex to be a central an-
choring point, both for the pore and for pore-associated pro-
teins.
Materials and methods
cDNA cloning and protein expression of Nup98 and Nup153
A partial rat Nup98 construct, 98-1 (aa 43–824) (Radu et al., 1995), was
converted to full-length Nup98 as follows: the complete rat Nup98 COOH
terminus was obtained by reverse transcription of total rat RNA, using an
oligo specific to the 3 end of the ORF in the Nup98 GenBank/EMBL/DDBJ
accession no. L39991, and spliced onto Nup98-1 to give Nup98 aa 1–935.
This was sequenced to verify authenticity. Oligonucleotides were used to
produce subfragments of Nup98, which were cloned as EcoR1–Xho1 frag-
ments into vector pET28B (In Vitrogen) for bacterial expression or as
Nco1–Xho1 fragments in pCS2MT for mammalian expression. A cDNA of
human Nup153 aa 1–800 was reverse transcribed from HeLa total RNA,
amplified by PCR, and cloned into pET28. Oligonucleotides were used to
amplify Nup153 aa 1–339 from this cDNA clone, whereas Nup153 aa
1–245 were cloned as an EcoR1 fragment from the original clone. These
Nup153 subfragments were separately cloned into a pET28 vector contain-
ing the Staphylococcus aureus protein A zz domain fused to a 6-His tag
(zz-pET28) to produce zz-tagged fragments. Cloning of Xenopus Nup153
N (Fig. 2 a) was described previously (Shah et al., 1998). Recombinant
protein was expressed in bacteria and isolated on Ni-NTA-agarose
(QIAGEN).
Nup98 and Nup153 pulldowns
Nup98 protein fragments were coupled to CnBr–Sepharose CL4B beads (Am-
ersham Pharmacia Biotech). Beads (20 l) containing Nup98 fragment (20 g)
were blocked with BSA (1 h; 20 mg/ml). Xenopus extract, diluted 1:50 in PBS,
1 mg/ml BSA, 1 g/ml aprotinin, and 1 g/ml leupeptin, was added and incu-
bated at 4C with tumbling. The beads were washed five times with PBS. We
found cleavage at HF876SKY to cause the Nup98 fragments I and II used here
to be truncated at aa 876 (unpublished data). For Nup153 pulldowns, each
fragment (5 g; zz-Nup153 1–339, zz-Nup153 N, or zz-Nup153 1–245) was
prebound to IgG Sepharose (Amersham Pharmacia Biotech) for 1 h at room
temperature in Nup153 PDB (150 mM NaCl, 1 mM MgOAc, 0.2% Tween-20,
50 mM Tris, pH 8.0, 1 mg/ml BSA, 1 g/ml aprotinin, and 1 g/ml leupeptin).
Xenopus extract (diluted 1:50 in PDB) was incubated with the beads at 4C
with tumbling. Pulldowns were washed (three times with PDB; one time with
PBS), and proteins were eluted with 100 mM glycine, pH 2.5. SDS-PAGE and
IB were performed as in Shah et al. (1998).
Protein purification, peptide sequence analysis, and
column chromatography
Proteins A–D were purified from Xenopus egg extract (diluted as above to
40 ml) by binding to Nup98 fragment II beads (6 ml), washing five times
with PBS, and eluting with 100 mM glycine, pH 2.5 (12 ml). A–D were
concentrated with a Millipore Centrifugal Filter (30-kd cutoff), and resolved
on a 7% SDS-PAGE gel. After transfer to polyvinylidene difluoride and
staining with amido black, A–D were excised individually and digested
with endoproteinase Asp-N. Peptides were sequenced by automated Ed-
man degradation on a PE Biosystems Procise 494 (Fischer et al., 1991). Pro-
teins A–D were purified from a large scale zz-Nup153 aa 1–339 pull-
down from Xenopus egg extract (2 ml); they proved to contain identical
peptides to proteins A–D (unpublished data). Peptides from proteins A, C,
and D are provided in Figs. 3 and 6. Protein B peptides and their mouse
and human matches include: Xenopus Peptide/mouse EST BE532781/hu-
man AK001754 peptides: DLAVNQISV/DKAVTQISV/558DRAVTQISV566;
Xenopus peptide 2/mouse EST AA536824/human AK001754 peptides:
VSLSSVSKSSRQAV/SQLKSLEKSSDQER/818SQLKSVDKSSNRER831; and Xe-
nopus peptide 3/mouse EST AA536824/human AK001754 peptides: DMN-
YAQKRS/EVEYLQKRS/836EMEYLQKRS844.
For gel filtration, Xenopus egg extract (200 l) was fractionated on a
Sephacryl S400-HR column (30  1 cm, 0.11 ml/min; Amersham Pharma-
cia Biotech) equilibrated in column buffer (150 mM NaCl, 50 mM Tris, pH
8.0, 1 mM MgCl2, 0.1% Tween-20). 54 fractions (400 l) were collected.
Even fractions (30 l) were fractionated on gels and immunoblotted (Fig. 7
a); odd fractions were subjected to Nup153 aa 1–339 pulldown and the
bound proteins were eluted, electrophoresed, and silver stained (Fig. 7 a;
BioRad Laboratories).
Transfection and IF
HeLa cells on coverslips were transfected with Nup98 or Nup153 sub-
clones in pCS2MT using TFX-20 transfection reagent (Promega) or
CaPO4. LMB was added as noted in Fig. 9. After 16 h, transfected
cells were fixed with 4% formaldehyde (5 min), permeabilized with
PBS/0.2% Triton X-100, and blocked 10 min with PBS/0.2% Triton
X-100/5% fetal calf serum. Transfected cells were stained with anti–
c-myc tag antibody (9E10; Calbiochem) (l hr, 1:500 dilution) and de-
tected with rhodamine-labeled goat anti–mouse IgG (l hr, 1:500; South-
ern Biotechnology Associates). Coverslips were mounted on Vectashield
(2 l; Vector Laboratories) and visualized with a Zeiss Axioskop 2 mi-
croscope. Digitonin and Triton X-100 permeabilization of HeLa cells
was performed as in Bastos et al. (1996).
Constructs, expression, and antibody production to Nup133
and Nup160
A cDNA clone encoding aa 777–1105 of putative human Nup133 (Fig. 4 d)
was prepared by reverse transcription of HeLa total RNA, amplification by
PCR, and cloning into pET28c. Recombinant protein was expressed in Esche-
richia coli BL21/DE3, purified on Ni-NTA resin (QIAGEN), and used to immu-
nize a rabbit. Antibodies were affinity purified against the same hNup133 pro-
tein fragment coupled to CnBr-Sepharose (Amersham Pharmacia Biotech).
The complete human Nup133 sequence was RT-PCR cloned using 5 and 3
oligos derived from GenBank/EMBL/DDBJ accession no. AK01676 and total
HeLa cell RNA. Antibody was raised to Xenopus Nup133 protein expressed
from Xenopus EST AW635680. Subclones of human Nup133 and Nup160
were prepared by using specific oligonucleotides and PCR or by restriction di-
gestion, followed by subcloning into pCS2MT. A globular protein structure for
Nup133 and Nup160 was predicted by PredictProtein through Columbia
University’s Bioinformatics Center (New York, NY).
A presumptive full-length Xenopus cDNA clone homologous to the
mouse 160-kd protein (GenBank/EMBL/DDBJ accession nos. AAD17922)
was prepared by reverse transcription of Xenopus total RNA using oligos de-
rived from the extreme 5 and 3 aa sequences of the GenBank Xenopus
clones (GenBank/EMBL/DDBJ accession no. BF048903 and BF049549) (Re-
sults), amplification by PCR, and cloning into pET28. After expression, anti-
bodies were raised and affinity purified against the same Xenopus protein.
Human Nup160 aa 317–697 was subcloned from GenBank/EMBL/DDBJ
accession no. KIAA0197 into pCS2MT for transfection and into pET28 for
production of antisera in rabbits. We note that the KIAA0197 clone lacks 88
aa in regions around the COOH terminus of mouse Nup160, but we found
human EST clones that contain these missing aa (Fig. 6 a).
Immunoprecipitation was done using Xenopus egg cytosol prepared as
in Shah et al. (1998). Affinity-purified IgG against xNup160, xNup133, and
xNup214, or preimmune antisera (1 g) was coupled to 10 l protein
A-Sepharose beads (Amersham Pharmacia Biotech). The antibody beads
were blocked (1 h; 4C; 20 mg/ml BSA). Blocked beads (10 l) were incu-
bated in egg extract (20 l) and 500 l PBS (2 h; 4C), washed five times in
PBS, and eluted with 0.1 M glycine, pH 2.5 (50 l). 5 l were loaded per
lane for Western blots; 15 l was loaded for silver-stained gels. SDS-PAGE
(7 or 8%) protein gels were used throughout. We note that not all verte-

Nup160 and Nup133 play a role in mRNA export | Vasu et al. 351
brate Nup133 may be contained in the Nup160 complex, as a greater
amount of Nup133 was observed in Fig. 7 c, lane 6 than in lane 5.
Poly[A] RNA accumulation, protein import, and protein
export assays
Poly[A] RNA accumulation was monitored after transfection of HeLa cells
with nucleoporin subclones, using fixation and permeabilization condi-
tions from Dr. Susan Wente (Washington University, St. Louis, MO), modi-
fied as below. Cells were grown on coverslips for 16 h, then transfected
for 16 h with control or nucleoporin gene fragments in pCS2MT using
QIAGEN Effectene. These were fixed (3% formaldehyde in PBS, 20, on
ice), permeabilized (0.5% Triton X-100 in PBS), incubated 5 min with 2 
SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7), and then prehybridized
with 50% formamide, 2  SSC, 1 mg/ml BSA, 1 mg/ml yeast tRNA (Be-
thesda Research Laboratories), 1 mM vanadyl ribonucleoside complexes
(GIBCO BRL), and 10% dextran sulfate (1 h, 37C). The cells were hybrid-
ized with Texas red–oligo[dT]50 (MWG Biotech) at 50 pg/l in the same
buffer (16 h, 37C), washed three times in 2  SSC (37C, 5 min each), then
refixed as above. Transfected expressed proteins were detected with FITC–
anti-myc antibody (1:500; Calbiochem). Effects on nuclear protein import
and export were tested by cotransfection (16 h) of a pXRGG plasmid ob-
tained from Dr. Bryce Paschal (University of Virginia, Charlottesville, VA)
with either control or nucleoporin-encoding plasmids. After transfection
cells were treated with 1 mM dexamethasone (Calbiochem; 60 min), the
cells were visualized by fluorescence microscopy for nuclear import of the
RGG fusion protein. Parallel cells were treated with dexamethasone for 60
min to allow RGG import, washed, then incubated with media lacking hor-
mone (2 hr, 37C) to allow export (Gustin and Sarnow, 2001).
Antibodies
The antibodies used were affinity-purified anti–human Nup133 aa 777–
1105 (1:100 for IB; 1:1,000 for IF); anti-Xenopus Nup133 (1:4,000, IB; 10
g/immunoprecipitation); anti–human Nup160 (1:100, IF); anti-Xenopus
Nup160 (1:1,000, IB and 5 g/immunoprecipitation); anti–rat Nup98 aa
43–470 (1:100, IF); anti–rat Nup155 aa 295–578 (1:1,000, IB); anti-
Nup153 and anti-Tpr (Shah et al., 1998); anti-Nup62 and anti-Nup214
(Macaulay et al., 1995); anti-Nup205 and anti-Nup93 (Miller et al., 2000);
anti–mouse Gle2 (1:100 IB), a gift of M. Powers (Emory University, Atlanta,
GA); mAb414 (1:500, IF; 1:2,000, IB; Babco); anti-importin  and , anti-
transportin and anti-Ran (1:2,000, IB; Transduction Laboratories); and
anti–lamin B and RCC1 from Dr. John Newport (University of California at
San Diego, San Diego, CA). Anti-Crm1 (1:5,000, IB) and RanQ69L were
gifts of D. Gorlich (Heidelberg University, Heidelberg, Germany). The
anti-sec13p antibody was the gift of Dr. Bill Balch and Jacques Weismann
(The Scripps Research Institute, La Jolla, CA) before publication. Rabbit an-
tibodies were detected using rhodamine or fluorescein goat anti–rabbit IgG
(1:200) for IF, and goat anti–rabbit HRP (1:2,000) for blots (Jackson Immu-
noResearch Laboratory).
Accession numbers
Nup160 homologues were non–full-length human KIAA0197 (GenBank/
EMBL/DDBJ accession no. BAA12110), a human EST (N53299), a full-
length 160-kd mouse protein (GenBank/EMBL/DDBJ accession no.
AAD17922), a 176-kd C. elegans protein (GenBank/EMBL/DDBJ accession
no. AAB37803.1), and a 160-kd Drosophila protein (GenBank/EMBL/DDBJ
accession no. AAF53075). A Xenopus EST with homology to mouse
Nup160 is encoded in GenBank/EMBL/DDBJ accession nos. BF048903
and BF049549. Nup133 homologues were: the full-length human gene
AK001676, related ESTs from Xenopus (AW635680), and mouse (Gen-
Bank/EMBL/DDBJ accession no. AA536824), Drosophila AAF56042, and
S. pombe GenBank clone CAB55845. (A partial human Nup133 clone,
GenBank/EMBL/DDBJ accession no. AK001754, was used in early analy-
sis.) Sequences were aligned and compared using Clustal W and SeqVu
with Kyte-Doolittle algorithms for homology. No homology was observed
between vertebrate Nup160 and Nup133, unlike that reported for the
yeast proteins Nup120p and anti-Nup133p (Aitchison et al., 1995).
Online supplemental material
To determine which Xenopus proteins bind to fragments of Nup98 or
Nup153, egg extract was added to beads conjugated to individual Nup98
or Nup153 fragments. Bound proteins were assessed by immunoblotting
(Table S1). Only Bands A–D, i.e., Nup160, Nup133, Nup96, and
Nup107, as well as sec 13, bound in significant amounts to the beads;
they did so only to Nup98 aa 470–876 and to Nup153 aa 1–339 beads.
Cotransfection (Fig. S1) was performed to assess the effect of Nup133 and
Nup160 fragments on the shuttling reporter protein RGG. No effect was
observed on the nuclear import or export of RGG.
The authors thank Kathy Wilson for excellent technical help, Amnon Harel
and Rene Chan for helpful discussions, and Jacques Weismann and Bill
Balch for the gift of anti-sec13p antibody; they regret if important work
was not cited.
The work was supported by an American Cancer Society Research grant
CB199 to D. Forbes, a National Institutes of Health grant RO1 GM33279
to D. Forbes, National Institutes of Health Shared Equipment grant (S10 RR
11404-01A1), and Foundation for Medical Research grants to W. Fischer.
Submitted: 1 August 2001
Revised: 13 September 2001
Accepted: 18 September 2001
Note added in proof. Recent studies by Belgareh published after accep-
tance of this work also identify the novel vertebrate nucleoporins Nup133
and Nup160, the latter of which they designate hNup120 (Belgareh, N., G.
Rabut, S.W. Bai, M. van Overbeek, J. Beaudouin, N. Daigle, O.V. Zat-
sepina, F. Pasteau, V. Labas, M. Fromont-Racine, J. Ellenberg, and V.
Doye. 2001. J. Cell Biol. 154:1147–1160). We have designated it verte-
brate Nup160, as it shows very slight homology to yeast Nup120 and is
larger, containing an additional 32 kd at its COOH terminus, as deter-
mined by the NCBI Blast 2 Sequences algorithm. We find Nup133 and
Nup160 inaccessible to antibody in digitonin-permeabilized cells, indicat-
ing that they are either localized to the nuclear pore basket or, if symmetri-
cally localized, their epitopes are masked on the cytoplasmic side of the
pore.
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