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INTRODUCTION |
The bidirectional exchange of macromolecules between the nucleus
and cytoplasm takes place through nuclear pore complexes (NPCs)1 that perforate the
nuclear envelope (reviewed by Refs. 1-4). The active transport of
large macromolecules through NPCs requires escort or carrier proteins
that shuttle rapidly between the two cellular compartments. Members of
the importin-
/karyopherin- family function as carriers for many
nuclear trafficking processes including nuclear protein import and
export and tRNA export (reviewed by Refs. 2 and 3). However, not all
nuclear trafficking is mediated by members of this family. For example,
the NXF family is important for mRNA export (reviewed by Refs. 5
and 6), and NTF2 mediates nuclear import of RanGDP (7, 8).
Transport by carrier proteins is thought to be a sequential stepwise
process. For example, importin- binds cargo molecules in the
cytoplasm, either directly or via an adapter such as importin-
, the
complex moves through the NPC, and then cargo is released in the
nucleus on importin- binding RanGTP. The
importin-·RanGTP complex is recycled to the cytoplasm where
it is dissociated by hydrolysis of the GTP on Ran, freeing importin-
for another round of import (reviewed by Refs. 9 and 10). Although the
actual mechanism of translocation through NPCs remains controversial, a
shared tenet of each model involves binding of carrier molecules to a
subset of NPC proteins (termed nucleoporins or "Nups") that contain
domains with repeating Phe-Gly (FG) sequence motifs (Refs. 11-14,
reviewed in Refs. 15 and 16). These repeats have hydrophobic cores
based on FG, GLFG, or FxFG motifs separated by hydrophilic linkers of
variable sequence and length. In vitro, FG-Nups
interact directly with a broad range of transport factors and there is compelling in vivo evidence that interaction between
carriers and FG-Nups is important for at least one stage of
translocation through NPCs (Ref. 11, and reviewed by Refs. 16 and 17). FG-Nups may function to concentrate carrier-cargo complexes at the NPC
entrance (18) or participate in a sequence of docking and undocking
interactions as the carrier-cargo complexes transit through NPCs (Refs.
11, 13, and 14, reviewed by Ref. 16). Alternatively, FG-Nups may form a
meshwork within the central channel that is permeable only to molecules
that interact with FG repeats (19).
Rout and Wente (15) suggested that FG-Nups may divide into different
subfamilies, based on differences in the primary core sequences between
different Nups (FG, GLFG, and FxFG) and on differences in linker
sequences. In addition, the distribution of different FG-Nup
subfamilies within NPCs is not the same (18, 20). The most complete
analysis has been possible in Saccharomyces cerevisiae where
there are 13 different FG-Nups. The GLFG-Nups are found on both sides
of the NPC, some FG-Nups are exclusively on the cytoplasmic side, and
some FxFG-Nups are exclusively on the nuclear side (18). Individual
FG-Nups show distinct binding preferences for different carriers (11),
suggesting that potentially some may have specialized functions. This
possibility is also supported by a range of genetic, biochemical, and
in vivo evidence implicating different FG-Nups in import or
export events (Refs. 1 and 21-24, reviewed in Refs. 11, 17, 25, and
26).
X-ray crystallographic studies (Refs. 27-29, reviewed in Ref. 30) have
shown that importin- is a helicoidal molecule constructed from 19 HEAT repeats, each formed by a pair of -helices (the A- and
B-helices). A combination of biochemical and structural studies have
indicated that, although FxFG-Nups may bind at several sites on
importin-, the primary site is located in the N-terminal half of the
molecule between the A-helices of HEAT repeats 5 and 6 (21, 31, 32).
However, fragments containing the C-terminal half of importin- do
show some binding to nuclear envelopes (31, 32). The crystal structure
of residues 1-442 of importin- complexed with FxFG repeats (21)
shows two putative FxFG sites, but the site between the A-helices of
repeats 5 and 6 has higher occupancy. Moreover, mutations at this site
(for example, I178D) weaken the binding of FxFG constructs
considerably, consistent with its representing the primary FxFG binding
site (21). The binding of importin- to FG-Nups as well as to cargo
and/or importin- is disrupted by RanGTP (11, 14, 33-35) and
comparison of different co-crystal structures suggests that RanGTP
binding may induce a conformational change in importin- that
occludes the primary FxFG binding site (21). It has not been
established unequivocally if such a mechanism contributes to vectorial
movement through NPCs or release of importin- from Nups.
Crystal structures of FxFG repeat cores bound to importin- and NTF2
(21, 36) and an FG core bound to the mRNA export factor Tap/NXF1
(37) indicate that, in each complex, the interaction is dominated by
repeat core Phe side chains being buried in a hydrophobic pocket.
Mutations in this pocket reduced the binding of these transport factors
to FG-Nups (21, 36, 37). In addition, the affinity of FxFG repeat
peptides for the UBA domain of Tap was dramatically reduced when the
second Phe, F2, was mutated to Ala (38). Overall, these data indicate
that the hydrophobic cores of FG repeats are crucial to their binding
to transport factors, although they do not rule out there also being
contributions from the linkers.
To determine the precise function of each interaction in a cycle of
productive nuclear transport, it is crucial to define how the FG
repeats are recognized by nuclear transport factors. To address this
question, we have investigated the interaction of GLFG- and FxFG-Nups
with importin-, and report here the 2.8-Å resolution crystal
structure of a complex between residues 1-442 of importin- and a
GLFG repeat containing peptide. The GLFG core binds to the same
hydrophobic site on importin- previously identified as the principal
FxFG binding site (21). I178D-importin-, which shows reduced
affinity for FxFG repeats, also shows reduced binding to GLFG repeats
and nuclear protein import is inhibited by soluble GLFG repeats.
Moreover, we show that the binding of GLFG repeats to importin- is
inhibited by soluble FxFG repeats confirming that, in solution, the
GLFG and FxFG repeat cores bind primarily to overlapping sites on
importin-. Similar competition between GLFG- and FxFG-Nups was
observed with Kap95.
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MATERIALS AND METHODS |
Protein Preparation--
The cloning, expression, and
purification of human importin- and its I178D mutant, Ib442, canine
Ran, and the S. cerevisiae Nsp1 FF18 (containing eighteen
FxFG repeats) have been described previously (21, 39, 40). GST-tagged
constructs of Nup100 residues 2-610 (41), Nup116 residues 161-730
(22), and the FxFG repeat containing domain of Nup1 residues 423-816
were purified on glutathione-Sepharose 4B as described by the suppliers
(Amersham Biosciences). GLFG peptide (DSGGLFGSK) was
dissolved in Milli Q water buffered to pH 7.4 with Tris (~2
mM).
Crystallization and Data Collection--
Crystals of the
Ib442-GLFG complex were obtained by vapor diffusion using hanging drops
of 6 µl composed of 3 µl of well buffer and 3 µl of Ib442/GLFG
mixture (15 mg/ml Ib442, 25 mM GLFG peptide). Well buffer
was composed of 100 mM ammonium acetate, pH 6.0, and 1.2 M ammonium sulfate. Crystals were slowly dehydrated by
transfer to a hanging drop containing 2 M ammonium sulfate,
100 mM ammonium acetate, pH 6.0, 25 mM GLFG
peptide above a well containing 3.2 M ammonium sulfate, 100 mM ammonium acetate, pH 6.0. After 2 days the crystals were
transferred to 3.2 M ammonium sulfate, 5% (v/v) glycerol,
100 mM ammonium acetate, pH 6.0, before plunge-freezing into liquid nitrogen. Ib442-GLFG crystals vitrified in this way diffracted to 2.8-Å resolution using 1.488-Å wavelength radiation and
a Mar345 detector on beamline 14.1 at the SRS (Daresbury, UK). Data
were integrated using MOSFLM (42) and reduced using SCALA (42).
Crystals of Ib442-FF5 with
P212121 symmetry were
grown under similar conditions to the
P21212 crystals (21) by vapor
diffusion using 7-µl hanging drops composed of 3 µl of drop buffer,
3 µl of Ib442 protein (of 4.5 mg/ml stock solution), and 1 µl of
FF5 (11 mg/ml stock solution). Reservoir buffer was 1.2-1.28
M ammonium sulfate, 100 mM ammonium acetate, pH
5.9, 30 mM DTT and drop buffer was 1.16-1.2 M
ammonium sulfate, 200 mM ammonium acetate, 25 mM DTT. Diffraction-quality crystals (dimensions 200 × 50 × 50 µm), which diffracted past 3 Å, were grown by
microseeding. Crystals were transferred to reservoir buffer containing
24% glycerol for less than 1 min and flash-frozen at 100 K. A native
data set was collected at 100 K using 0.934-Å wavelength radiation on
beamline ID14-EH1 at ESRF (Grenoble, France) using a MarCCD detector.
Structure Solution--
Molecular replacement and refinement
used the CNS package (43). The structures of the crystals of Ib442
complexed to either GLFG or FxFG peptides were determined by molecular
replacement using residues 1-442 of importin- bound to the IBB
domain (28) as a model. Although the coordinates of Ib442 when bound to
FF5, in the original Ib442-FF5 crystal form, was a more similar
starting model than the IBB-bound structure, we thought it prudent to
avoid the possibility of introducing bias arising from a Nup-bound
Ib442 as a model. The Ib442 chains were rebuilt locally and refined using simulated annealing before examining F0 
Fc difference maps to locate putative bound Nup
repeats. In both cases, characteristic tubes of difference density were
observed from which there were bulky protrusions that could be
identified as Phe and Leu side chains. Structural figures were made
using MOLSCRIPT (44), BOBSCRIPT (45), and Raster3d (46).
Solution Binding Assays--
Saturating amounts of GST-Nup were
incubated with 200 µl of packed glutathione-Sepharose beads (Amersham
Biosciences) for 1 h in binding buffer (phosphate-buffered saline
(PBS) supplemented with 0.05% Tween 20, 2 mM
MgCl2, and 1 mM DTT). Unbound material was
removed by washing four times with binding buffer. For each binding
reaction, 40 µl of bead slurry (50% suspension) in 1 ml of binding
buffer was incubated with 10 µg of wild type or I178D importin-
for 40 min at 4 °C. For competition assays, 100 µg of FF18 or 60 µg of importin- were included in the incubation. After incubation,
beads were washed twice with 1 ml of binding buffer, resuspended in
sample buffer (47), and analyzed by SDS-PAGE.
Microtiter Plate Binding Assay--
Solid phase binding assays
were carried out on Immulon II microtiter plates (Dynex) essentially as
described by Ben-Efraim and Gerace (34). Plates were coated with 25 nM Nsp1 FF18, GST-Nup100 (residues 2-610), GST-Nup116
(residues 161-730), or GST per well for 16 h at 4 °C in
coating buffer (PBS supplemented with 2 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride). After adsorption the
plates were washed three times by immersion in PBS and incubated overnight at 4 °C in binding buffer (coating buffer supplemented with 3% BSA and 0.1% Tween 20). Binding reactions were carried out
for 2 h at 4 °C with 100 µl/well of the indicated amounts of
S-tagged importin- (wild-type or I178D mutant) or Kap95 proteins in
binding buffer. After binding, plates were washed three times by
immersion in binding buffer without BSA and proteins were cross-linked for 15 min at room temperature by incubation in 1 mg/ml
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Pierce) in the
same buffer. The wells were then washed for 20 min in PBS-T (PBS
supplemented with 0.2% Tween 20), 10 min with PBS-T containing 100 mM ethanolamine, and finally incubated for 10 min in PBS-T
containing 3% BSA. The bound S-tagged proteins were detected by
incubation with S-protein-horseradish peroxidase conjugate (Novagen) in
coating buffer containing 1% BSA and 0.1% Tween 20. After 1 h at
4 °C, the plates were washed three times by immersion in PBS.
Horseradish peroxidase substrate (100 µg/ml 3,3',5,5'-tetramethylbenzidine (Vector Laboratories), 0.006%
H2O2 in 100 mM sodium acetate, pH
6.0) was added for 10 min at room temperature and the reaction was
stopped by the addition of an equal volume of 0.5 M
H2SO4. The signal was determined at 450 nm with
a Molecular Devices plate reader. For competition experiments, free Nup
proteins, RanGTP, or RanGDP were added to the wells along with a fixed
concentration of S-tagged importin- or Kap95. Ran was loaded with
either GTP or GDP as previously described (34, 48).
Yeast Manipulations--
KAP95, importin-, and
I178D-importin- were fused in-frame behind LexA in pCH432 (gift of
C. Hardy, Vanderbilt University Medical Center, Nashville, TN) to
generate pSW1381, pSW1371, and pSW1449, respectively. The plasmids were
transformed into SWY561 (Mata ade2-1 ura3-1
his3-11,15 trp1-1 leu2-3,112 can1-100 kap95::HIS3
pSW271 (KAP95 URA3 CEN), Ref. 12) by the lithium acetate method
(49). To test for complementation, yeast transformants were struck to
synthetic complete media supplemented with 2% glucose and 1 mg/ml 5-fluoroorotic acid. Growth was assayed at 23 °C for 7 days.
Nuclear Protein Import Assay--
Nuclear protein import was
assayed using digitonin-permeabilized HeLa S3 cells essentially as
described (21, 32, 36) using fluorescein-labeled BSA coupled to an NLS
peptide (CGYGPKKKRKVED). Purified bacterially expressed proteins (1.2 µM importin-, 2.25 µM Ran, and 2.7 µM NTF2) were combined in a final volume of 8 µl of
transport buffer (20 mM HEPES-KOH, pH 7.5, 120 mM potassium acetate, 5 mM Mg acetate, 250 mM sucrose, 0.5 mM EGTA, 5 mg/ml BSA, 0.5 mM GTP, 0.5 mM ATP, 10 mM creatine
phosphate, and 0.05 mg/ml creatine kinase). Importin- was used at a
concentration of 250 nM, and cells were examined using
identical microscope settings. In competition studies, the Nup proteins
Nsp1 FF18 or GST-Nup100-(2-610) were included at a final
concentration of 10 or 25 µM. After 30 min, cells were
fixed and examined using a MRC1024 confocal microscope. All fluorescent
images were captured using 488 nm excitation and with identical laser
intensity, iris, and gain settings. Six-frame Kalman averaging was used
for all images. Low-power micrographs were recorded for a number of
fields in each sample and the number of cells in which fluorescent
substrate had been imported into the nucleus was quantitated.
Approximately 100 cells were counted for each sample.
Coordinates--
Coordinates were deposited in the Protein Data
Bank with accession codes 1o6o and 1o6p.
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RESULTS |
Crystal Structure of Ib442 Bound to a GLFG Peptide--
We
investigated the structural basis for the interaction between
importin- and GLFG repeats using crystals of importin- residues
1-442 (Ib442) complexed with a peptide of the sequence DSGGLFGSK. By analogy with the binding to FxFG repeats (50),
we anticipated that the binding of GLFG repeats to importin- would
probably be mediated largely by their hydrophobic cores, and so we
employed ammonium sulfate as the precipitant. In this way we obtained
crystalline plates, measuring ~0.1 × 0.05 × 0.02 mm,
which had P21 symmetry and diffracted past
2.8-Å resolution (Table I) using
synchrotron radiation. We also obtained crystals of putative Ib442-GLFG
complexes using several repeats derived from Nup116, but none
diffracted to high resolution.
Molecular replacement identified only two Ib442 chains in the
asymmetric unit. Although this gave an unusually high (77.3%) solvent
content (51), rigid body refinement gave an R-factor of
37.1% that was reduced to 29.3% (Rfree 31.7%)
after simulated annealing and local model rebuilding. These statistics
indicated that it was extremely unlikely that there was an additional
Ib442 chain present and there was no Fo Fc electron density indicative of a third Ib442
chain. The crystal lattice contained large square solvent channels
formed by the intersection of approximately perpendicular Ib442 chains.
This configuration exposed a large proportion of the surface of each
chain to solvent and, by extension, to the GLFG peptide. After
refinement, both 2Fo Fc and
Fo Fc 
A weighted electron
density maps showed tubes of additional density associated with the
Ib442 chains in two different positions that could be modeled as GLFG
peptide cores (Fig. 1A).
Including these peptides reduced the R-factor to 23.6%
(Rfree 27.3%) after refinement and rebuilding.
Overall, the final structure had good stereochemistry (Table I).
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Fig. 1.
Binding of GLFG and FxFG repeats to
Ib442. Annealed omit Fo Fc maps contoured at 2.5 at the primary GLFG
binding site (A) (site 1, see C) and the primary
FxFG binding site (B). C, the GLFG peptide
showed difference density located at two sites on Ib442. Site 1 (black) was located between the A-helices of HEAT repeats 5 and 6 and was also the primary site at which FxFG cores bound, whereas
site 2 (red) was located at a contact between two Ib442
chains in the crystal and was thought to be a crystallization
artifact.
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One putative GLFG repeat core was located between the A-helices of HEAT
repeats 5 and 6 (site 1, Figs. 1C and 2A) at the
same location as the primary FxFG binding site observed in Ib442-FF5 (21). A second GLFG core was bound at a crystal contact between two
Ib442 chains (site 2, Fig. 1C, Fig.
2, C and F). The
GLFG peptide at the crystal packing interface contacted residues in HEAT repeats 2 and 3 from one chain and repeats 6 and 7 from the other.
However, the site 2 binding pocket in which the hydrophobic core was
buried was formed partially from each Ib442 chain and neither
individually buried the hydrophobic Phe and Leu side chains effectively
(Fig. 2, C and F). A similar conformation was
adopted by the GLFG peptide at both importin- binding sites, and in
each case the interaction was primarily hydrophobic. The buried surface area (Table II) and extensive molecular
contacts between the GLFG core and the two Ib442 molecules at the
crystal packing interface (site 2) were comparable with those at site
1. However, because the GLFG peptide at site 2 interacts with two
importin- chains, it is unlikely to contribute to nucleoporin
binding in solution to the same extent as the primary site. Indeed, the
surface buried in the interface between the GLFG core at site 2 and
either of the two individual importin- chains was substantially
smaller (Table II). Moreover, the Leu of the core interacted with one importin chain while the Phe of the core interacted with the other. Thus, the interaction observed at site 2 would only be of physiological significance in the context of an importin- dimer. Importin- itself has not been reported to dimerize and so we concluded that the
interaction observed at site 2 was most probably a crystallization artifact. In contrast, site 1 is not located near a crystal contact, and, as demonstrated by several lines of evidence presented below, constitutes a bona fide binding site for GLFG repeats on importin-.
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Fig. 2.
Details of the interaction between GLFG and
FxFG cores and Ib442. Stereo views of GLFG (A,
black) and FxFG (B, red) cores show that each
binds in the hydrophobic pocket (yellow) formed between HEAT
repeats 5 (light green) and 6 (light blue) by the
side chains of Leu174, Thr175,
Ile178, Glu214, Phe217, and
Ile218. In addition to the hydrophobic interaction,
putative H-bonds are formed between the main chain and
Glu214. Thr175 forms a H-bond to the FxFG core
but not to the GLFG core. Surface views (D and E)
illustrate the intimacy of the contact between the repeat cores and
Ib442. In contrast, the contact between the GLFG peptide at site 2 involved a pocket formed by two Ib442 chains (green and
orange in C and F) and neither Ib442
chain alone buried a significant amount of the core, consistent with
site 2 representing a crystallization artifact and site 1 representing
the true GLFG binding site. G, although the
conformation of GLFG and FxFG cores bound to Ib442 was different, they
had a similar outline.
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Comparison between the Binding of GLFG and FxFG Repeat Cores to
Ib442--
Although our original
P21212 Ib442-FxFG crystals (21) were
adequate to identify both the location of the FxFG binding site on
importin- and the key residues involved in the interaction, their
diffraction was highly anisotropic. Although data extended past 2.8 Å along one axis, diffraction was weak past 3.4 Å along the others. To
enable a more precise comparison to be made of the binding of FxFG and
GLFG cores, we obtained a second Ib442-FxFG crystal form that yielded
higher quality diffraction data (Table I). These crystals had
P212121 symmetry and
produced a complete 2.8-Å resolution data. They were solved by
molecular replacement to give an R-factor of 23.6%
(Rfree 26.7%) after refinement and contained
three Ib442 chains in the asymmetric unit, each of which bound a FxFG
core essentially as observed in the original
P21212 crystals (Fig. 2,
B and E).
The GLFG peptide at site 1 followed a path similar to that adopted by
FxFG repeats bound to the same site (Ref. 21 and Fig. 2G).
Thus, the Phe-Gly moiety of the GLFG core adopted a position
within the binding site closely matching the analogous FG in
the FxFG peptide. As observed in the FxFG construct (Ref. 21
and see below), the aromatic Phe residue was inserted into a
hydrophobic cavity formed by the side chains of residues
Leu174, Thr175, Ile178,
Glu214, Phe217, and Ile218 of
importin- (Fig. 2, A and B). The GLFG core
adopted a different backbone conformation from the FxFG core (Fig. 2).
Whereas the FxFG core adopted a -conformation, with its two Phe
rings pointing into the groove between the A-helices of HEAT repeats 5 and 6 of importin- and the "X" residue
(here Ser) pointing away from the surface, the GLFG core formed a loop
with both central residues (LF) of the GLFG
repeat pointing into the groove between HEAT repeats 5 and 6. The Leu
side chain of the repeat followed a path similar to the peptide
backbone of the FxFG cores (Fig. 2G). Overall, the different
conformations adopted by the two repeat types produced for each the
most efficient burying of their hydrophobic residues into the binding
site on importin-.
The interaction of GLFG repeats with the primary site of importin-
buried slightly less surface area than was buried by the FxFG
interaction (Table II). The FxFG cores appeared to fit to the
importin- primary site more effectively than GLFG, as shown, for
example, in Fig. 2, where the first Phe, F1, of the FxFG core clearly occupies more of the volume of the site than the Leu of
the GLFG. Moreover, the Leu side chain is less involved in
molecular contacts with importin-, whereas in FxFG repeats both Phe
are in contact. Also, because of the different backbone conformation of
GLFG repeats, the core is only able to form one of the two putative
H-bonds observed for FxFG cores. The GLFG peptide was positioned so
that it could form a putative H-bond with Glu214, but it
was not positioned close enough to Thr175 to form a H-bond
with it as well (Fig. 2). In summary, although, the binding site on
importin- was able to recognize both classes of Nup core, it formed
a more intimate contact with those from FxFG-Nups.
Of the two large hydrophobic residues in each FG repeat, the side chain
of the second (i.e. the Phe of the FG-dipeptide) formed a
more intimate contact with the surface of importin- than the side
chain of the first (i.e. the Leu of the GLFG
repeat, or F1 of the FxFG repeat). Thus, whereas the side
chain of the second Phe, F2, is wholly buried in a hydrophobic
pocket, the Leu/F1 side chains were more exposed to solvent. Thus the molecular recognition of FG repeats by importin- appears to involve primarily the FG-dipeptide. Although a contribution to the interface is
also made by "Leu/F1," which interacts with hydrophobic components of the binding site, its primary function appears to be to form a cap
or seal over F2 that further shields it from solvent. This capping
feature was observed in both FxFG and GLFG complexes and may be a
general feature of FG repeat binding to importin-. The Pro of a PSFG
repeat (see, for example, Ref. 1) may perform a similar function.
Although the crystal data indicate that both FxFG and GLFG repeat cores
bind in an analogous way to the hydrophobic groove formed between the
A-helices of importin- HEAT repeats 5 and 6, these results were
obtained with an importin- fragment and a peptide with a single GLFG
sequence. To ensure that these results reflected genuine interaction
sites, we conducted biochemical binding studies using full-length
importin- or Kap95 and domains from GLFG and FxFG nucleoporins that
contain large numbers of repeats (see below).
I178D-Importin- Binds Less Strongly to Both FxFG and GLFG
Repeats--
Previous work (21) indicated that Ile178,
located between the A-helices of HEAT repeats 5 and 6 (Fig. 2), was a
crucial component of the primary FxFG binding site on importin- and
showed that the I178D mutant reduced the binding of FxFG repeats to
below detectable levels. This mutant retained wild type affinity for RanGTP and importin- (mutant/wild-type Kd values
measured in microtiter plate binding assays (34) were 8.9 and 5.8 nM, respectively (data not shown)) indicating that it had
not introduced a major, global conformational change in importin-.
To test whether the mutant also perturbed interactions with GLFG
repeats, binding experiments were conducted. Initially, bacterially
expressed GST fusions containing the GLFG regions of Nup100 (residues
2-610) and Nup116 (residues 161-730) were coupled to
glutathione-Sepharose and used in pull-down assays with wild-type- or
I178D-importin-. Both GLFG domains (Nup100 and Nup116) and a control
FxFG-containing protein (GST-Nup1, residues 423-816), bound wild-type
importin- effectively (Fig. 3,
lanes 1, 5, and 9). Consistent with
the GLFG and FxFG cores binding to overlapping sites,
I178D-importin- showed negligible binding to both GST-GLFG proteins
(Fig. 3, lanes 7 and 11) and to GST-Nup1. In
control experiments, no binding of the soluble proteins
(i.e. wild-type or I178D-importin-, importin- or
Nsp1-FF18) to GST-glutathione-Sepharose was observed (data not
shown).
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Fig. 3.
Importin- binds both
FxFG and GLFG nucleoporins at overlapping sites. GST-Nups
containing FxFG repeats (Nup1 residues 423-816, lanes 1-4)
or GLFG repeats (Nup100 residues 2-610, lanes 5-8, and
Nup116 residues 161-730, lanes 9-12) were coupled to
glutathione-Sepharose 4B (Amersham Biosciences), washed and incubated
with 10 µg of wild type (lanes 1, 5, and
9) or I178D (lanes 3, 7, and
11) importin-. Bound proteins were eluted with Laemmli
sample buffer and analyzed by SDS-PAGE. Binding of wild type
importin- to GST-Nups was also done in the presence of 100 µg of
soluble Nsp1 FF18 (residues 262-603, lanes 2, 6,
and 10) or 60 µg of importin- (lanes 4,
8, and 12).
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We complemented the solution pull-down binding studies with solid phase
binding assays (34) to determine the relative affinities of the
interactions. In this assay, bacterially expressed proteins corresponding to the FxFG repeats from Nsp1 (residues 262-603, Nsp1
FF18) or Nup1 (residues 423-816), or the GLFG repeats from Nup100 or
Nup116, or GST alone were adsorbed onto microtiter plates. In contrast
to GST control wells, where no binding of importin- was observed,
both FxFG and GLFG constructs showed saturable binding to S-tagged
importin- (Fig. 4). The apparent
dissociation constants calculated from these data (Table
III) were similar to those reported by
Ben-Efraim and Gerace (34) for importin-/FxFG interactions. The
~2-fold weaker binding of the GLFG constructs compared with FxFG
might explain previous reports that failed to detect an interaction between importin- and GLFG-Nups under conditions where the
importin-/FxFG repeat interaction was observed (24). Nonetheless,
the binding of importin- to GLFG repeats is stronger than that
reported for the NTF2/FxFG repeat interaction (Kd
1.2 µM; see Refs. 52 and 53) that is essential for NTF2
to import Ran into the nucleus. Therefore, the interaction between
importin- and GLFG repeats is within the range expected for a role
in nuclear trafficking. In agreement with the pull-down assays (Fig.
3), importin- I178D bound much more weakly to either FxFG or GLFG
repeats compared with wild-type importin- (Fig. 4, A and
B, Table III). The strength of the interaction between
importin- and Nups is severely reduced by RanGTP (11, 14, 34).
Moreover, we observed that binding of importin- to both Nsp1-FF18
and GST-Nup100-(2-610) was inhibited by RanGTP (but not RanGDP), thus
demonstrating the specificity of the interaction (data not shown).
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Fig. 4.
Importin- and Kap95
bind both FxFG and GLFG nucleoporins. The affinity of importin-
and Kap95 for FxFG and GLFG constructs was measured using solid phase
binding assays (34). Binding of S-tagged importin- ( ) or
I178D-importin- ( ) to Nsp1 FF18 (A) (residues
262-603) or GST-Nup116 (B) (residues 161-730). Binding of
S-tagged Kap95 ( ) to Nsp1-FF18 (C) or GST-Nup116
(D) (residues 161-730). Lineweaver-Burk plots for
importin-/Nsp1 FF18 (E), importin-/Nup116
(F), Kap95/Nsp1 FF18 (G), and Kap95/Nup116
(H). These binding isotherms were used to calculate the
apparent dissociation constants (see Table III).
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Table III
Dissociation constants for nucleoporins and importin- or Kap95
Data represent the mean ± range from separate experiments, each
of which was performed in triplicate. Numbers in parentheses indicate
the number of separate experiments.
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GLFG and FxFG Repeats Compete for Overlapping Sites on Importin-
and Kap95--
Cross-competition studies were conducted between FxFG
and GLFG-containing Nups for binding importin- in both pull-down
assays (Fig. 3) and solid-phase assays (Fig.
5A). FxFG repeats from Nsp1 displaced wild-type importin- bound to either GST-Nup1 (FxFG) or
GST-GLFG recombinant protein derived from Nup100 or Nup116 (Fig. 3,
lanes 2, 6, and 10, respectively; Fig.
5A). This competition between different repeat types
confirms that the FxFG and GLFG repeats bind to overlapping sites on
importin-.
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Fig. 5.
Competition of GLFG and FxFG nucleoporins for
binding to importin- and Kap95.
A, displacement of S-tagged importin- () or Kap95
() bound to GST-Nup116 (residues 161-730) by Nsp1-FF18 (residues
262-603). B, different GLFG cores compete for binding to
importin- and Kap95. S-tagged importin- () or Kap95 ()
bound to His-Nup116 (residues 181-725) is displaced by GST-Nup100
(residues 2-610).
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Because our binding studies used a vertebrate transport factor with
GLFG and FxFG repeat domains derived from S. cerevisiae proteins, we also investigated the extent to which these interactions might be influenced by the heterologous origin of the proteins employed. In one set of experiments, we tested whether the S. cerevisiae importin- homologue, Kap95, bound FxFG and GLFG
repeats in a similar manner as importin-. As shown in Fig. 4 and
Table III, S-tagged Kap95 bound the FxFG repeats of Nsp1 and Nup1 and also the GLFG repeats of Nup100 and Nup116 in a comparable manner to
importin-, albeit Kap95 showed a marginally greater affinity for
GLFG- than FxFG-containing proteins. Significantly, Nsp1-FF18 was able
to displace Kap95 bound to immobilized Nup116 (Fig. 5A), consistent with Kap95, like importin-, binding both FxFG- and GLFG-Nups at common sites. In addition, we found that Nup100 could displace importin- or Kap95 bound to Nup116 (Fig. 5B).
These results are entirely consistent with those obtained using
vertebrate importin-, indicating that the binding of both types of
repeat to common sites is a general feature of at least this subset of nuclear transport factors rather than being species-specific.
We also tried to use the vertebrate GLFG-nucleoporin Nup98 (13, 23) to
probe interactions with importin-, because both we (21) and others
(for example, Refs. 13 and 54) had observed that this protein bound
importin- in blot overlays. However, other studies had either failed
to detect an interaction between Nup98 and importin- (24) or had
indicated that the interaction was nonspecific and nonsaturable (34).
When we investigated this binding more thoroughly, we too found that
the binding of importin- to both native Nup98 from rat liver nuclear
envelopes and bacterially expressed GST-Nup98 (residues 43-518) was
nonspecific. For example, Nup98 bound S-protein-horseradish peroxidase
just as effectively in the absence of S-tagged-importin-, and in
solid-phase assays, importin- failed to show saturable binding to
the bacterially expressed GLFG-containing domain of Nup98 (data not
shown). We also attempted to use the yeast two-hybrid system to
investigate interactions between Nup98 and either importin- or
Kap95, but the expression levels of Nup98 were low, and we were not
able to detect any interaction in this way (data not shown).
Consequently, it was not possible to assay the binding of Nup98 to
importin-.
The binding of FxFG and GLFG repeats to overlapping sites on Kap95
appeared inconsistent with previous studies analyzing a mutant
Kap95-L63A (22). In the two-hybrid assay, Kap95-L63A failed to interact
with the GLFG regions of Nup116 and Nup100 but retained interaction
with the Nup1-FxFG repeat region. To try to resolve this issue, we
examined the binding of Kap95-L63A to FxFG and GLFG repeat regions
using solution pull-down and solid phase binding assays. Bacterially
expressed Kap95-L63A bound both GST-Nup116-GLFG and Nsp1 FF18 in
vitro at 4 °C (data not shown). Furthermore, the apparent
dissociation constants for the binding of Kap95-L63A to both types of
repeat regions were similar to wild-type Kap95 under these assay
conditions. Therefore, the L63A mutant likely indirectly perturbs
nucleoporin binding in the context of the two-hybrid assay. This may be
a reflection of the RanGTP environment in the nucleus wherein the
two-hybrid interactions are measured and will require future analysis.
Importin- Requires FG Nup Binding for Function in Yeast--
To
examine whether the similar FG binding interactions for Kap95 and
importin- reflected identical functional properties, we tested if
expression of importin- could complement the lethal phenotype of a
kap95 null yeast strain. Plasmids expressing either wild-type LexA-importin- or I178D mutant were transformed into a
kap95 null tester strain harboring plasmid borne
(URA3/CEN) wild-type KAP95. The assay for
complementation of lethality was conducted on media containing
5-fluoroorotic acid, lethal to cells that cannot survive without the
KAP95/URA3 plasmid. Interestingly, we found that
LexA-importin-, but not the corresponding I178D mutant, was able to
complement the kap95 null strain (Fig.
6). This further supports the idea that
Kap95 and importin- interact with components of the nuclear
trafficking machinery in very similar ways.
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Fig. 6.
LexA-importin-, but
not LexA-I178D-importin-, is able to
complement kap95 yeast. A
kap95 null strain harboring a URA3 plasmid
expressing Kap95 was transformed with plasmids containing LexA,
LexA-importin-, LexA-I178D-importin-, or LexA-KAP95.
Complementation was assayed on synthetic complete media containing 2%
glucose and 5-fluoroorotic acid at 23 °C for 7 days. Expression of
the LexA constructs and the absence of Kap95 in cells expressing
LexA-importin- were confirmed by immunoblot.
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Soluble GLFG Repeats Inhibit Nuclear Protein Import--
Previous
studies have shown that soluble FxFG repeats inhibit
importin--mediated nuclear protein import in permeabilized cells
(21, 55). If importin- interactions with GLFG repeats are required
in a similar manner, we predicted that exogenous GLFG repeats should
also inhibit in vitro import. As illustrated in Fig.
7, soluble GST-Nup100 (residues 2-610)
inhibited the import of a fluorescein-labeled NLS-BSA substrate in a
similar manner to that observed for soluble FxFG repeats. These data
are also consistent with the observation of a protein import defect in a nup116GLFG yeast strain (12). However, whereas a
40-fold excess of Nsp1-FF18 was sufficient to inhibit nuclear protein import in this system by 85%, a 100-fold excess of GST-Nup100 was
required to produce a comparable level of nuclear import inhibition. The lower efficiency of the GLFG construct to inhibit nuclear protein
import is consistent with the weaker interaction of importin- with
GST-Nup100 compared with Nsp1 FF18 (Figs. 4 and 5, Table III). In
summary, our data indicate that importin- binds both FxFG- and
GLFG-containing nucleoporins at an overlapping site in vitro
and that both interactions are likely to be significant for nuclear
import in vivo.
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Fig. 7.
Soluble GLFG inhibits nuclear protein import
in permeabilized cells. Soluble FxFG (Nsp1 FF18) at 10 µM (B) and GLFG (GST-Nup100-(2-610)) at 25 µM (C) both inhibit
importin--dependent import of a fluorescein-labeled
NLS-BSA substrate (A). Bar is 25 µm.
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DISCUSSION |
The preferential association of individual transport factors with
different FG-Nups has raised the possibility that there are
functionally different pathways for movement through NPCs (Ref. 11,
reviewed in Ref. 17). Data obtained using the S. cerevisiae
importin- homologue Kap95 suggested that GLFG- and FxFG
repeat-containing Nups may be involved in different steps of the
transport pathway (22). To address the extent to which these different
classes of Nups might contribute to different trafficking pathways, we
have explored the way in which each biochemically interacts with
importin-.
The crystal structures of the Ib442-GLFG and Ib442-FxFG complexes (Fig.
2), the dramatically reduced affinity of I178D-importin- for both
FxFG and GLFG repeats (Figs. 3 and 4), and the ability of FxFG repeats
to displace GLFG repeats from importin- (Figs. 3 and 5) are all
consistent with both classes of FG-Nups binding to overlapping sites on
importin-. Moreover, both the crystal structures and the reduced
affinity for both FxFG and GLFG repeats shown by the I178D mutant
indicate that a common binding site is located in the hydrophobic
pocket between the A-helices of HEAT repeats 5 and 6. Finally, the
binding data and competition experiments with Kap95 (Figs. 4 and 5)
were analogous to those obtained with importin-.
There are unexpected similarities in the way in which each type of
repeat binds importin-. The structural data indicate that the core
hydrophobic residues of both types of repeat make major contributions
to the interaction with importin-. The Phe side chain of the FG
portion of both FxFG and GLFG cores is inserted deep into the
hydrophobic pocket formed from Leu174, Thr175,
Ile178, Glu214, Phe217, and
Ile218 (Fig. 2 and Ref. 21). In contrast, the hydrophobic
residue in the first half of the repeat core (Leu or Phe) appears to
function primarily to shield the Phe side chain of the FG dipeptide
from solvent. Although our structural data do not exclude the
possibility that there may be additional contributions to the binding
to importin- from residues in the hydrophilic linkers between the
GLFG and FxFG cores, especially in the regions immediately flanking the cores, these are probably not major determinants of binding. However, by analogy to the results obtained with G-proteins binding to their
effectors (see, for example, Ref. 56), interactions with these flanking
regions might make contributions to the selectivity of interactions or
to modulating the affinity of interactions between carriers and Nups.
For example, this could explain why different carrier molecules may
have preferences for different portions of the Nup116 GLFG repeat
region (41).
The structural data were obtained using only residues 1-442 of
importin- and a GLFG peptide, which inevitably raises the question
of whether the binding site observed is an artifact associated with the
use of fragments or with crystallization. However, these results were
completely validated by the binding and competition data using
full-length importin- or Kap95 and Nup GLFG and FxFG domains that
contained a large number of repeats. These data certainly do not
exclude the possibility of additional FxFG or GLFG binding sites on
importin- or Kap95. However, the competition binding experiments
indicate that any such binding sites would be of lower affinity than
the site between the A-helices of HEAT repeats 5 and 6. Previous
deletion mutagenesis studies have indicated that a major NPC binding
site on importin- is located between residues 152 and 352 (and thus
between the A-helices of HEAT repeats 5 and 6), but at least one
additional NPC binding site, probably with lower affinity, may exist
between this site and residue 618 of importin- (21, 31, 32). Based
on our new in vitro binding analysis, the previous
observations of differential FG nucleoporin two-hybrid interactions
with the Kap95-L63A mutant (22) may not reflect differences in FG
nucleoporin binding sites. In the absence of high-resolution structural
information for either Kap95 or a complex of Kap95 with FG Nup repeats
and further analysis of FG-binding mutants, it is not possible to be
certain that the GLFG/FxFG site in Kap95 is directly equivalent to the
site characterized in importin- (Fig. 2 and Ref. 21).
The binding of both FxFG and GLFG repeats to overlapping sites on
importin- raises the question of the extent to which there is a
functional difference between FxFG- and GLFG-Nups, at least in the
context of importin--mediated nuclear protein import. A number of
studies have indicated that GLFG and FxFG repeats are not simply
interchangeable in vivo and at least some of their functions
in trafficking pathways may be distinct. For example, in S. cerevisiae, the FxFG region of Nsp1 cannot substitute for the GLFG
region of Nup116 (12). Furthermore, different transport factors appear
to preferentially associate with different Nups (Ref. 11, reviewed in
Ref. 17), and genetic approaches show that some Nups are required for
certain classes of transport, whereas other Nups are dispensable
(reviewed by Ref. 17). One way in which GLFG- and FxFG-Nups could share
common binding sites on importin-/Kap95 and yet be functionally
different, would be if the different FG-Nup classes were associated
with different steps of the nuclear protein import process. In this
context, it may be important that different classes of FG-Nups are
thought to be distributed differently throughout S. cerevisiae NPCs, with PSFG-Nups located in the cytoplasmic
fibrils, FxFG-Nups (except for Nsp1p) located at the nucleoplasmic
face, and GLFG-Nups in the central region between them (11, 18). This
would be consistent with a model in which the different functions of
FxFG- and GLFG-Nups may not be linked so much to their differential
interaction with the carrier molecule but rather with their location in
the NPC and spatial relationship to the translocation mechanism.
Alternatively, the putative lower affinity binding sites may be more
functionally significant in vivo than they are in
vitro.
Recent work has indicated that, in addition to its central function in
nuclear protein import, importin- also plays an important role in
in vitro nuclear envelope assembly with Xenopus
egg extracts (57). Based on the observation that the I178D mutant is
unable to substitute for wild-type importin- in this system, it has been proposed (57) that one function of importin- in nuclear envelope assembly depends on its binding FxFG nucleoporins. However, our present data demonstrate that the I178D mutant also inhibits the
binding of GLFG nucleoporins, and so it is therefore not clear which
class of nucleoporin is actually critical for nuclear envelope assembly
in the Xenopus system.
In summary, we have shown that GLFG and FxFG nucleoporins both bind to
a site between HEAT repeats 5 and 6 of importin- and that Kap95
similarly binds both classes of Nups at overlapping sites. Although
further work will be required to determine whether other importin-
superfamily members also bind different types of nucleoporin FG repeats
at common sites, the present results indicate that functional
differences between different FG-Nups may arise primarily from
differences in their spatial organization.
*
§¶,
,
TOP
ABSTRACT
INTRODUCTION
