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Vol. 14, Issue 5, 1923-1940, May 2003
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, S-17177 Stockholm, Sweden
Submitted September 30, 2002;
Revised December 13, 2002;
Accepted January 23, 2003
Monitoring Editor: Jennifer Lippincott-Schwartz
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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;
Fahrenkrog et al.,
2001; Rout and Aitchison,
2001; Vasu and Forbes,
2001). Fibrils emanating from the outer ring appear as short tufts
of 
35–50 nm in length that have free distal ends. In contrast, the
eight rectilinear fibrils of 60–80 nm that are attached to the
inner ring have been reported to be laterally interconnected at their distal
ends by another ring-like structure, sometimes described as the
"terminal ring." Together, terminal ring and fibrils proximal to
the NPC proper are considered to represent a structural and functional entity,
called the nuclear "fishtrap" or "basket" (Ris,
1989,
1991;
Jarnik and Aebi, 1991;
Goldberg and Allen, 1992).
NPC proteins (nucleoporins) located at the nuclear side of the vertebrate
NPC comprise Nup153 (Sukegawa and Blobel,
1993), Nup98 (Powers et
al., 1995; Radu et
al., 1995), Nup 50 (Fan
et al., 1997; Guan
et al., 2000;
Smitherman et al.,
2000), the components of the Nup160-Nup133-Nup96-Nup107 subcomplex
(Radu et al., 1994;
Fontoura et al.,
1999; Belgareh et al.,
2001; Vasu et al.,
2001), the Nup93-Nup188-Nup205 subcomplex
(Grandi et al., 1997;
Miller et al., 2000),
and a 267-kDa protein termed Tpr (Byrd
et al., 1994; Cordes
et al., 1997;
Zimowska et al.,
1997). However, because different antibodies and immunolabeling
procedures have been used in different investigations, resulting in partly
inconsistent findings, the exact localization of most of these proteins
remains unknown. Although some have been attributed to the basket, it is
uncertain which of them are in fact components of the inner coaxial ring, the
associated fibrils, or the terminal ring.
Furthermore, in certain cell types additional fibers can be found emanating
from the terminal ring region and projecting further into the nuclear
interior. In amphibian oocytes this conspicuous fibrous material appears to
form hollow cylinders several hundred nanometers in length that occasionally
connect the NPCs with the cortex of amplified nucleoli (e.g.,
Ris and Malecki, 1993;
Cordes et al., 1993;
Arlucea et al., 1998).
In the stage VI oocytes of the South African clawed frog, Xenopus
laevis, at least two proteins, Nup153 and Tpr, reside not only in
proximity to the NPC but are found associated with these distal fibers as well
(Cordes et al., 1993,
1997). These filamentous
structures might be specific for oocytes, known to stockpile individual
proteins in large quantities and various forms. In fact, it has been
considered possible that they might represent a common stockpile of Tpr,
Nup153, and perhaps even some other nucleoporins, later to be used during
embryogenesis (Hase et al.,
2001).
The functions of Tpr are controversially discussed; propositions include
roles in intranuclear and nucleocytoplasmic transport and as a scaffolding
element of the nuclear interior and the NPC (e.g.,
Fontoura et al.,
2001; Frosst et al.,
2002, Shibata et al.,
2002; Zimowska and Paddy,
2002). Although different metazoan species have been shown to
contain only one Tpr ortholog, two probable homologues exist in both
Saccharomyces cerevisiae and Schizosaccharomyces pombe
(Kuznetsov et al.,
2002); in the budding yeast, these paralogs are called Mlp1 and
Mlp2 (Strambio-de-Castilia et
al., 1999; Kosova et
al., 2000). Although overproduction of Mlp1 has been shown to
cause nuclear accumulation of poly[A]+ RNA
(Kosova et al.,
2000), disruption of both Mlp genes is not lethal and
does not notably affect any type of nucleocytoplasmic transport
(Strambio-de-Castilia et al.,
1999; Kosova et al.,
2000). However, null mutants show increased sensitivity toward
UV-induced DNA damage (Kosova et
al., 2000), an impairment in DNA double-strand break repair,
and alterations in the spatial organization of telomeric heterochromatin
(Galy et al.,
2000).
Tpr and Mlps are proteins forming coiled-coil dominated homodimers of
extended rod-like shape (Kosova et
al., 2000; Hase et
al., 2001). Homodimerization occurs via the large N-terminal
domain, which also includes a short sequence segment found to be essential for
mediating binding of recombinant versions of Tpr to the NPC of transfected
cells (Bangs et al.,
1998; Cordes et al.,
1998, Hase et al.,
2001). However, binding partners responsible for positioning Tpr
at the nuclear periphery have remained elusive. One mammalian nucleoporin,
Nup98, has been described as an interaction partner of Tpr
(Fontoura et al.,
2001), but whether Nup98 serves as an NPC-attachment site for Tpr
or whether Tpr itself provides an anchor site for Nup98 remained
unanswered.
Amphibian NPCs reassembled from Xenopus egg extracts that had been
immuno-depleted of Nup153 have been reported to be devoid not only of Nup153
but also of all other presumptive basket proteins investigated in that study,
namely Nup93, Nup98, and Tpr (Walther
et al., 2001). Accordingly, the authors suggested that
Nup153 might be required for the formation or the structural integrity of a
large part of the nuclear basket. However, whether Nup153, which seemingly
lacks a yeast homolog, might serve as a direct binding partner for any of
these proteins was not determined.
In yeast again, several nucleoporins have been found associated with either
Mlp1 or Mlp2. Synthetic lethality screens had pointed at interactions between
Nup145p-C, the probable yeast homolog of Nup96, and both Mlp1 and Mlp2
(Galy et al., 2000).
On the other hand, in vivo cross-linking followed by affinity-purification of
Nic96p, the yeast homolog of Nup93, resulted in cofractionation of Mlp2
(Kosova et al.,
2000). Finally, in a yeast two-hybrid screen a cDNA clone encoding
Mlp2 had been isolated by using Nup60p as bait, a mammalian homolog of which
is not immediately apparent (Feuerbach
et al., 2002). However, whether these proteins interact
directly with Mlps, or whether binding is mediated by additional linker
proteins, remained uncertain.
In the present study, we have identified nucleoporin Nup153 as the direct binding partner that links Tpr to the NPC in mammals. In addition, we have addressed the question whether Tpr itself might function as a central architectural element of the NPC and as a scaffold for other nucleoporins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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,
1997), rabbit antibodies
against aa 1–14 and aa 604–615 of hTpr
(Kuznetsov et al.,
2002), mAb (mAb) 203-37 against hTpr
(Cordes et al., 1997;
Hase et al., 2001),
mAb PF190x7A8 against an epitope located between aa 439 and 611 of rat Nup153
(Cordes et al.,
1993), guinea-pig antibodies against aa 21–36 and
391–404 of hNup153 (Ferrando-May
et al., 2001) and against murine p62
(Cordes et al.,
1991), and mAb 9E10 for human c-Myc (ATCC CRL 1729) have been
described. MAb R27 against mammalian lamin A and mAb X223 against B-type
lamins (Höger et al.,
1991) were kindly provided by G. Krohne. Novel guinea-pig and
rabbit antibodies were raised against synthetic peptides corresponding to (i)
aa 1–21 and (ii) aa 126–147 of hNup 50 (accession no. NM_007172
[GenBank]
)
and (iii) aa 1743–1763 of hNup196 (AAL56659
[GenBank]
). Additional guinea-pig
antibodies were raised against (iv) aa 350–369, (v) 371–389, and
(vi) 586–606 of hNup93 (BAA07680
[GenBank]
); (vii) aa 206–219 and (viii)
596–618 of hNup98; (ix) aa 33–51 of hNup 107 (NM_020401
[GenBank]
); (x) aa
53–67 and (xi) 1784–1803 of the partial hNup 205 sequence
(D86978
[GenBank]
); and (xii) aa 993-1009 and (xiii) 1437–1458 (C at position 1447
exchanged for amino butyric acid) of hNup196. Peptide synthesis and coupling
to KLH (Cordes et al.,
1997) via an additional cysteine residue at the peptide's
N-terminus (ii, viii, ix, xii) or C-terminus (i, iii, iv, v, vi, vii, x, xi,
xiii) was done at Peptide Specialty Laboratories (Heidelberg, Germany).
Affinity purification of Igs on immobilized peptides followed standard
procedures (Cordes et al.,
1997). Additional guinea-pig antibodies were raised against
bacterially expressed His-tagged aa 8–203 of hNup93. Species-specific
secondary antibodies coupled to fluorochromes and horseradish peroxidase (HRP)
were from Jackson ImmunoResearch (West Grove, PA) and DAKO (Glostrup,
Denmark).
Expression Vectors and cDNA Cloning
pRC/CMV expression vectors myc.hTpr.1-775/SV40-NLS, myc.hTpr.1-775/SV40-NLS
(L458P, M489P) and myc.hTpr.1-775/SV40-NLS (L458D, M489D) have been described
(Cordes et al., 1998;
Hase et al., 2001);
in all constructs, silent point mutations (G/T) were introduced by in vitro
mutagenesis at positions corresponding to nt 94, 97, and 100 of the human cDNA
sequence (accession no. U69668
[GenBank]
). Yeast two hybrid expression vectors encoding
segments of Tpr (pAS/pACT-hTpr.1-398, hTpr.76/172–651,
hTpr.76/172-651[L458P/M489P; L458D/M489D], hTpr.72/305-775, hTpr.774-1370, and
hTpr.1178-1640) have been described (Hase
et al., 2001). Novel two-hybrid constructs based on
vectors pAS2–1 and pACT-2 (Clontech, Palo Alto, CA) are summarized in
Table 1; aa sequences are
deduced from ORFs downstream of the vector's NcoI site. DNA segments
encoding nucleoporins were amplified by PCR using Pfu polymerase
(Stratagene, La Jolla, CA) or subcloned from the following constructs kindly
provided by B. Burke (pCMV HA-hNup153;
Bastos et al., 1996),
J. Borrow (pCDNA3 Flag-hNup196), B. Fontoura (pAlter-MAX Myc-rNup197;
Fontoura et al.,
1999), F. Müller-Pillasch (pSport1 hNup107, NM_020401
[GenBank]
), T.
Nagase (hNup93, KIAA 0095; hNup205, KIAA 0225), and the German Human Genome
Project (pT7T3D-Pac hNup50, IMAGE clone 1352973). Novel derivatives of the
bacterial expression vector pGEX6P1 (Amersham Pharmacia Biotech, Uppsala,
Sweden) encoding hTpr aa 172–651 with and without aa substitutions L458D
and M489D, and aa 228–611 of hNup153 are described in
Table 2.
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Immunofluorescence Microscopy, Posttranscriptional Gene Silencing,
and Cell Synchronization
Culture conditions for HeLa cells (ATCC CCL-2) of low and high passage
numbers and immunofluorescence (IF) microscopy of cells were as described
(Kuznetsov et al.,
2002). Confocal microscopy and image processing was performed with
an LSM 510 and the release 2.3 software (Zeiss, Oberkochen, Germany). Tpr
siRNA duplexes and transfection conditions used for posttranscriptional
silencing of the tpr gene have been described
(Elbashir et al.,
2001; Kuznetsov et
al., 2002). In brief, HeLa cells were split 1 d before
transfection using Oligofectamine reagent (Invitrogen, Groningen,
Netherlands), and transfected at 15–30% cell density; the degree of
density depending on whether cells were cotransfected with expression vectors
later. SiRNA duplexes against Tpr, lamin A, and Nup153
(Harborth et al.,
2001) were applied at 15–25 pM/cm2 of culture
dish surface and at 15–50 pM/cm2 for controls with
single-stranded sense and anti-sense RNAs. Immunoblotting and IF microscopy
was performed with cells harvested 24, 48, 72, 96, and 120 h after
transfection with Tpr and lamin A siRNAs; Nup153 siRNA-treated cells were
analyzed 72–120 h posttransfection. Additional transfection of Tpr
siRNA-treated cells with mammalian expression vectors using Fu-Gene 6
Transfection Reagent (Roche, Mannheim, Germany) was performed 3 or 4 d after
initial transfection with siRNAs; cells were fixed 14–18 h later. For
cell cycle phase synchronization of Tpr siRNA-treated cells, 2.5 mM thymidine
was added to the culture medium 3 d after the initial transfection with
siRNAs. After an incubation of 24–26 h, the medium was replaced with
fresh thymidine-free medium. Cells were analyzed at different time points
0–28 h later. In some experiments, a second 24-h thymidine block was
performed 12 h after release from the first.
In Vivo Protein Cross-linking and Immunoprecipitation
HeLa cells grown to near confluency in 8-cm culture dishes were washed
briefly with hand-warm PBS; subsequent steps were with ice-cold solutions.
Cells were permeabilized with 0.5% Triton X-100 in PBS for 3 min, briefly
washed and then covered with PBS.
3,3'-dithio-bis[sulfosuccinimidyl proprionate] (DTSSP;
Pierce, Rockford, IL) dissolved in PBS was added at a final concentration of
0.2 mM. Cross-linking was performed on ice for 90 min, with cells remaining
adherent to the culture dish. After further washes with PBS, cells were
quenched with 0.5 M Tris, pH 7.5, for 30 min and then scraped off from the
culture dish into 10 ml RIPA-buffer (40 mM HEPES, pH 7.5, 150 mM NaCl, 1%
NP-40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with Complete Protease
Inhibitor Cocktail (Roche), and homogenized. Lysates were cleared by
centrifugation at 20,000 x g and 2°C for 15 min. The
resulting supernatants were first incubated with mAb 203-37 against hTpr at
2°C for 12 h, then supplemented with 50 µl bedvolume of Protein A
Sepharose (Sigma-Aldrich, Stockholm, Sweden), and again incubated at 2°C
for 4 h. Beads were sedimented by mild centrifugation, washed three times in
RIPA-buffer, resuspended in SDS protein sample buffer containing 1.4 M
-mercaptoethanol, and boiled for 5 min, resulting in cleavage of
cross-links. SDS-PAGE and immunoblotting were as described
(Kuznetsov et al.,
2002).
Bacterial Expression and Purification of Recombinant
Polypeptides
Synthesis of GST-tagged Tpr polypeptides in Escherichia coli
BL21-CodonPlus (DE3)-RIL (Stratagene) was induced in cultures of logarithmic
growth phase by addition of 0.1 mM IPTG. Cells were grown further at 30°C
for 3 h, harvested, and incubated in lysis buffer I (PBS, 0.1% Triton X-100,
0.1 mg/ml lysozyme, and Complete Protease Inhibitors) at 2°C for 30 min.
After brief sonication, lysates were cleared by centrifugation. In contrast,
synthesis of GST-tagged Nup153 polypeptides was induced in cultures in early
stationary growth phase. After further incubation at 30°C for 3 h, cells
were incubated in lysis buffer II (PBS, 0.1% Triton X-100, 10 mM
MgCl2, 0.1 mg/ml lysozyme, Complete Protease Inhibitors, and 10
U/ml bovine pancreas deoxyribonuclease I; Pharmacia) at 2°C for 3 h. The
resulting lysate was supplemented with 2 mM ATP, incubated at 37°C for 10
min, and centrifuged at 20000 x g and 4°C for 20 min.
Cleared lysates were incubated with glutathione Sepharose 4B (Pharmacia) at
2°C for 2–4 h. The slurry containing GST-Tpr fusion proteins was
washed with PBS containing 0.1% Triton X-100; bound proteins were eluted with
10 mM reduced glutathione in 100 mM Tris, pH 8.0, containing 120 mM NaCl, 5 mM
DTT, and 0.1% Triton X-100, and stored at 4°C or dialyzed against PBS for
further use. GST-Nup153 beads were washed with PBS first and then with
PreScission buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 8.0)
and then incubated with PreScission Protease (30 U/ml; Pharmacia) at 2°C
for 12–16 h. Nup153 polypeptides of which GST tags had been
proteolytically removed were eluted in PreScission buffer and stored at
4°C or dialyzed against PBS for further use.
Protein-Protein Interactions
For interaction studies between purified GST-Tpr and tag-free Nup153
polypeptides, the recombinant proteins in PBS were mixed in different molar
ratios, supplemented with 5 mM MgCl2, and incubated by rotation at
2°C for 12 h. After addition of glutathione Sepharose 4B (30 µl/ml),
the suspension was incubated further at 2°C for 1 h. The beads were washed
three times with PBS and boiled in SDS protein sample buffer. For pull-down
studies using mammalian cell extracts, purified GST or GST-Tpr polypeptides in
PBS were first reimmobilized on glutathione Sepharose 4B by incubation at
2°C for 4 h. In parallel, human PLC cells (ATCC CRL 8024) grown to
near-confluency were homogenized in PBS containing 500 mM NaCl, 0.5% Triton
X-100, and Complete Protease Inhibitors. The homogenate was cleared by 20,000
x g centrifugation at 4°C for 20 min. The supernatant was
diluted 1:5 in PBS without NaCl, resulting in a final concentration of 100 mM
NaCl, supplemented with 1 mM MgCl2, and incubated with the GST or
GST-Tpr beads at 2°C for 4 h. Beads were first washed with 10 vol PBS and
then with 10 vol PBS containing 1 M MgCl2. Bound proteins were
eluted stepwise with 10 and 20 mM reduced glutathione in 100 mM Tris-HCl, pH
8.0, containing 120 mM NaCl and 5 mM DTT. For RNase treatment of cell extracts
before affinity-chromatography on GST and GST-Tpr beads, extracts were
incubated with 2 mg/ml RNase A (Qiagen, Hilden, Germany) at 4°C for 60
min; efficiency of mRNA and tRNA digestion was controlled by gel
electrophoresis. PBS used for washings of beads was supplemented with 1 mg/ml
RNase A as well. Proteins from all wash and elution steps were precipitated
with 80% methanol at –20°C, boiled in sample buffer, and separated
by SDS-PAGE. Image analysis of protein gels stained with Coomassie Brilliant
Blue R250 and quantification of protein band intensities was by using the
Fujifilm Science Lab 99 Image Gauge software version 3.2 (Fuji Photo Film,
Tokyo, Japan).
Yeast Two-Hybrid Assays
S. cerevisiae strains Y190 (Mat a) and Y187 (Mat 
; both
from Clontech) were used for all assays. Cell cultures, transformation
procedures, filter lift assays, and control experiments with empty two-hybrid
vectors were as described earlier (Hase
et al., 2001). Liquid -galactosidase activity
assays using the fluorescent substrate
o-nitrophenyl--D-galactopyranoside (ONPG; Sigma)
were performed according to the Yeast Protocols Handbook PT3024–1 from
Clontech.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
;
Hase et al., 2001;
our unpublished results). Reassociation of Tpr to the reassembling NPC in
telophase is preceded by incorporation of several nucleoporins of the
cytoplasmic side (RanBP2/Nup358, Can/Nup214) and the central NPC channel (p62,
Pom121), but also by that of at least one presumptive protein of the nuclear
basket, namely Nup153 (Byrd et
al., 1994; Bodoor et
al., 1999; Haraguchi
et al., 2000). The sequel by which other basket
nucleoporins are recruited back to the NPC and whether their presence might be
essential for reincorporation of Tpr was not known. Further, it could not be
excluded that Tpr itself might act as a scaffold for other basket proteins and
that their recruitment would follow that of Tpr. It was similarly unknown
whether NPC disassembly during prophase occurs in a sequential manner too and
whether some nucleoporins are released after or before Tpr. Because such knowledge was expected to provide insight into Tpr's role in NPC architecture, we raised antibodies against the human orthologues of Nup50, Nup93, Nup96, Nup98, Nup107, and Nup205. Raised in different species, some of these antibodies, and others available for Nup153 and Tpr, allowed to study mitotic HeLa cells by double-labeling IF microscopy.
In prophase, complete release of Nup98 is well in advance of Tpr
dissociation (Figure 1),
indicative that this protein is not required for tethering Tpr to the NPC.
Similarly, release of Nup50 appears to begin earlier than that of Tpr, whereas
Nup96 parallels the dissociation of Tpr or follows it shortly afterwards.
Nup107 again is found to be released late in prophase and still be present at
remnants of the nuclear envelope (NE) already negative for Tpr. Different from
the other nucleoporins, disassociation of Nup153 appears to occur in a
stepwise manner: a first pool is rapidly detached from the NE early during
prophase (unpublished data), whereas the remaining pool of NPC-attached Nup153
disappears only later, concomittant to the release of Nup96 and Tpr
(Figure 1; see also
DISCUSSION). The disassembly of Nup93 during prophase could not be studied by
IF microscopy, despite having raised Nup93 antibodies against different parts
of the protein: When applying standard immunolabeling procedures, no Nup93 or
only traces thereof were detectable at the NPCs of interphase and prophase
cells. Instead, visualization of the NPC-bound form of Nup93 required
detergent-extraction of cells before fixation (see below; also
Grandi et al., 1997).
However, this procedure resulted in near-quantitative extraction of Nup93 and
several other nucleoporins from cells in prophase till anaphase. In
nonextracted cells, accessibility of NPC-associated Nup93 was noted only in
cells in telophase (see below). Similarly, the antibodies we have raised
against Nup205 so far did not yet allow the detection of the NPC-bound protein
by standard IF microscopy.
|
Tpr is recruited back to the nucleus only late in telophase when
chromosomes are mostly enclosed by a continuous NE
(Figure 2; Bodoor et al., 1999)
and nuclear import activity has been recovered
(Haraguchi et al.,
2000). In striking contrast, all the other presumptive basket
nucleoporins are already present at the periphery of the newly segregated
chromatids in anaphase, only preceded by even earlier association of Nup107 to
kinetochores in metaphase (unpublished data; but see
Belgareh et al., 2001)
and of Nup96 to other structures proximal to prometaphase and metaphase
chromatids (unpublished data). In early telophase, all these nucleoporins are
largely incorporated into the newly formed NE. At this time point, Tpr is
still found dispersed throughout the cytoplasm.
|
Taken together these observations indicated that Tpr might not act as an anchoring site for other basket proteins at the NPC; on the contrary, most of these nucleoporins had to be rated as potential candidates through which Tpr itself might be tethered to the nuclear periphery.
Posttranscriptional tpr Gene Silencing Leads to Cellular
Depletion of Protein Tpr, Which Does Not Prevent Other Basket Nucleoporins
from Binding to the NPC
Similar conclusions as outlined above can be drawn from the analysis of
HeLa cells in which tpr synthesis has been suppressed by RNA interference
(RNAi).
Use of small interfering RNA duplexes (siRNAs;
Elbashir et al.,
2001) is known to allow posttranscriptional silencing of the
tpr gene, resulting in cellular depletion of the Tpr protein
(Kuznetsov et al.,
2002) without reducing the cellular content of other presumptive
basket nucleoporins (Figure
3A). Some variability in the degree of the RNAi response can be
noted between different established cell lines and also between their
different laboratory substrains. For HeLa cells, the most striking RNAi
effects can be seen in the original strain of low passage number (ATCC CCL-2)
and in one of several laboratory HeLa substrains of high passage number
(Chen, 1988). In these,
near-complete loss of Tpr staining at the nuclear periphery of transfected
cells can be noted 3.5 d after the initial transfection with siRNAs. From
then on, Tpr-deficient cells still remain viable for at least a few more days.
This behavior closely resembles that of HeLa cells in which we have silenced
the nonessential lamin A gene by use of siRNAs
(Harborth et al.,
2001; our unpublished results). In our RNAi experiments, a small
percentage of cells was generally found to have remained untransfected in
siRNA-treated cultures, thus providing an internal subpool of Tpr-positive
reference cells.
|
Here we show that Tpr deficiency neither alters the NPC distribution within
the plane of the NE nor prevents NPC association of other known nucleoporins
in late mitosis and interphase (Figure
3B). Moreover, using antibodies specific for both lamin A and
B-type lamins (Höger et al.,
1991), lamina staining in both Tpr-deficient and control nuclei
was found to be indistinguishable (unpublished data).
In both synchronized and asynchronous cell populations, no differences in NPC labeling for Nup96 and Nup358 were observed between Tpr-deficient and control nuclei. Likewise, Nup93 staining at the NE of control and Tpr-deficient cells that had been detergent-extracted before fixation was indistinguishable as well. Similarly, in most Tpr-deficient nuclei Nup153 labeling also remained unchanged; only few nuclei showed a minor reduction of Nup153 staining intensity at the NE. Also no reduction was noted for Nup50 and Nup98; in fact, in some Tpr-deficient nuclei NPC labeling with these antibodies appeared slightly more intense than in control cells. A striking difference between Tpr-deficient and control nuclei was only noted for Nup107; in the absence of Tpr, Nup107 labeling at the nuclear periphery was generally far more intense (Figure 3B).
In summary, these results indicate that Tpr does not act as a central scaffold for these presumptive nuclear basket proteins.
In Vivo Cross-linking Confirms Proximity between Presumptive Basket
Proteins and Tpr
Several vertebrate nucleoporins form stable subcomplexes (e.g.,
Kita et al., 1993;
Fornerod et al.,
1997, Bastos et al.,
1997), some of which also remain associated during mitosis
(Grandi et al., 1997;
Matsuoka et al.,
1999; Miller et al.,
2000; Belgareh et al.,
2001; Vasu et al.,
2001). In contrast, the mitotic form of Tpr does not remain bound
to other nucleoporins, and extraction conditions required for releasing
NPC-bound Tpr from interphase nuclei result in the release of soluble Tpr
homodimers (our unpublished results; also
Shah et al., 1998;
Hase et al., 2001).
Therefore, in order to stabilize the interaction between Tpr and its NPC
binding partner(s), and thus allow coimmunoprecipitation of the proteins, HeLa
cells were treated with DTSSP, a homo-bifunctional and thiol-cleavable
cross-linker with a short spacer arm of 1.2 nm. After immunoprecipitation of
the cross-link products with Tpr antibodies, the cross-linkers were cleaved
and proteins analyzed by immunoblotting
(Figure 4).
|
Although p62, a component of the central pore channel did not become cross-linked to Tpr, we could coimmunoprecipitate all but one of the presumptive basket proteins, pointing at close spatial relationships to Tpr. Only the distribution of Nup50 remained uncertain, because of technical problems that were a consequence of cross-linker treatment, preventing unambiguous evaluation of the Nup50 results (unpublished data).
However, despite variations in DTSSP concentration and length of incubation, these and other cross-link approaches did not allow to distinguish unequivocally between nucleoporins that were bound directly to Tpr and those located only in close proximity.
Amino Acid Substitution Mutations That Abrogate NPC Binding of
Recombinant Tpr Directly Affect the Interaction between Tpr and Another
Nucleoporin
Recently we have shown that certain Tpr amino acid (aa) substitution
mutations can abolish NPC binding of recombinant Tpr polypeptides in
transfected cultured cells in which the natural tpr gene is expressed
in parallel (Hase et al.,
2001). To use these mutants in another approach aiming at
identifying Tpr's NPC binding partner, we first had to verify that these
mutations indeed abolish the interaction between Tpr and other nucleoporins.
Though considered unlikely (Hase et
al., 2001), we could not exclude definitively that the NPC
binding observed in such transfected cells merely reflected the result of
potential homo-oligomeric interactions between recombinant and wild-type Tpr
homodimers and that such interactions might be sensitive for the mutations we
had introduced.
To clarify this issue, we made use of our finding that cultured cells
depleted of wild-type Tpr by RNAi can be readily posttransfected with
expression vectors encoding a broad range of different proteins and that
synthesis of these proteins is not impaired (our unpublished results).
However, to allow synthesis of recombinant Tpr variants in the presence of
siRNAs that target the wild-type Tpr mRNA, concomitant degradation of the
recombinant mRNA had to be prevented. To this end, we introduced silent point
mutations into the nucleotide sequence encompassing the start codon
(Figure 5A), thus preventing
"RNAi initiation" by siRNA duplexes that efficiently target the
corresponding wild-type sequence (Elbashir
et al., 2001). Second, all original 5'-UTR
sequences of the Tpr mRNA upstream of these mutations were exchanged for
Tpr-unrelated sequences to prevent potential "transitive RNAi
effects" known to occur in some nonmammalian species (e.g.,
Nishikura, 2001;
Hutvágner and Zamore,
2002).
|
These sequence alterations allowed expressing recombinant versions of Tpr
despite on-going silencing of the wild-type tpr gene. Recombinant
polypeptides possessing an intact NPC binding domain (NBD;
Hase et al., 2001)
were found to bind to the NPC even though the wild-type protein was no longer
present (Figure 5B). In
contrast, Tpr polypeptides with aa substitution mutations L458P and M489P, or
with L458D and M489D, bound neither to the NPCs of control cells nor to those
of wild-type Tpr-depleted cells. Instead, these mutants accumulated within the
nuclear interior (Figure 5B)
from where they could be quantitatively extracted by brief
detergent-permeabilization of cells (unpublished data; but see also
Hase et al., 2001).
However, polypeptides with an intact NBD remained stably bound to the NPC
despite detergent-extraction of cells
(Figure 5C).
The NPC Binding Domain of Tpr Attracts Nup153
Having established that certain Tpr aa substitution mutations abolish NPC
binding, we searched for nucleoporins whose interaction with Tpr would depend
on sequence integrity of Tpr's NBD
To this end, recombinant GST and GST-Tpr fusion proteins with and without
aa substitution mutations L458D and M489D were immobilized on glutathione
Sepharose beads and used for affinity chromatography of human cell extracts.
Bound and unbound nucleoporins were detected by immunoblotting. Only Nup153
was found to bind specifically to the intact NBD of Tpr
(Figure 6). RNase treatment of
cell extracts before affinity chromatography did not affect this interaction
(unpublished data; see MATERIALS AND METHODS), indicative that RNA molecules
possibly bound to Nup153 (Dimaano et
al., 2001) are not likely to be involved in the binding of
Tpr to Nup153. Moreover, this interaction clearly depended on sequence
integrity of Tpr's NBD because only traces of Nup153 were found associated
with the Tpr mutant. GST alone did not bind Nup153.
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Yeast Two-hybrid Analysis Confirms a Specific Interaction between Tpr
and Nup153 That Depends on Sequence Integrity of Tpr's NPC Binding Domain
As an independent approach to study interactions between Tpr and other
nucleoporins, we made use of the yeast two-hybrid methodology
(Fields and Song, 1989). To
this end we constructed two-hybrid expression vectors encoding the full-length
sequences and segments of presumptive basket nucleoporins. These were then
studied for their ability to engage in two-hybrid interactions with the intact
or mutated forms of Tpr's NBD.
Only coexpression of Nup153 with Tpr polypeptides containing an intact NBD
caused lacZ reporter gene activation as determined by -galactosidase
filter lift assays (Figure 7A).
Remarkably, Tpr amino acid substitutions L458P and M489P that abolish Tpr's
ability to bind to the NPC but do not inhibit Tpr homodimerization
(Hase et al., 2001)
near-completely abolished binding to Nup153
(Figure 7B).
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The results of liquid -galactosidase assays
(Figure 7C) that allow to
assess the relative strength of two-hybrid interactions
(Estojak et al.,
1995) further emphasized the specific binding between Nup153 and
Tpr. Fused to the activation domain (AD) of GAL4, the Tpr NBD did not interact
with any of the nucleoporins except for Nup153
(Figure 7C, left diagram).
Similarly, in the reciprocal combination in which the GAL4 DNA binding domain
(BD) was fused to Tpr, high levels of -galactosidase activity were only
noted when Tpr was coexpressed with Nup153
(Figure 7C, right diagram).
Such -galactosidase activity was significantly reduced by Tpr amino acid
mutations L458D and M489D and was near-quantitatively suppressed by the L458P
and M489P mutations (Figure
7D).
Tpr's NBD was found to interact not only with Nup153 aa 228–611 but
also with a shorter Nup153 segment comprising aa 228–439
(Figure 7E). In contrast, no or
only traces of -galactosidase activity were detected in cells
coexpressing Tpr and Nup153 segments comprising aa 1–244 and aa
337–611.
Specific Binding between Purified Polypeptides Reveals Direct
Interaction between Nup153 and the NPC Binding Domain of Tpr
Yeast two-hybrid interactions and affinity chromatography of cell extracts
indicated that Nup153 might be the direct binding partner of Tpr at the NPC.
To rule out the possibility that another, yet undiscovered protein acts as a
linker between both proteins, we investigated whether purified Tpr and Nup153
polypeptides that had been synthesized in bacteria were capable of direct
interaction. To this end, a molar surplus of tag-free Nup153 polypeptide
228–611 was incubated with smaller amounts of GST-Tpr fusion protein
172–651 with and without aa substitutions L458D and M489D. As a further
control, Nup153 was incubated with GST alone. The L458P/M489P mutant, however,
could not be used because expression in bacteria went along with enhanced
degradation, preventing purification of sufficient amounts of the full-sized
polypeptide.
After incubation in PBS supplemented with 5 mM MgCl2, potential complexes were bound to glutathione Sepharose via GST, eluted, and analyzed by SDS-PAGE and Coomassie staining (Figure 8A) and immunoblotting (Figure 8B). Nup153 polypeptides and GST-Tpr were bound to glutathione in seemingly stoichiometric amounts. Image analysis of protein gels from two different experiments, and quantification of the amount of Coomassie dye bound to the Tpr and Nup153 bands revealed an average Tpr:Nup153 staining ratio of 2.1:1. In contrast, the relative amount of Nup153 bound to the Tpr aa substitution mutant was significantly reduced; the average ratio of Tpr:Nup153 staining being 9.1:1. Once again, GST alone did not bind Nup153. Interestingly, no or only dramatically reduced binding between Tpr and Nup153 was observed in buffers lacking MgCl2 (unpublished data).
|
Posttranscriptional Nup153 Gene Silencing Leads to
Mislocalization of Tpr and Nup50 to the Nuclear Interior but Does Not Prevent
Other Nucleoporins from Binding to the NPC
Recently, silencing of the Nup153 gene in HeLa cells has been
reported to lead to cell growth arrest several days after transfection with
Nup153 siRNAs (Harborth et al.,
2001). Here we now wanted to investigate to which extent Nup153
deficiency in a cultured mammalian cell might affect NPC composition and
distribution in general, and the localization of Tpr in particular.
Near-complete loss of Nup153 staining at the nuclear periphery was noted 3–4 d after the initial transfection of nonsynchronized HeLa cells with Nup153 siRNAs. NPC distribution within the plane of the NE of such Nup153-deficient cells was indistinguishable from that seen in control cells.
At 4 d posttransfection, bright staining for Nup96 and Nup107 at the
nuclear periphery appeared largely unaltered in most Nup153-deficient cells
(Figure 9). In fact, similar as
in Tpr-deficient cells, intensity of Nup107 staining at the NE was often noted
to be more intense than in neighboring control cells, perhaps reflecting
facilitated Nup107 accessibility for antibodies in the absence of Nup153.
Similarly, Nup93 and Nup98 remained stably bound to the Nup153-deficient NE,
even after detergent extraction of cells before fixation
(Figure 9). Staining intensity
for these nucleoporins was found to diminish only later, concomittant to
morphological and physiological alterations of the Nup153-deficient nuclei
(unpublished data). Such late decrease of NE staining intensity was
accompanied by the appearance of numerous variably-sized dot-like structures
in the cytoplasm, reminiscent of the staining for annulate lamellae (AL) seen
in certain cell lines (Cordes et
al., 1996).
|
In contrast, the nuclear localization of Nup50 and Tpr was strikingly
altered in Nup153-deficient cells already at 3 to 4 d posttransfection with
siRNAs. Nup50 staining at the nuclear periphery was largely diminished;
instead, the staining for Nup50 deep within the nuclear interior (see also
Figure 3 and
Guan et al., 2000)
often appeared more intense than in control cells. This intranuclear pool of
Nup50 could be extracted by brief detergent treatment of cells before
fixation, resulting in near-complete loss of Nup50 staining from
Nup153-depleted cells, but not affecting the Nup50 staining at the NE of
control cells (Figure 9).
Similarly, on-going depletion of Nup153 went along with a progressive decrease
of Tpr staining at the NE. Although minor residual amounts of Tpr could still
be observed at the nuclear periphery of some Nup153-deficient cells (see also
DISCUSSION), a striking accumulation of Tpr in several detergent-resistant
intranuclear aggregates was quite frequently observed 4 d after the initial
transfection with siRNAs (Figure
9). In addition, loss of Nup153, and release of Tpr from the NPC,
was also paralleled by a gradual decrease of the total cellular content of Tpr
(unpublished data). Furthermore, in contrast to the other nucleoporins, Tpr
and Nup50 in Nup153-depleted cells were never found to accumulate in
AL-reminiscent cytoplasmic structures.
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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267 kDa,
which locates near-exclusively at the NPC in most somatic cell types
(Kuznetsov et al.,
2002). In the present study, we have assessed Tpr's role as an
architectural element of the NPC in HeLa cells. In addition, we have
identified the NPC binding partner that links Tpr to the nuclear
periphery.
Tpr and NPC Architecture
Tpr is shown to be only peripherally attached to the NPC and not to act as
a scaffold onto which several other nucleoporins need to be assembled. In
Tpr-deficient cells, binding of these nucleoporins to the NPC was found to be
neither impaired in G1 phase nor in G2 when NPC numbers have doubled
(Maul, 1977). These findings
are in harmony with recent studies in which Tpr has been prevented from
reincorporation into the NPC by injection of Tpr antibodies into mitotic
cells, resulting in sequestration of Tpr into large, mostly cytoplasmic
aggregates at or near the nuclear periphery in G1 cells
(Frosst et al., 2002;
Shibata et al., 2002;
our unpublished results). NPC staining intensity for Nup153 and Nup98 in such
injected cells has been reported to remain largely similar to that seen in
noninjected controls (Frosst et
al., 2002). Here we show that Tpr is also dispensable for NPC
binding of Nup50, Nup93, Nup96, and Nup107. In fact, we noted that NPC
staining for some of these presumptive basket proteins was even more intense
in Tpr-deficient nuclei than in control nuclei. However, it remains to be
clarified whether this reflects an actual increase in protein copy numbers at
the NPC or whether it simply reflects better antibody accessibility to some
nucleoporins when Tpr is absent.
Furthermore, NPC distribution within the NE remains unaffected by lack of
Tpr, demonstrating that Tpr is not an essential anchoring element for the NPC
as proposed earlier. Apart from Tpr, only Nup98 has been shown to be
dispensable for correct NPC positioning in vertebrates
(Wu et al., 2001).
Here, we also observe that NPC positioning in Nup153-depleted cells is
indistinguishable from that seen in control cells. NPCs, however, that lack at
least four presumptive basket components at the same time, namely Nup93,
Nup98, Nup153, and Tpr, have been shown to be mobile and form clusters within
the plane of the NE (Walther et
al., 2001), suggesting that several nucleoporins in concert
might be involved in holding the NPCs in place.
Tpr and Nucleoporin Binding Partners at the NPC
Binding of Tpr to the NPC is mediated by only a short segment of its
aminoterminal domain. Aa substitution mutations introduced into this NBD
abolish Tpr's ability to bind to the NPC, rendering the protein soluble and
resulting in its accumulation in the nuclear interior
(Hase et al., 2001).
None of the Tpr regions outside the NBD are sufficient to stably tether the
protein to the nuclear periphery (Bangs
et al., 1998; Cordes
et al., 1998; Hase
et al., 2001).
In the present study, we have searched for those proteins that specifically interact with the intact version of Tpr's NBD but disdain the mutated form. This objective was tackled by different experimental approaches in parallel, each of them of different probative force. Nevertheless, all approaches consistently lead to the identification of Nup153 as a specific binding partner for the NBD of Tpr. This finding does not rule out the possibility that still other nucleoporins might bind to other regions of Tpr and that such additional interactions might help stabilize Tpr's association with the NPC. It is equally well imaginable that once Tpr has been linked to the NE, this localization might be stabilized further by interactions with also other, non-NPC molecules located at the nuclear periphery. On their own, however, none of these additional interactions can be expected to suffice to stably and durably secure Tpr at the NPC. Cellular depletion of Nup153 by RNAi clearly caused a mislocalization of Tpr to the nuclear interior but at the same time had no apparent effect on the localization of several other nucleoporins.
A previous study has reported a direct binding between in vitro translated
full-length Tpr and Nup98 in reticulocyte lysates; however, the region of Tpr
involved in such binding was not determined
(Fontoura et al.,
2001). Because Nup98 does not bind to Tpr's NBD, it is unlikely
that Nup98 is directly involved in the attachment of Tpr to the NPC. On the
other hand, the possibility remains that other regions of Tpr might contain
binding sites for Nup98. However, because staining for Nup98 at Tpr-deficient
NPCs remains unaltered or sometimes is even slightly more pronounced than in
control cells, the majority of binding sites for Nup98 at the NPC must be
provided by proteins other than Tpr.
Affinity purification of the yeast homolog of Nup93, Nic96p, from
glutaraldehyde-treated yeast nuclei has been shown to result in copurification
of one of the probable yeast homologues of Tpr, Mlp2p, together with several
other nucleoporins (Kosova et
al., 2000). The possibilities that Nic96p could serve as an
NPC-attachment site for Mlp2p or that Mlp2p itself might provide an anchor
site for Nic96p at the nuclear basket were proposed as equally likely
alternatives. However, whether Mlp2p and Nic96p interact directly with each
other or via other nucleoporins remained uncertain
(Kosova et al.,
2000). NPC binding of mammalian Nup93 in any case does not depend
on the presence of Tpr. And because Nup93 does not interact with Tpr's NBD, a
direct role for Nup93 in tethering Tpr to the mammalian NPC basket seems
unlikely too. However, the consistent finding that Mlp2 and Nic96p as well as
Tpr and Nup93 can be coenriched from nuclei treated with cross-linkers points
at similarly close, though not necessarily direct spatial relationships in
both lower and higher eukaryotes.
Again in yeast, potential NPC docking sites for Mlp proteins have also been
searched for by performing synthetic lethality screens. This resulted in the
identification of C-Nup145p, the probable homolog of mammalian Nup96. In cells
lacking C-Nup145p, both Mlp1p and Mlp2p were found to be mislocalized to the
nuclear interior (Galy et al.,
2000). Although the experimental approach did not allow to argue
for direct interactions between these proteins, it clearly pointed at an
essential role of C-Nup145p in positioning the Mlps at the NPC. Although our
present study did not reveal any direct interaction between the mammalian
Nup96 homolog and Tpr's NBD (see also
Fontoura et al.,
2001), the possibility remains that also Nup96 might act as a
chain link, perhaps via its interaction with Nup153
(Vasu et al., 2001),
and might therefore be essential for indirectly tethering Tpr to the NPC. Our
observation that Nup153-depletion of cells leads to mislocalization of Tpr,
but has no direct and immediate effect on the localization of Nup96 at the
nuclear periphery, supports this point of view.
Most recently, Mlp2p has also been identified in a yeast two-hybrid screen
as a binding partner of yeast Nup60p; the latter having been used as the bait.
Subsequent deletion of the Nup60 gene led to mislocalization of both Mlps to
the nuclear interior, similar to what had been seen in cells negative for
C-Nup145p. Additional results then indicated that Nup60p provides a link
between C-Nup145p and Mlp proteins
(Feuerbach et al.,
2002). Supposing Nup60p might be the direct binding partner that
tethers Mlp2p to the NPC, its function as an Mlp anchor site would correspond
to that of Nup153 in Tpr binding. At first sight, however, Nup153 and Nup60p
exhibit no apparent sequence homology. Most strikingly, the central Zn-finger
domain present in Nup153 (Sukegawa and
Blobel, 1993) does not have any equivalent in Nup60p. Because
other candidates that might represent possible counterparts of Nup153 and
Nup60p in the respective other species are not at hand either, their similar
function in binding Tpr and Mlps might be considered an example of convergent
evolution. Closer inspection of both protein sequences, however, reveals some
interesting similarities between the N-terminal segment of Nup60p covering aa
1–300 and the Nup153 segment comprising aa 1–600, the latter
including the binding sites for Tpr and other nucleoporins. Although overall
sequence homology between both segments is rather poor, secondary structures
are predicted to be very similar (Garnier
et al., 1996; at
http://npsa-pbil.ibcp.fr/NPSA).
Furthermore, both segments are similarly charged (pI of 9.6 for Nup60p, 9.28
for hNup153), are rich in hydroxy amino acids (23.7% for Nup60p, 24.3% for
hNup153), and include a few regions with similarly positioned proline
residues. Although such crude sequence similarities can also be found in pairs
of analogous proteins, the additional occurrence of short but seemingly
conserved sequence segments within these and other regions of the two proteins
might indicate a common ancestor. Moreover, both proteins share further common
properties. For example, Nup153 interacts with Nup50
(Guan et al., 2000;
Smitherman et al.,
2000), and Nup60p represents a binding partner for Nup2p, the
probable yeast homolog of Nup50. The binding site for Nup2p is located within
the N-terminal half of Nup60p (aa 1–388;
Denning et al.,
2001), resembling the Nup50 binding region of Nup153, located
between aa 337 and 611 (our unpublished results). Moreover, the Nup153 region
(aa 228–439) that binds Tpr also contains the binding sites for the
Nup160 complex including Nup96 (Vasu
et al., 2001); this region is also essential for
anchoring Nup153 to the NPC itself
(Enarson et al.,
1998). Because Nup50 and Tpr are dispensable for NPC binding of
Nup153 (Smitherman et al.,
2000; this study), it might be the Nup160 complex that provides
the anchor site for Nup153. This scenario would then again resemble the
situation in yeast where Nup60p provides a link between C-Nup145p and Mlps.
Finally, Nup153 contains binding sites for nuclear transport factors,
including importin beta and Ran-GTP (e.g.,
Saitoh et al., 1996;
Moroianu et al.,
1997; Shah et al.,
1998; Nakielny et
al., 1999), as does Nup60p for the corresponding yeast
homologues Kap95p and Gsp1p-GTP (Denning
et al., 2001). In view of these similarities, Nup153 and
Nup60p might be considered blown-up respectively truncated versions of each
other; the Zn-finger domain present in the one and missing in the other might
have been acquired or lost later during evolution, possibly concomittant with
the acquirement or loss of additional functions.
Wild-type Tpr in vivo and tag-free recombinant Tpr polypeptides in vitro
have recently been shown to readily form homodimers; such homodimers have been
proposed to represent the units that bind to the NPC
(Hase et al., 2001;
see also below). Here we have noted that the GST-tagged NBD of Tpr binds
Nup153 in seemingly stoichiometric amounts. Although we do not exclude that
the dimerization property of GST may contribute to the homodimerization of the
GST-Tpr fusion protein, Tpr's NBD on its own is already well capable of
homodimerizing (Hase et al.,
2001). Binding of recombinant Tpr to Nup153 in an approximate 2:1
ratio would therefore be consistent with the conception of Tpr homodimers
being bound to the NPC. In this context it might be interesting to note that
the molar content of Nup153 in at least some mammalian cell lines can exceed
that of Tpr 2.5-fold (our unpublished results). Assuming that one Nup153
monomer acts as the binding partner for one Tpr homodimer, only 20% of
the cellular content of Nup153 would be required for NPC-binding of Tpr in
such cells. In this case, the majority of Nup153 molecules would be free to
fulfill other functions, including those that do not necessarily require all
Nup153 polypeptides to be stable structural components of the NPC (e.g.,
Nakielny et al.,
1999; Dimaano et al.,
2001; Daigle et al.,
2001). In fact, we consider it possible that HeLa cells contain at
least two distinct pools of protein Nup153: a major more dynamic one (e.g.,
Nakielny et al.,
1999; Daigle et al.,
2001; our unpublished results) and a smaller pool that comprises
those Nup153 molecules that represent intrinsic components of NPC structure
(e.g., Walther et al.,
2001; our unpublished results) and act as Tpr binding partners.
Tpr
P), or permeabilized before fixation (P