From the
The Ras-related nuclear protein, Ran, has been implicated in
nuclear transport. By screening a HeLa cell
Bidirectional traffic of macromolecules (proteins,
ribonucleoproteins, and deoxyribonucleoproteins) into and out of the
nucleus proceeds through the nuclear pore complex (NPC).
The requirement for soluble transport factors for protein
import into nuclei has been demonstrated in an in vitro system
(4) . This system consisted of
digitonin-permeabilized cells that retained import-competent nuclei
with intact nuclear envelopes (NEs) but that had lost most of their
soluble cytosolic proteins. Protein import into these nuclei depended
on exogenously added cytosol. Subfractionation of the cytosol yielded
two fractions (A and B) with distinct activities (5). Fraction A
activity recognized a nuclear localization sequence (NLS)-containing
transport substrate and docked it at the NPC, whereas fraction B
activity mediated translocation into the nucleus. The active component
of fraction A has been purified and shown to consist of a
stoichiometric complex of two proteins referred to as karyopherin
In overlay assays, Ran-GTP (but not
Ran-GDP) has been shown to bind specifically to a number of cellular
proteins
(13, 14) . One of these proteins, termed RanBP1
(for Ran Binding Protein), has previously been
characterized
(13, 15) . Here we report the molecular
characterization and cellular localization of a second Ran binding
protein. Its molecular mass of 358 kDa was calculated from its
cDNA-derived primary structure. Immunoelectron microscopy localized the
protein to the NPC, specifically to its cytoplasmically exposed fibers.
We therefore termed this protein Nup358. In addition to four RanBP1
homologous domains, Nup358 contains eight zinc finger motifs of the
Cys
Ran binding to
isolated NEs was detected using recombinant Ran
(13) conjugated,
as described by the manufacturer (Amersham Inc.), directly to 10 nm
gold. A standard binding reaction consisted of: 20 µl of isolated
NEs (derived from 3
We have probed a
Striking ring-like surface decorations of NPCs can be seen
after isolated rat liver NEs were reacted with Ran-gold, deposited on a
grid, and slightly stained with uranyl acetate (Fig. 5,
bottom).
Nup358 is the largest nucleoporin that has so far been
molecularly characterized. It has previously been detected in urea
extracts of NEs as a potential nucleoporin that reacted with monoclonal
antibody 414 but not with wheat germ agglutinin
(2) . Most
importantly, in an overlay assay in which NE proteins separated by
SDS-PAGE were probed with NLS-containing substrate in the presence of
karyopherin, Nup358 (previously referred to as p270
(2) ) was
found to be one of several docking site nucleoporins for
karyopherin-mediated binding of an import substrate
(2) . The
repeat-containing domains of the nucleoporins have been proposed to
form an array of multiple docking sites that extend from the
cytoplasmic to the nucleoplasmic ends of the NPC along which the
transport substrate would be moved in a factor-mediated fashion by
guided diffusion
(3, 12) . That overlay assay result
provided direct biochemical evidence of a link between a subgroup of
nucleoporins (functioning as a stationary phase) and one of the
transport factors, karyopherin, functioning as a mobile phase. The data
here show that Nup358 can interact with a second transport factor,
namely Ran-GTP. A Ran-GTP/GDP cycle has been proposed to affect the
interaction between the stationary and the mobile
phase
(3, 12) .
Our immunoelectronmicroscopic
sublocalization data indicate that Nup358 is located at or near the
tips of the cytoplasmic fibers that extend from the NPC into the
cytoplasm. Thus, Nup358 could function as a port of entry into a
multiple docking site pathway across the NPC (and/or as a port of exit
in nuclear export).
The functions of the other domains of Nup358
remain to be elucidated. The leucine-rich region at the N-terminal
region could function in protein-protein interaction
(24) .
Likewise, the zinc finger domain could function in protein-protein,
protein-RNA, or protein-DNA interaction. Nup358 is the second
nucleoporin with zinc finger domains that has so far been identified.
The other, Nup153, contains four zinc finger domains (also of the
Cys
The striking decoration of isolated NEs by
Ran-coupled gold at only the cytoplasmic side of NPC is consistent with
the localization of Nup358. Quantitative analysis indicates that Ran
bound to the NPC at a distance of 46 nm from the midplane of the NE and
that the
The availability of a
nucleoporin with both karyopherin and Ran binding sites should now
allow a more detailed biochemical analysis of how a GTP/GDP cycle may
affect karyopherin-mediated docking of transport substrate.
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank
We thank Philip Bernstein for recombinant Ran and
Aurelian Radu for help and advice. We also want to thank Helen Shio for
preparation of the thin section EM specimens and members of the
Rockefeller University Biopolymer Facility, especially Joseph
Fernandez, for protein sequencing.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
expression library
with Ran-GTP and sequencing overlapping cDNA clones, we have obtained
the derived primary structure of a protein with a calculated molecular
mass of 358 kDa. Using antibodies raised against an expressed segment
of this protein, we obtained punctate nuclear surface staining by
immunofluorescence microscopy that is characteristic for nucleoporins.
Electron microscopy of immunogold-decorated rat liver nuclear envelopes
sublocalized the 358-kDa protein at (or near) the tip of the
cytoplasmic fibers of the nuclear pore complex (NPC). In agreement with
current convention, this protein was therefore termed Nup358 (for
nucleoporin of 358 kDa). Nup358 contains a leucine-rich region, four
potential Ran binding sites (i.e. Ran binding protein 1
homologous domains) flanked by nucleoporin-characteristic FXFG
or FG repeats, eight zinc finger motifs, and a C-terminal cyclophilin A
homologous domain. Consistent with the location of Nup358 at the
cytoplasmic fibers of the NPC, we found decoration with Ran-gold at
only the cytoplasmic side of the NPC. Thus, Nup358 is the first
nucleoporin shown to contain binding sites for two of three soluble
nuclear transport factors so far isolated, namely karyopherin and
Ran-GTP.
(
)
The NPC consists of a central opening of
40 nm in
diameter surrounded by a scaffold that is attached to the pore
membrane. Fibers emanate from the NPC for at least 50 nm into the
cytoplasm, and a nuclear basket extends into the nucleoplasm (for
review, see Ref. 1). A number of NPC proteins (collectively termed
nucleoporins) have now been molecularly characterized. Of particular
interest is a subgroup of nucleoporins that contain domains with short
peptide repeats (for review, see Ref. 1). These peptide
repeat-containing nucleoporins have been shown to function as docking
sites in transport factor-mediated protein import (2), and, for one of
these nucleoporins, Nup98, the docking site has indeed been mapped to
its peptide repeat-containing domain
(3) . These data provide a
direct biochemical link between nucleoporins and soluble transport
factors.
and
(2, 6, 7, 8) . Karyopherin
(synonymous with NLS receptor or importin) recognizes the NLS,
whereas karyopherin
functions as an adapter that links the
karyopherin
-NLS substrate complex to the repeat-containing
nucleoporins
(2, 3, 8) . The active components of
the B fraction are the small GTPase Ran
(9, 10) and a
10-kDa Ran interactive protein
(11) . A Ran-GTP/GDP cycle has
been proposed to mediate docking and release of the
karyopherin-substrate complex along an array of repeat-containing
nucleoporins extending from the tips of the cytoplasmic fibers to the
nucleoplasmic basket, resulting in guided diffusion across the
NPC
(3, 12) .
-Cys
type, several FXFG and FG
repeats, a leucine-rich region, and a C-terminal cyclophilin A-like
domain. Electron microscopy (EM) of isolated NEs that were incubated
with Ran coupled to gold showed decoration of only the cytoplasmic side
of the NPC, consistent with the localization of Nup358.
Molecular Cloning
Recombinant Ran was prepared
and loaded with [-
P]GTP as
described
(13) . A HeLa cell cDNA library was made in
EXlox
vector (Novagen) as described
(16) and was screened with
Ran-[
-
P]GTP
(13) . Of 1.2 million
plaques screened, 69 positive clones were identified. Among them, 32
were RanBP1
(13) and 28 were Nup358. Clones 7-1, 13-2, 7-4,
11-2, 14-1, 23-2, and 6-2, spanning most of Nup358, were characterized.
To obtain sequences further to the 5` end, the same library was
screened by DNA hybridization with a probe of about 380 base pairs from
the 5` end of the clone 7-4. Nine positive clones were identified,
among which one clone, 5A, contained sequences extending further to the
5` terminus. 5` End sequences were also obtained through two rounds of
PCR amplification of the library with two pairs of nested primers. The
upstream nested primers annealed to the vector directly upstream of the
cloning site, and downstream nested primers annealed to sequences of
clone 5A, corresponding to amino acids 93-100 and 84-92.
The amplified PCR fragment was cloned into TA cloning vector
(Invitrogen) and sequenced. Standard molecular biology techniques were
used for all analyses
(17) .
Production of a Recombinant Protein and Antibodies and
Immunological Analyses
A DNA fragment encoding amino acids
2550-2837 was obtained by PCR using clone 6-2 as a template and
was inserted into Escherichia coli expression vector pQE-30
(Qiagen). Recombinant protein (called C-288) was induced by
isopropyl-1-thio--D-galactopyranoside and was purified
using a Nickel-NTA agarose column according to the manufacturer
(Qiagen). Antibodies were produced by immunizing mice with the purified
recombinant protein C-288
(18) . The resulting anti-Nup358
antiserum was used at a dilution of 1:200 for immunoblotting
(18) and immunofluorescence microscopy
(19) .
Microsequencing of the Rat Homolog of Nup358
NEs
from rat livers were extracted with 2 M urea and 1 mM
EDTA as described previously
(20) . The proteins were separated
by SDS-PAGE (3.75-7.50% acrylamide) and transferred to
polyvinylidene difluoride membrane. The 350-kDa band was cut out,
digested with the endoproteinase Glu-C (Sigma), and peptides were
separated and sequenced as described
(21) .
Electron Microscopy
Rat liver NEs were prepared as
described previously
(22) , with slight
modifications.(
)
For immunogold localization of
Nup358, isolated NEs were fixed for 15 min in 2.5% formaldehyde in STM
(10% sucrose, 20 mM triethanolamine-HCl (pH 7.5), 0.1
mM MgCl
) and pelleted at 2,000
g for 5 min onto 35-mm plastic dishes. Subsequent antibody
incubations and washes were performed in the dishes with buffer A (1%
bovine serum albumin, 68 mM NaCl, 13 mM KCl, 15
mM KH
PO
, 40 mM
Na
HPO
, 0.5 mM phenylmethylsulfonyl
fluoride). Attached NEs were washed three times, samples were incubated
with anti-Nup358 polyclonal mouse serum diluted 1:400, and binding was
detected with goat anti-mouse IgG conjugated to 10 nm gold (Amersham
Inc.) and diluted 1:50. Processing for thin sectioning and EM were
performed as described previously
(23) .
10
nuclei) suspended in STM
plus 0.5 mM phenylmethylsulfonyl fluoride, and 1 µg/ml
each of leupeptin, pepstatin A, and aprotinin, 14 µl of buffer B
(20 mM Hepes (pH 7.3), 110 mM potassium acetate, 2
mM Mg(OAc)
, 1 mM EGTA), 2 µl of 20
mM GTP in buffer B, 2 µl of 20 mg/ml bovine serum albumin
in buffer B, and 2 µl of Ran-gold in buffer B. Reactions were
incubated for 15 min at room temperature and terminated by the addition
of 40 volumes of reaction buffer (50% STM, 50% buffer B) plus 2.5%
glutaraldehyde. After 30 min on ice, samples were spun onto plastic
dishes and processed as above for thin sectioning and EM. To detect the
binding of Ran to the surface of intact isolated NEs, fixed samples
were pelleted at 2,000
g, washed once with distilled
water, and, after resuspending in 10 µl of distilled water, bound
to glow-discharged Formvar carbon-coated copper grids. Samples were
viewed by EM after staining with 2% uranyl acetate.
expression library of HeLa cell cDNAs
with Ran-GTP. Sequencing of overlapping cDNA clones yielded the deduced
primary structure for a protein with a calculated molecular mass of 358
kDa (Fig. 1, A and B). Because this protein was
localized to the NPC (see below), it was named Nup358. Consistent with
the ability of Nup358 to bind Ran-GTP, it possesses four sites that
showed similarity to RanBP1 (Fig. 1C). Nup358 also
contains repeats of FG and FXFG that are among the signature
motifs of peptide repeat-containing nucleoporins. In addition, Nup358
has eight similar zinc finger motifs of the Cys
-Cys
type (Fig. 1D) as well as a C-terminal cyclophilin
A-like domain and an N-terminal leucine-rich region
(Fig. 1B).
Figure 1:
Sequence and analysis of
human Nup358. A, amino acid sequence of human Nup358 deduced
from the cDNA sequence. Amino acid residues are numbered on
the right. The region between the two arrows indicates a leucine-rich region (LRR), regions with
thin underlines indicate four RanBP1 homologous domains
(RBH), eight zinc finger domains (Zn), and a
cyclophilin A homologous domain (CycH), the region with a
thick underline indicates the segment of the protein used to raise
antibodies; residues with overhead dots represent FG or
FXFG repeats. B, a schematic representation of human
Nup358 domains and cloning strategy. Human Nup358 domains detailed in
A are shown here schematically with boxes. Vertical lines indicate locations of FG or FXFG repeats. Horizontal
bars below the boxes indicate cDNA clones obtained by Ran
screening, DNA hybridization screening, or PCR amplification.
C, an alignment of four RanBP1 homologous domains
(RBH) of Nup358. Identical residues are shown in shaded
areas. Residues of RanBP1 (13) identical with those found in at
least three of the Nup358 RBHs are shown at the top. D, an
alignment of eight zinc finger domains (Zn) of Nup358.
Identical residues are shown in shaded areas. Overhead asterisks indicate conserved cysteines.
A 288-amino-acid segment corresponding to
residues 2550-2837 of Nup358 (Fig. 1A) was
expressed in E. coli, purified (using its His tag), and
injected into mice for antibody production. Probing SDS-PAGE separated
proteins of rat liver NEs with the antibodies yielded decoration
primarily of one protein of 350 kDa (Fig. 2, lane
4). There were also weaker reactions with bands below and above
the 200-kDa marker. These bands are likely to be degradation products
of Nup358 since they are not consistently seen, although they could
also represent distinct cross-reactive proteins. A polypeptide of
similar mobility as the antibody-reactive 350-kDa band can be seen
among the Amido Black-stained polypeptides of the rat liver NEs
(Fig. 2, lane 1). As expected, a protein of similar
mobility was also one of the major Ran-GTP-reactive polypeptides
(Fig. 2, lane 3) and reacted with monoclonal antibody
414 (Fig. 2, lane 2), most likely because of the
presence of FXFG repeats. To determine whether this rat NE
protein is the homolog of human Nup358, we did partial amino acid
sequencing of the 350-kDa band. We found that the obtained peptide
sequences of this rat protein indeed matched the corresponding
cDNA-deduced amino acid sequence of human Nup358 (Fig. 3).
Figure 2:
Characterization of the rat homolog of
Nup358. Proteins of rat liver nuclear envelopes were separated on 10%
SDS-PAGE, transferred to nitrocellulose membrane, and stained with
Amido Black (lane 1) or probed with 414 monoclonal antibody
(lane 2), Ran-[-
P]GTP as described
(13) (lane 3), or anti-Nup358 antibody (lane 4).
Arrow indicates Nup358.
Figure 3:
Comparison of partial amino acid sequences
of the rat homolog of Nup358 (rhNup358) with human Nup358. The
numbers indicate the position of human Nup358 residues.
Vertical lines indicate identical amino acids; dots indicate similar amino acids.
Immunofluorescence microscopy of paraformaldehyde-fixed HeLa cells
showed the nucleoporin-characteristic punctate staining when focusing
on the nuclear surface (Fig. 4) and occasional punctate staining
in the cytoplasm.
Figure 4:
Localization of Nup358 by
immunofluorescence microscopy. HeLa cells were grown on coverslips,
fixed with paraformaldehyde, solubilized with Triton X-100, and probed
with anti-Nup358 antibodies (1:200 dilution) and fluorescein-labeled
goat anti-mouse IgG. Focusing on the nuclear surface yielded a punctate
staining pattern that is typical for nucleoporins. Bar equals
5 µm.
To sublocalize Nup358 within the NPC, immunogold
EM was done using isolated rat liver NEs. Nup358 appears to be
associated with the tips of the cytoplasmic fibers emanating from the
NPC (Fig. 5, top). Quantitative analysis of the
distribution of gold particles (n = 87) showed them to
peak at a mean distance of 59 nm from the midplane of the NE (data not
shown). These data suggest that Nup358 is located at or near the tip of
the cytoplasmically exposed NPC fibers.
Figure 5:
Nup358 and Ran binding sites localize to
the cytoplasmic face of the NPC. Top panel, isolated rat liver
NEs were fixed and incubated with anti-Nup358 antibodies followed by
gold-conjugated goat anti-mouse IgG. Samples were processed for thin
sectioning and observation by EM as described under ``Materials
and Methods.'' Middle panel, isolated rat liver NEs were
incubated with Ran-gold conjugate, fixed, and processed for thin
sectioning and observation by EM as described under ``Materials
and Methods.'' Shown in the top and middle panels are views along a single NE, as well as four individual NPCs
depicting typical patterns of gold labeling. In each case, the
cytoplasmic face of the NE (as determined by double labeling with an
anti-lamin B antibody (data not shown)) is oriented toward the top of
the figure, and gold was localized exclusively to the fibers associated
with the cytoplasmic face of the NPC. Bottom panel, isolated
rat liver NEs were incubated with Ran-gold conjugate, fixed, and
deposited onto EM grids. Samples were stained with 2% uranyl acetate
and observed by EM. Up to eight gold particles can be seen forming
rings around individual NPCs. Bars equal 0.1
µm.
As Nup358 binds Ran-GTP,
NPCs in nuclear envelopes could be expected to bind to it as well.
Using Ran directly coupled to 10 nm gold, we found gold decoration of
NPCs, exclusively on their cytoplasmic side (Fig. 5,
middle), consistent with the localization of Nup358.
Quantitative analysis (n = 65) showed Ran-gold to peak
at a mean distance of 46 nm from the midplane of the NE (data not
shown).
-Cys
type), is localized at the
nucleoplasmic side of the NPC
(25, 26, 27) , and
has been shown to bind DNA in a zinc-dependent fashion
(25) . The
cyclophilin A homologous domain at the C terminus could function as a
peptidyl-prolyl cis-trans isomerase or a
chaperone
(28) .
-Nup358 antibodies bound at a distance of 59 nm. Given
the size of Nup358, the 13 nm difference between binding sites is also
consistent with the binding of antibody and Ran to the same protein.
Nevertheless, these data do not prove that the Ran-gold is in fact
binding to Nup358, as there could be other Ran-binding nucleoporins.
Furthermore, whether the bound Ran is in a GTP-bound form will require
further experiments with Ran mutants and GTP analogs. A definitive
identification of the observed Ran binding sites (and other potential
Ran binding sites not detected under these conditions) at the NPC is
the focus of current investigation.
/EMBL Data Bank with accession number(s)
L41840.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.