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J. Biol. Chem., Vol. 277, Issue 20, 18161-18172, May 17, 2002
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From the Department of Biological Sciences, Stanford University,
Stanford, California 94305-5020
Received for publication, December 21, 2001, and in revised form, February 23, 2002
Transport of macromolecules across the nuclear
pore complex (NPC) occurs in seconds and involves assembly of a
karyopherin·cargo complex and docking to the NPC, translocation of
the complex across the NPC via interaction with nucleoporins (Nups),
and dissociation of the complex in the nucleoplasm. To identify
rate-limiting steps in the Kap95p·Kap60p-mediated nuclear import
pathway of Saccharomyces cerevisiae, we reconstituted key
intermediate complexes and measured their rates of dissociation and
affinities of interaction. We found that a nuclear localization
signal-containing protein (NLS-cargo) dissociates slowly from Kap60p
monomers and Kap60p·Kap95p heterodimers with half-lives
(t1/2) of 7 and 73 min, respectively; that Kap60p
and Kap60p·NLS-cargo complexes dissociate slowly from Kap95p
(t1/2 = 36 and 73 min, respectively); and that
Kap95p·Kap60p·NLS-cargo complexes and Kap95p·Kap60p heterodimers
dissociate rapidly from the nucleoporin Nup1p (t1/2 Nucleocytoplasmic protein transport across nuclear pore complexes
(NPCs)1 proceeds at the rate
of 1-1000 molecules/NPC/second (1-3) and requires the assembly and
disassembly of several multiprotein intermediates (4). In general,
mobile receptors termed karyopherins (or Kaps; these include importins,
exportins, and transportins) bind the nuclear localization signal (NLS)
or nuclear export signal (NES) of a cargo molecule and interact with
nucleoporins (or Nups; nuclear pore complex proteins) to mediate
translocation across the NPC. In the case of the Kap95p·Kap60p
heterodimer of Saccharomyces cerevisiae, Kap60p (Srp1p)
binds the NLS of a cargo molecule (NLS-cargo), and Kap95p tethers the
Kap60p·NLS-cargo complex to Nups (docking), mediating translocation
across the NPC via an unknown mechanism (see Fig. 1). Upon entering the
nucleus, the NLS-cargo is released from Kap60p, and Kap95p and Kap60p
are recycled separately to the cytoplasm for further rounds of import
(5, 6).
Kap95p·Kap60p heterodimers can deliver NLS-cargoes to the nucleoplasm
against a concentration gradient of "free" cargo molecules (7),
implying the existence of a mechanism at the NPC that imparts
directionality to nuclear import. Structural features of the NPC may
bias the direction of movement of Kap95p·Kap60p·NLS-cargo complexes
within the NPC. In S. cerevisiae, the nuclear pore complex is composed of ~30 distinct nucleoporins (8). A subset of the Nups
(FG Nups) contains repeats of the dipeptide motif FG, which bind Kaps
(9, 10). Each of the 13 FG Nups of yeast is present in eight or more
copies per NPC, and together FG Nups may contribute up to 200 docking
sites for karyopherins within the NPC. Eight FG Nups are distributed
symmetrically within the NPC (e.g. Nup100p and Nup116p),
whereas the rest are localized exclusively to either the cytoplasmic
fibers (Nup42p and Nup159p) or the nuclear basket structure (Nup1p,
Nup2p, and Nup60p) (8, 11, 12). In vivo, the FG Nups of the
nuclear basket (Nup1p, Nup2p, and Nup60p) are required for efficient
Kap95p·Kap60p-mediated nuclear import (12-15). These same three
Nups, plus the exportin Cse1p in complex with Gsp1p-GTP, are also
required for efficient nuclear export of Kap60p in vivo (11,
12, 14-16).
The directionality of karyopherin-mediated transport is also governed
by the Gsp1p GTPase (the Ran GTPase in vertebrates) (4). Binding of
Gsp1p-GTP to importins (karyopherins dedicated to nuclear import)
disrupts their ability to bind cargoes and Nups and thus terminates
nuclear import reactions in the nuclear basket structure of the NPC or
in the nucleoplasm (9, 17, 18). Conversely, exportins (karyopherins
dedicated to nuclear export) bind cargoes and Gsp1p-GTP cooperatively
to initiate export reactions in the nucleoplasmic side of the NPC (19,
20). Export reactions are terminated when Yrb1p stimulates dissociation
of Gsp1p-GTP from exportins, and Rna1p stimulates the hydrolysis of GTP
by Gsp1p (4). Because Rna1p is localized to the cytoplasm of yeast (21)
and the Gsp1p guanine nucleotide exchange factor (Prp20p) is localized
to the nucleoplasm (22, 23), a steep concentration gradient of
Gsp1p-GTP across the NPC likely exists.
Although mechanistic details of Kap95p translocation across the NPC are
lacking, it is clear that Kap95p orthologues in vertebrates (importin
Preparation of Recombinant Proteins--
Genes encoding the
proteins or protein fragments used were amplified from S. cerevisiae genomic DNA using Taq- or
Pfu-driven polymerase chain reactions with designed
oligonucleotides that incorporate restriction enzyme sites
compatible for ligation into pGEX-2TK in-frame with the 3' end of the
gene encoding glutathione S-transferase (GST). For Kap60p,
Kap95p, Cse1p, and Nup2p the entire open reading frame was amplified.
For Nup1p, the gene fragment encoding the entire FG repeat region was
used to generate Nup1p Solution Binding Assay--
All assays were performed using
recombinant proteins in binding buffer (20 mM Hepes, pH
6.8, 150 mM KOAc, 2 mM Mg(OAc)2, 2 mM DTT, and 0.1% Tween 20). For each experiment, the
GST-Nup, -Kap, or -NLS-cargo was incubated in batch with
glutathione-Sepharose beads (1-2 µg of GST fusion/10 µl of packed
beads; Amersham Biosciences) in 1 ml of binding buffer for 15 min at
4 °C. The beads were collected by centrifugation at 2,000 × g for 30 s and washed six times by resuspension in 0.5 ml of binding buffer and sedimentation as before. Two washes were done
at room temperature and contained 100 µM ATP to remove
any E. coli heat shock proteins that bound to the GST
chimera. Washed collected beads were resuspended in a 50% slurry, and
the bead slurry was aliquoted in 20-µl portions into siliconized
0.5-ml microtubes (Sigma) that contained protein additions for a total
volume of 40 µl. Tubes were then tumbled for 1 h at 4 °C. At
the end of the incubations, beads were sedimented at 2000 × g for 30 s, and unbound proteins in the supernatant fractions were collected by removing 30 µl from the meniscus; this
constitutes the unbound fraction. Beads were washed twice and were
resuspended with 30 µl of buffer. All samples were finally processed
by adding 10 or 12 µl of 6× sample buffer with Equilibrium Affinity Assay--
Protein affinities were
conducted as described previously (14). Purified Kap95p and Kap60p were
phosphorylated at an engineered site in their NH2 termini
using bovine heart kinase and [32P]ATP (PerkinElmer Life
Sciences), as described in the Amersham Biosciences GST handbook.
GST-Kap60p, GST-Nup1p Molecular Dissociation Assay--
Dissociation rates were
assayed using purified radiolabeled proteins in the bead-based solution
binding assay. Kap95p and Kap60p were radiolabeled as above.
GST-Kap60p, GST-Nup1p There is ample cell biological and genetic evidence that the
nucleoporins Nup1p and Nup2p, the GTPase Gsp1p, and the exportin Cse1p
perform essential functions in the Kap95p·Kap60p-mediated nucleocytoplasmic transport pathway, and there is evidence of biochemical interactions among subsets of these proteins (9, 11, 12,
14-17, 24-26). However, the basic mechanism underlying the function
of these proteins in the pathway is not known. Specifically, it has not
been resolved whether these proteins function in a passive manner to
modulate the interaction of Kap60p and/or Kap95p with other proteins or
whether they function in an active manner to trigger allosteric
mechanisms in Kap60p and/or Kap95p that accelerate capture or release
of bound partners (for example see Fig. 5A). Here we
addressed this issue directly by conducting a quantitative analysis of
the dynamics of assembly and disassembly of key nuclear import intermediates.
An NLS-cargo molecule was constructed as a fusion of the NLS of
S. cerevisiae Cbp80p (an mRNA cap-binding protein; amino
acids 1-30) and the MBP. We chose the NLS of Cbp80p because it may be the highest affinity endogenous NLS recognized by Kap60p in yeast. This
is based on the observation that Cbp80p is one of only two proteins in
yeast extracts that remain bound to Kap60p when all other proteins have
been removed by extraction (27) (data not shown). The second protein is Nup2p.
Nup1p was chosen for this analysis because (i) it binds Kap95p·Kap60p
heterodimers with the highest affinity in comparison to other FG Nups
tested (9) (data not shown), (ii) it functions in the Kap95p·Kap60p
transport pathway in vivo (12, 26), and (iii) it resides in
the nuclear basket structure of the yeast NPC (8, 12) where disassembly
of import complexes is presumed to occur. Indeed, Nup1p may function as
one of the last "stepping stones" (docking sites) in the path of
Kap95p·Kap60p-mediated transport (Fig.
1). In addition to its serving as a
stepping stone for transport, we demonstrate here that Nup1p
accelerates the rate of dissociation of NLS-cargo from Kap60p·Kap95p
heterodimers. We used a version of Nup1p that lacks the NH2
terminus (Nup1p Purified proteins and a solution binding assay were used to
reconstitute: (i) the binding of Kap60p to a protein that contains a
classic nuclear localization signal (NLS-cargo) in the presence or
absence of Kap95p (Fig. 2A),
(ii) the binding of Kap95p to its heterodimeric partner Kap60p (Fig.
2B), and (iii) the binding of Kap95p·Kap60p heterodimer
(with and without NLS-cargo) to Nup1p (Fig. 2C). Kap60p
bound directly to NLS-cargo, and Kap95p was recruited to the complex in
a Kap60p-dependent manner as expected (Fig. 2A)
(17). Also, note that purified recombinant Kap60p was ~100% active
in binding NLS-cargo judging from its absence from the unbound fraction
(open arrowhead, Fig. 2A, bottom panel). Kap95p
bound Kap60p as expected (Fig. 2B) (17) and was ~100% active in binding Kap60p judging from its absence from the unbound fraction (open arrowhead, Fig. 2B, bottom panel).
When combined, Kap95p·Kap60p heterodimers were able to dock the
NLS-cargo to Nup1p
Accelerating the Rate of Disassembly of Karyopherin·Cargo
Complexes*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
21 s) and other Nups. A search for factors that accelerate
disassembly of the long-lived intermediates revealed that Nup1p and
Nup2p accelerate 16- and 19-fold the rate of dissociation of NLS-cargo from Kap60p·Kap95p heterodimers; that Gsp1p-GTP accelerates 
447-fold the rate of dissociation of Kap60p·NLS-cargo from Kap95p; and that Nup2p and the Cse1p·Gsp1p-GTP complex independently
accelerate 22- and 39-fold the rate of dissociation of NLS-cargo
from Kap60p. We suggest that Nup1p, Nup2p, Cse1p, and Gsp1p accelerate disassembly of Kap95p·Kap60p·NLS-cargo complexes by triggering allosteric mechanisms within Kaps that cause rapid release of binding
partners. In that way, Nup1p, Nup2p, Cse1p, and Gsp1p may function as
karyopherin release factors (or
KaRFs) in the nuclear basket structure of the S. cerevisiae
NPC.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and transportin) move at a very rapid rate through the NPC (1-1000
molecules/NPC/second) (1-3). It follows that each intermediate step in
the nuclear import pathway (e.g. assembly of the
Kap95p·Kap60p·NLS-cargo complex, interaction of this complex with
Nups, and disassembly of the complex) must occur as fast or faster than
the overall rate of transport. Here, we have identified potential
rate-limiting steps in the Kap95p·Kap60p-mediated nuclear import
pathway by measuring the rate of dissociation of key intermediates. We
demonstrate that the nucleoporins Nup1p and Nup2p, the exportin Cse1p,
and the GTPase Gsp1p accelerate the disassembly of long-lived intermediates in the pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N (amino acids 332-1076), which is missing
the NH2 terminus. Full-length Nup1p was not used because it
could not be expressed in Escherichia coli. For NLS-cargo,
we used the nuclear localization signal of Cbp80p (amino acids 1-30)
fused to the COOH terminus of a chimera between GST and the
maltose-binding protein (MBP). Recombinant proteins were expressed in
E. coli strains BLR or BL21 Codon Plus (Novagen) and were
purified on glutathione-coated Sepharose beads (Amersham Biosciences).
In each case, 40 ml of a bacterial cell extract prepared from 1000 A600 units of cells was incubated with 1 ml of glutathione-coated Sepharose beads in batch for 1 h at 4 °C (as described in Ref. 9). After extensive washing of the column, GST-proteins were eluted with elution buffer (50 mM
Tris, pH 8, 110 mM KOAc, 2 mM
Mg(OAc)2, 2 mM DTT, and 0.1% Tween 20 plus 15 mM reduced glutathione), and proteins were concentrated in
a Centricon 30 unit (Amicon). Concentrated proteins were aliquoted in
1-mg portions, frozen in liquid nitrogen, and stored at -70 °C. To
remove GST, thawed GST fusion proteins were cleaved with thrombin (Calbiochem) at room temperature for specific times. After
adding hirudin (Calbiochem) to neutralize thrombin, the samples were
applied separately to fast protein liquid chromatography Superdex 200 or Superose 6 columns (Amersham Biosciences), which were equilibrated
in 20 mM Hepes, pH 6.8, 150 mM KOAc, 2 mM Mg(OAc)2, and 2 mM DTT. Peak
fractions containing the purified karyopherin, nucleoporin, or
NLS-cargo were pooled. Tween 20 was added to 0.1%, and aliquots were
frozen in liquid nitrogen and stored at 
70 °C. His-tagged Gsp1p
was purified and charged with GTP as described previously (9, 17).
-mercaptoethanol to the unbound and bound fractions, respectively. Samples were heated
at 95 °C for 10 min, and proteins in one-half of each sample were
resolved by SDS-PAGE and stained with Coomassie Blue.
N, and GST-NLS-cargo were immobilized
separately on beads and incubated with increasing amounts of
radiolabeled Kap95p or Kap60p at room temperature in 20 mM
Hepes, pH 6.8, 150 mM KOAc, 2 mM
Mg(OAc)2, 1 mM DTT, 0.1% Tween 20, protease
inhibitors (leupeptin, pepstatin, aprotinin, and phenylmethylsulfonyl
fluoride), and 1 mg/ml BSA. After incubation, beads were washed
quickly, and bound radiolabeled proteins were quantified by liquid
scintillation. Binding curves were fit to the data using GraphPad
PrismTM software (Biosoft).
N, and GST-NLS-cargo were immobilized
separately on beads and incubated for 2 h with radiolabeled Kap95p
or Kap60p at room temperature in 20 mM Hepes, pH 6.8, 150 mM KOAc, 2 mM Mg(OAc)2, 1 mM DTT, 0.1% Tween 20, and 10 mg/ml BSA. Unlabeled Kap95p
or Kap60p were also present when indicated. 12 µl of the bead mix was
diluted into 1.2 ml of 20 mM Hepes, pH 6.8, 150 mM KOAc, 2 mM Mg(OAc)2, 1 mM DTT, 0.1% Tween 20, and 10 mg/ml BSA containing various
concentrations of competitor proteins. Beginning 10 s after
dilution, beads were flash-collected on filters using a vacuum
manifold. Bound radiolabeled proteins were eluted with 1% SDS and
quantified by scintillation counting. Single exponential dissociation
curves were fit to the data using GraphPad PrismTM software (Biosoft).
Half-lives of complexes (t1/2) were calculated using
the equation t1/2 = ln2/koff.
Mean residence time was calculated using the equation
1/koff. Association rate constants were
calculated using the equation kon = KD/koff.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N; amino acids 332-1076) because we could not obtain
full-length Nup1p as a recombinant protein. Nonetheless, Nup1pN
contains all of the FG repeats in Nup1p (9).
View larger version (30K):
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Fig. 1.
Diagram of the molecular dynamics of
Kap95p·Kap60p-mediated nuclear import pathway of S. cerevisiae.
Kap60p captures the nuclear localization signal of a cargo
molecule (NLS-cargo) and binds Kap95p. The Kap95p·Kap60p·NLS-cargo
complex then moves between FG-containing nucleoporins (FG
Nup) using fast cycles of association and dissociation that
facilitate its translocation across the NPC. The mechanism of
translocation remains unsolved. Upon arrival at the nucleoplasmic side
of the NPC, the Kap95p·Kap60p·NLS-cargo complex encounters the
nuclear basket nucleoporins Nup1p and Nup2p, the GTPase Gsp1p, and the
exportin Cse1p. These proteins function as KaRFs that accelerate the
rate of disassembly of Kap95p·Kap60p·NLS-cargo complexes in two
possible order of events (top and bottom). The
top half of the diagram depicts Nup1p and Nup2p accelerating
the release of NLS-cargo from a Kap95p·Kap60p·NLS-cargo complex and
the subsequent effect of Gsp1p-GTP on the resulting Kap95p·Kap60p
heterodimer. Gsp1p binds Kap95p and accelerates the release of Kap60p.
The bottom half of the diagram depicts Gsp1p-GTP
accelerating the release of Kap95p from a Kap95p·Kap60p·NLS-cargo
complex and the subsequent effect of Nup2p or Cse1p on the resulting
Kap60p·NLS-cargo complex. Cse1p (with its bound cofactor Gsp1p-GTP)
or Nup2p bind Kap60p and accelerate release of the NLS-cargo. The
top right legend key lists the names (and alternate names)
of the yeast (S. cerevisiae) proteins used in this study, as
well as the names of their vertebrate homologues. The lower
legend key explains the significance of the
arrows.
N (Fig. 2C).
View larger version (18K):
[in a new window]
Fig. 2.
Reconstitution of key intermediate complexes
in the Kap95p·Kap60p-mediated nuclear import pathway. The
diagrams depict reconstituted intermediate complexes. The gray
tether symbols mark the immobilized protein used in each case.
A, reconstitution of the Kap60p·NLS-cargo complex in the
presence and absence of Kap95p. GST-MBP-NLSCbp80p
(GST-NLS-cargo) immobilized on glutathione-coated Sepharose
beads (1 µg/10 µl of beads; 1.4 µM within the bead
volume) was mixed with purified Kap60p and Kap95p as indicated (1 µg
each for final concentrations of 417 and 263 nM,
respectively). After 2 h at 4 °C, beads were collected by
centrifugation, and the supernatant containing unbound proteins was
removed. Beads were washed twice, and bound proteins were extracted
with SDS. Proteins in the bound and unbound fractions were resolved by
SDS-PAGE and visualized with Coomassie Blue stain. B, reconstitution of
the Kap60p·Kap95p heterodimer. GST-Kap60p immobilized on beads (1 µg/10 µl of beads; or 1.1 µM within the bead volume)
was mixed with purified Kap95p (1 µg; final concentration of 263 nM). After 2 h at 4 °C, reactions were processed
and analyzed as described for A. C,
reconstitution of karyopherin·nucleoporin complexes in the presence
and absence of NLS-cargo. GST-Nup1p N immobilized on beads (1 µg/10
µl of beads; 0.9 µM within the bead volume) was mixed
with purified Kap60p, Kap95p, and NLS-cargo (MBP-NLSCbp80p)
as indicated (1 µg each; final concentrations of 416, 273, and 566 nM, respectively). After 2 h at 4 °C, the reactions
were processed and analyzed as described for A.
Equilibrium Affinities of Nuclear Import
Intermediates--
Binding affinities of the nuclear import
intermediates were quantified using radiolabeled karyopherins in a
solution binding assay (Fig. 3). For each
affinity experiment, GST-Nups, -Kaps or -NLS-cargo were immobilized on
beads and incubated with increasing concentrations of purified
radiolabeled Kap95p or Kap60p. Kap95p and Kap60p were labeled with
32P at an engineered tag in their NH2 termini,
which does not interfere with their functioning (see
"Experimental Procedures") (28). After 2 h at 25 °C, beads
were collected by centrifugation, and the amount of bound radiolabeled
karyopherin was quantified by liquid scintillation. The results are
shown in Fig. 3; the diagrams depict the immobilized protein bound to
cross-hatched surface, the radiolabeled ligand marked by the
radioactivity symbol, and other proteins present in each incubation.
Not shown is bovine serum albumin, which was present at 1 mg/ml in
these reactions.
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Kap60p bound to an immobilized NLS-cargo with KD = 2.8 nM (Fig. 3A and Table
I). This affinity is similar to the affinity of Kap60p for the SV40 T-antigen NLS (KD = 2 nM) (29). Notably, the presence of Kap95p increased by
18-fold the affinity of Kap60p for the NLS-cargo (from
KD = 2.8 nM to KD 149 pM) (Fig. 3, A and B). This is
consistent with previous results (17, 30) and is likely due to the
binding of Kap95p to the IBB domain of Kap60p, which otherwise serves as an intramolecular inhibitor of NLS binding (31). The affinity value
obtained for the interaction between Kap95p·Kap60p and
NLS-cargo (KD 149 pM) is near
the limit of detection of our assay. The actual KD
may be lower (indicated by the symbol) because reduction of the
concentration of immobilized NLS-cargo yielded a lower apparent
KD (data not shown). However, loss of signal at
reduced concentrations of Kap95p·Kap60p prevented unambiguous
quantitation. In such cases, we have reported the most accurate value
we could measure.
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Kap95p bound immobilized Kap60p with KD 153 pM (Fig. 3C and Table I). This value was near
the limit of detection of our assay (as explained above), and so the
actual KD for the Kap95p·Kap60p complex may be
lower. The interaction of Kap95p with Kap60p is often referred to as a
typical karyopherin·cargo interaction because Kap95p binds an
NLS-like sequence in Kap60p (the IBB domain). However, it is possible
that Kap60p modulates Kap95p function in ways that other cargoes that
bind Kap95p directly do not.
Kap95p·Kap60p·NLS-cargo complexes bound immobilized Nup1pN with
KD = 400 pM (Fig. 3D and
Table I). Kap95p·Kap60p heterodimers (without
NLS-cargo) bound Nup1pN with KD 51 pM (Fig. 3E). The latter value was at the limit
of detection of our assay (as explained above), and thus the reported
number is an upper bound on the actual KD. As both
Kap95p and Kap60p can bind independently to Nup1pN (Fig.
2C), in principle both karyopherins could mediate the
interaction when binding in heterodimeric form. This may explain the
exceptionally high affinity of Kap95p·Kap60p toward Nup1pN. The
presence of an NLS-cargo bound to Kap60p·Kap95p lowered by 8-fold
the affinity of Kap95p·Kap60p toward Nup1pN (from
KD 51 pM to KD = 400 pM (Fig. 3, D and E; Table I).
Because it is possible that different NLSs have
different effects on Kap60p·Kap95p interactions
with Nups or other proteins, we conducted experiments in the absence and presence of NLS-cargo to offer a comparison between cargo-bound and
cargo-free Kap95p·Kap60p.
The nuclear import intermediates examined above exhibit high affinities
of interaction. These affinities reflect the ratio between the
dissociation rate constant (koff) and the
association rate constant (kon) of the
interacting pairs (KD = koff/kon). Assuming
typical association rate constants for protein-protein interactions
(107-108
M
1s1) (32), the high affinities
of the nuclear import intermediates measured here predict interactions
on a time scale of many seconds to minutes. However, a high affinity of
binding is not necessarily incompatible with fast rates of
dissociation. In cases in which proteins can bind each other at rates
that approach the limit set by molecular diffusion
(kon = 108-109
M1s1) (32), a molecular
interaction of very high affinity (e.g. KD = 0.5 nM) could dissociate with a
half-life of less than 2 s.
Dissociation Rate Constants of Nuclear Import
Intermediates--
The solution binding assay used above was modified
to quantify molecular dissociation rates between Nups, Kaps, and
NLS-cargo (Fig. 4). Immobilized
NLS-cargo, Kap60p, or Nup1p was incubated with radiolabeled Kap95p or
Kap60p as depicted in the diagrams. After 2 h at 25 °C, binding
reactions were diluted 100-fold into buffer containing unlabeled
Kap95p, Kap60p, or NLS-cargo as competitors. At intervals of seconds or
minutes, aliquots from the diluted mix were removed and their beads
flash-collected in a manifold filter. The amount of radiolabeled
protein bound to beads was quantified by liquid scintillation.
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The inclusion of unlabeled proteins as competitors in the molecular dissociation assays played an important role. Excess unlabeled Kaps prevented rebinding of radiolabeled Kaps to sites in immobilized proteins that become available when radiolabeled Kaps dissociate following the dilution step. The amount of competitor needed to prevent significant rebinding of radiolabeled Kaps to immobilized proteins in each case was determined experimentally by conducting dissociation assays in the presence of increasing concentrations of unlabeled competitor until the apparent rate of dissociation showed no further change (Fig. 4).
The dissociation of radiolabeled Kap60p from immobilized NLS-cargo was
slow (koff = 1.8 × 103
s1) (Table I) with an apparent t1/2 of
the complex of 394 s in the presence of saturating amounts of
unlabeled competitor Kap60p (200 nM) (Fig. 4A).
Increasing the concentration of competitor Kap60p from 200 nM to 1.6 µM caused no further increase in
the apparent rate of dissociation of radiolabeled Kap60p (Fig.
4A and data not shown). Therefore, 200 nM
unlabeled Kap60p was sufficient to prevent significant rebinding of
radiolabeled Kap60p to immobilized NLS-cargo. Under such conditions of
no rebinding, the rate measured for Kap60p dissociation from NLS-cargo
is accurate.
Kap95p reduced the rate of dissociation of Kap60p from NLS-cargo (Fig.
4B). In the presence of Kap95p, dissociation of radiolabeled Kap60p from immobilized NLS-cargo occurred 10 times more slowly (koff = 1.6 × 104
s1) than in the absence of Kap95p
(koff = 1.8 × 103
s1), with a half-life of the complex of > 1 h (Fig.
4B and Table I). Inclusion of 200 or 800 nM
unlabeled competitor Kap60p·Kap95p produced similar rates of
dissociation, indicating that significant rebinding of radiolabeled
Kap60p had been prevented. Thus, the rate measured for
Kap60p·Kap95p dissociation from NLS-cargo is accurate.
Radiolabeled Kap95p dissociated very slowly from immobilized
Kap60p·NLS-cargo (koff = 1.6 × 104 s1) with a half-life of 73 min (Fig.
4C and Table I). Increasing concentrations of competitor
Kap95p up to 800 nM produced no further increase in the
rate of dissociation of radiolabeled Kap95p from Kap60p·NLS-cargo,
indicating that significant rebinding of radiolabeled Kap95p was
prevented and that the rate of dissociation measured is accurate. In
the absence of NLS-cargo, Kap95p dissociated from immobilized Kap60p
twice as fast as when NLS-cargo was present (koff = 3.2 × 104
s1) with a half-life of the complex of 36 min (Fig.
4D and Table I). Increasing the concentration of unlabeled
competitor Kap95p up to 800 nM produced no further increase
in the apparent rate of dissociation of radiolabeled Kap95p from
Kap60p. Thus the measured rate of Kap95p dissociation from
Kap60p·NLS-cargo is also accurate.
Surprisingly, Kap95p·Kap60p·NLS-cargo complexes and Kap95p·Kap60p
heterodimers dissociated very rapidly from Nup1pN with half-lives of 21 s (Fig. 4, E and F; Table I). Rapid
dissociation occurred despite the very high affinity of Kap95p·Kap60p
for Nup1pN in the presence and absence of NLS-cargo
(KD = 400 and 51 pM, respectively)
(Fig. 3, D and E). Increasing the concentration of unlabeled competitors revealed an even faster dissociation of the
Kap·Nup complexes, at rates beyond the limit of detection of our
assay (data not shown). Thus, the reported 21 s half-lives for the
interactions of Kap95p·Kap60p·NLS-cargo and Kap95p·Kap60p with
Nup1pN represent the slowest dissociation rate exhibited by these
complexes. Similar experiments using immobilized FG regions of Nup100p
and Nup116p revealed even faster dissociation of Kap95p·Kap60p from
those FG Nups (data not shown).
Accelerating Disassembly of Long-lived Nuclear Import
Intermediates--
Among the nuclear import intermediates
reconstituted and characterized above, only the Kap·Nup complexes
appear to spontaneously disassemble fast enough
(t1/2 21 s) to serve as intermediates in a
rapid nuclear import mechanism. In contrast, the dissociation of
NLS-cargo from Kap60p·Kap95p (t1/2 = 73 min) and
the dissociation of Kap95p from Kap60p·NLS-cargo
(t1/2 = 73 min) were extremely slow and represent
potentially rate-limiting steps for nuclear import in vivo.
Factors may exist within the NPC or nucleoplasm that accelerate the
dissociation of these long-lived complexes.
Gsp1p may function to accelerate molecular dissociation, as Gsp1p-GTP
binding to Kap95p disrupts its association with Kap60p and with
Kap60p·NLS-cargo complexes (17).
Structural studies suggest that Ran-GTP (Gsp1p-GTP) may cause local
conformational changes in importin (Kap95p) that progressively
loosen importin 
(Kap60p) and lead to its release (33). However,
direct evidence of such a mechanism has not been obtained. Previous
studies did not distinguish between two possible mechanisms of
Gsp1p-GTP function (see Fig. 5A): (i) a passive mechanism in
which Gsp1p-GTP must wait for Kap95p to spontaneously dissociate from
Kap60p before binding Kap95p (thus preventing its re-association with
Kap60p); or (ii) an active mechanism in which Gsp1p-GTP binds Kap95p in Kap95p·Kap60p complexes forming a transient trimeric intermediate that triggers rapid release of Kap60p.
|
We tested whether Gsp1p-GTP could accelerate the rate of dissociation
of Kap95p from Kap60p in the presence and absence of NLS-cargo (Fig. 5,
B and C). The dissociation of radiolabeled Kap95p
from immobilized Kap60p or Kap60p·NLS-cargo complex was monitored in
the presence of increasing concentrations of Gsp1p-GTP as a possible
effector or in the presence of excess unlabeled competitor Kap95p as a
control. The amount of competitor needed to block rebinding of
radiolabeled Kap95p was determined experimentally in Fig. 4. We
expected to find either (i) no increase in rate of dissociation after
addition of Gsp1p-GTP, consistent with a passive mechanism of Gsp1p
function (Fig. 5A), or (ii) an increase in the rate of
dissociation after addition of Gsp1p-GTP, consistent with an active
mechanism for Gsp1p function (Fig. 5A). 200 nM Gsp1p-GTP stimulated 447-fold the apparent rate of dissociation of
Kap95p from Kap60p·NLS-cargo complexes relative to the intrinsic rate
of dissociation of this complex in the absence of Gsp1p-GTP (Fig.
5B, compare curves 1 and 3; Table
II). In the absence of NLS-cargo, 50 nM Gsp1p-GTP stimulated the rate of dissociation of Kap95p
from Kap60p by 65-fold (Fig. 5C and Table II). Greater concentrations of Gsp1p-GTP allowed radiolabeled Kap95p to dissociate even faster from immobilized Kap60p·NLS-cargo or Kap60p, at rates that were too fast to measure with our assay. The data are consistent with an active mechanism of Gsp1p function in which Gsp1p-GTP binds to
Kap95p in a Kap95p·Kap60p·NLS-cargo complex (or in a Kap95p·Kap60p complex) and triggers an allosteric mechanism in Kap95p
that causes rapid release of Kap60p (and its bound NLS-cargo) (17, 33).
The concentration of Gsp1p-GTP in the yeast nucleoplasm is estimated at
1-10 µM (14). Thus, it is expected that Gsp1p-GTP will
bind Kap95p and trigger the release of Kap60p·NLS-cargo at a rate
much faster in vivo than that measured here in
vitro.
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Following the Gsp1p-GTP-mediated dissociation of Kap60p·NLS-cargo from Kap95p, the remaining Kap60p·NLS-cargo complex is stable and dissociates with a half-life of 394 s (Fig. 4A and Table I). It has been suggested that Nup2p promotes dissociation of NLS-cargo from Kap60p (12) and that Nup1p promotes dissociation of NLS-cargo from Kap95p·Kap60p heterodimers (17), but the distinctions between passive or active mechanisms of function have not been established experimentally. Cse1p, the exportin for Kap60p, may also promote dissociation of NLS-cargo from Kap60p because the binding of Cse1p and NLS-cargo to Kap60p is mutually exclusive (6). We therefore tested whether Cse1p, Nup1p, or Nup2p could accelerate the rate of dissociation of NLS-cargo from Kap60p.
First, we tested whether Cse1p-Gsp1GTP could accelerate release of
NLS-cargo from Kap60p (Fig.
6A). The dissociation of
radiolabeled Kap60p from immobilized NLS-cargo was monitored in the
presence of increasing concentrations of Cse1p·Gsp1p-GTP as a
possible effector or in the presence of excess unlabeled Kap60p as a
control. The amount of competitor Kap60p needed to block significant
rebinding of radiolabeled Kap60p was determined experimentally in Fig.
4A. Gsp1p-GTP is a co-factor for Cse1p function (6, 25) and
does not bind Kap60p. 800 nM Cse1p·Gsp1p-GTP increased by 39-fold the rate of dissociation of Kap60p from NLS-cargo (from
koff = 1.8 × 103
s1 to 6.9 × 102
s1; or t1/2 = 394 to 10 s) (Fig. 6A, compare curves 1 and 3; Table II).
It is possible that higher concentrations of Cse1p·Gsp1p-GTP
accelerate further the rate of dissociation of Kap60p from NLS-cargo,
but our assay could not monitor faster rates. The data obtained are
consistent with an active mechanism of Cse1p function whereby Cse1p (in
a Cse1p·Gsp1p-GTP complex) binds Kap60p (in a Kap60p·NLS-cargo
complex) to form a transient tetrameric intermediate that triggers an
allosteric mechanism in Kap60p, which causes rapid release of the
NLS-cargo (Fig. 6A, diagram). In this role, the exportin
Cse1p functions as a nuclear import factor that dissociates NLS-cargo
from the importin Kap60p.
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The presence of both Cse1p and Gsp1p-GTP was required to accelerate the release of NLS-cargo from Kap60p (Fig. 6B). In the presence of either 200 nM Cse1p or 200 nM Gsp1p-GTP alone, the amount of radiolabeled Kap60p that remained bound to immobilized NLS-cargo after 5 min was unaffected in comparison with a control incubation containing 200 nM unlabeled competitor Kap60p. This indicates that neither Cse1p nor Gsp1p-GTP alone could accelerate Kap60p dissociation from the NLS-cargo. However, when 200 nM Cse1p and 200 nM Gsp1p-GTP were combined, nearly all of the bound Kap60p dissociated from the NLS-cargo within 5 min (Fig. 6B). This demonstrates cooperation between Cse1p and Gsp1p in accelerating release of NLS-cargo from Kap60p, which is consistent with the finding that Cse1p binds Kap60p in the presence of Gsp1p-GTP (6, 25).
We next tested whether the nucleoporins Nup1p and Nup2p could
accelerate disassembly of Kap60p·NLS-cargo complexes (Fig.
7). Dissociation of radiolabeled Kap60p
from immobilized NLS-cargo was monitored as described above, in the
presence of Nup1pN or Nup2p as possible effectors or in the presence
of competitor unlabeled Kap60p as a control. The concentration of
competitor Kap60p sufficient to prevent significant rebinding of
radiolabeled Kap60p was determined experimentally in Fig.
4A. 800 nM Nup2p accelerated 22-fold the
disassembly of Kap60p·NLS-cargo complexes relative to the intrinsic
rate of Kap60p·NLS-cargo dissociation (from
koff = 1.8 × 103
s1 to 3.9 × 102
s1; or t1/2 = 394 to 18 s) (Fig. 7,
compare curves 1 and 5; Table II). In comparison,
the concentration of Nup2p in the nuclear basket structure of the NPC
is estimated to be ~10 µM based on the approximate
number (~16) of Nup2p molecules/NPC (15) and the volume occupied by
the nuclear basket structure where it resides. Together the data
suggest that Nup2p binds Kap60p in Kap60p·NLS-cargo complexes,
forming a trimeric intermediate that triggers an allosteric mechanism
in Kap60p, which causes rapid release of NLS-cargo (Fig. 7, diagram).
Binding of Nup2p to Kap60p is a prerequisite for the observed effect,
as a mutant of Nup2p that does not bind Kap60p does not stimulate
release of NLS-cargo from Kap60p (data not shown). Nup1pN also bound Kap60p directly (Fig. 2C), but it did not stimulate release
of NLS-cargo from Kap60p. The addition of Nup1pN at concentrations ranging from 40 to 800 nM caused no increase in the rate of
dissociation of Kap60p·NLS-cargo complexes (Fig. 7 and data not
shown).
|
Finally, we tested whether Nup1p and Nup2p could increase the rate of
dissociation of NLS-cargo from Kap60p·Kap95p heterodimers (Fig.
8), as opposed to release of NLS-cargo
from Kap60p monomers. Dissociation of Kap60p·Kap95p from an
immobilized NLS-cargo was monitored in the presence of Nup1pN or
Nup2p as possible effectors or in the presence of excess unlabeled
competitor Kap60p·Kap95p as control. The amount of competitor
Kap60p·Kap95p sufficient to prevent rebinding of radiolabeled
Kap60p·Kap95p to immobilized NLS-cargo was determined experimentally
in Fig. 4B. 40 nM Nup1pN caused a 16-fold
increase in the dissociation rate of Kap95p·Kap60p from immobilized
NLS-cargo (from koff = 1.6 × 104 s1 to 2.5 × 103
s1; or t1/2 = 4380 to 276 s)
(Fig. 8 and Table II). Likewise, 40 nM Nup2p caused a
19-fold increase in the rate of dissociation of Kap95p·Kap60p from
the immobilized NLS-cargo (from koff = 1.6 × 104 s1 to 3 × 103 s1; or t1/2 = 4380 to 228 s (Fig. 8 and Table II). Higher concentrations of Nup2p or
Nup1pN did not further stimulate the rate of Kap95p·Kap60p
dissociation from the NLS-cargo (data not shown). The effect of
Nup1pN and Nup2p on Kap95p· Kap60p·NLS-cargo complexes may
involve a reaction in which Nup1pN and Nup2p bind directly to
Kap95p, Kap60p, or both, forming a tetrameric intermediate that changes
the structure of Kap60p to weaken its interaction with the NLS-cargo
(Fig. 8, diagram). The ability to exert an allosteric
effect on Kap60p may be unique to Nup1p and Nup2p, as other FG Nups
such as Nup100p and Nup116p cannot accelerate the dissociation of
NLS-cargo from Kap60p·Kap95p even when added at concentrations well
above the affinity of Kap95p·Kap60p·NLS-cargo toward these FG Nups
(data not shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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We describe several molecular mechanisms by which slow, potentially rate-limiting steps in the Kap95p·Kap60p-mediated nuclear import pathway may be overcome in S. cerevisiae. We find that resident proteins of the nuclear basket structure of the NPC (namely, the nucleoporins Nup1p and Nup2p, the exportin Cse1p, and the GTPase Gsp1p in its GTP-bound form) accelerate the stepwise disassembly of Kap95p·Kap60p·NLS-cargo complexes by triggering allosteric mechanisms in Kap95p and/or Kap60p that cause the rapid release of a bound protein. Therefore, Nup1p, Nup2p, Cse1p, and Gsp1p-GTP function as karyopherin release factors (or KaRFs). We hypothesize that these reactions occur in vivo immediately upon arrival of the Kap95p·Kap60p·NLS-cargo import complex to the nucleoplasmic side of the NPC, as depicted in Fig. 1.
The Gsp1p-stimulated release of Kap60p from Kap95p via binding to
Kap95p (Fig. 5) likely reflects a general mechanism of action of Gsp1p
on karyopherin orthologues dedicated to nuclear import (importin
s). Indeed, Gsp1p-GTP also accelerates the release of Nab2p (an
NLS-cargo) from its importin Kap104p (another importin orthologue)
(data not shown). Thus, Gsp1p likely functions as a generic KaRF for
importin s. In contrast, the Nup2p- and Cse1p-stimulated release of
NLS-cargo from Kap60p seems specific for this karyopherin/importin orthologue. Thus, Nup2p and Cse1p function as karyopherin -specific KaRFs.
The high affinity of interaction between Kap60p and NLS-cargo in the
presence and absence of Kap95p (KD 149 pM and KD = 2.8 nM,
respectively), between Kap60p and Kap95p (KD 153 pM), and between Nup1pN and Kap95p·Kap60p with or
without NLS-cargo (KD = 400 pM and
KD 51 pM, respectively) (Fig. 3)
predict long interaction times assuming typical rates of molecular
association (kon = 107-108
M1s1) (32). In fact, the rate
of disassembly of Kap60p·NLS-cargo complexes
(koff = 1.8 × 103
s1), Kap95p·Kap60p complexes
(koff = 3.2 × 104
s1), and Kap95p·Kap60p·NLS-complexes
(koff = 1.6 × 104
s1) is slow, with half-lives of minutes to hours as
expected (Fig. 4 and Table I). Clearly, these reactions occur too
slowly on their own to support rapid growth in S. cerevisiae, which divide every 2 h. In contrast the
Kap95p·Kap60p·NLS-cargo complex and the Kap95p·Kap60p heterodimer
associate and dissociate very rapidly from Nup1p (Table I) and other FG
Nups such as Nup100p and Nup116p (data not shown). Such fast rates of
molecular interaction are more compatible with fast transport across
the NPC.
The long interaction times between Kap95p·Kap60p and NLS-cargo (mean interaction time of 104 min) and between NLS-cargo·Kap60p and Kap95p (104 min) are ideal to ensure karyopherin-cargo complex integrity during transport through the NPC. However, they also present a problem, as spontaneous disassembly of the Kap95p·Kap60p·NLS-cargo complex (Fig. 4, Table I) is too slow as a mechanism for rapid cargo release at the nucleoplasm. Natural selection may have favored the increased stability offered by high affinity interactions while overcoming the disadvantage of slow dissociation by placing "accelerator" molecules at strategic locations within the NPC, such as the nuclear basket structure.
Gsp1p-GTP accelerates the disassembly of
Kap95p· Kap60p·NLS-cargo complexes by increasing by 447-fold
the rate of dissociation of Kap95p from Kap60p·NLS-cargo (from
koff = 1.6 × 104
s1 to 7.7 × 102
s1) (Fig. 5 and Table II). Gsp1p-GTP also accelerates
disassembly of Kap95p·Kap60p heterodimers by increasing 65-fold
the rate of dissociation of Kap95p from Kap60p (from
koff = 1.6 × 104
s1 to 2.1 × 103
s1) (Fig. 5, Table II). Gsp1p-GTP likely concentrates at
the nuclear basket structure of the NPC because it binds directly to
the nuclear basket nucleoporins Nup60p and Nup2p (14). Also, the
Gsp1p-specific guanine exchange factor Prp20p binds directly to Nup60p
and Nup2p (14). This implies that Gsp1p-GTP is generated at a site of action in the nuclear basket structure. Altogether these data suggest
that Gsp1p-GTP disassembles Kap95p·Kap60p heterodimers (with or
without cargo) at the nuclear basket structure of the NPC by triggering
an allosteric change in Kap95p structure that causes rapid release of
Kap60p. This suggestion is supported by structural studies of similar
complexes in crystals, which show partially overlapping binding sites
for Ran-GTP (Gsp1p-GTP) and importin (Kap60p) on importin
(Kap95p) (33).
The Gsp1p-GTP-accelerated release of Kap60p from Kap95p (Fig. 5C) is the reverse of the reported reaction in which Kap60p releases Gsp1p-GTP from Kap95p (34). We speculate that in vivo the equilibrium of this reaction is determined by the cellular compartment in which it takes place. In the cytoplasm, Kap60p is expected to be present in molar excess over free Gsp1p-GTP, thus favoring removal of Gsp1p-GTP from Kap95p and formation of the Kap95p·Kap60p heterodimer. In the nucleus, Gsp1p-GTP is likely more abundant than free Kap60p and may therefore drive the disassembly of the Kap95p·Kap60p heterodimer (Figs. 1 and 5).
Cse1p is the exportin for Kap60p in S. cerevisiae (6, 24,
25). In complex with Gsp1p-GTP, Cse1p also functions as a nuclear
import factor because it accelerates disassembly of the Kap60p·NLS-cargo complex by increasing 39-fold the rate of
dissociation of NLS-cargo from Kap60p (from koff = 1.8 × 103 s1 to 6.9 × 102 s1) (Fig. 6 and Table II). Cse1p
concentrates at the nuclear basket structure of the NPC (12), and there
it may perform its function as a Kap60p-specific KaRF. Our data suggest
that Cse1p in complex with Gsp1p-GTP binds Kap60p in Kap60p·NLS-cargo
complexes and triggers an allosteric mechanism in Kap60p that causes
rapid release of the NLS-cargo (Fig. 6A,
diagram). This mechanism may employ the Kap60p intrasteric
autoregulatory sequence (IBB domain), which resembles an NLS (31).
Nup2p is localized to the nuclear basket of the NPC (11, 12, 15), binds
Kap60p with KD = 0.3 nM (data not
shown), and accelerates the disassembly of the NLS-cargo·Kap60p
complex (Fig. 7) by increasing more than 22-fold the rate of
dissociation of NLS-cargo from Kap60p (from koff = 1.8 × 103 s1 to 3.9 × 102 s1) (Fig. 7 and Table II). The nuclear
basket is an ideal location for Nup2p to encounter and disassemble
Kap60p·NLS-cargo complexes that dissociate from Kap95p after
Gsp1p-GTP action (see Figs. 1 and 5). Our data suggest that Nup2p binds
Kap60p in Kap60p·NLS-cargo complexes, forming a trimeric intermediate
that triggers an allosteric change in Kap60p structure, which causes
rapid release of the NLS-cargo molecule (Fig. 7). This mechanism may
employ the Kap60p intrasteric autoregulatory sequence (34). Because
Nup2p is not essential for the survival of S. cerevisiae
(35), its observed effect in disassembling Kap60p·NLS-cargo complexes
may serve as a backup or replacement for Cse1p·Gsp1p-GTP during times
of reduced concentrations of nuclear Gsp1p-GTP. Under such conditions,
Nup2p dissociates from Nup60p at the NPC and becomes mobile throughout the cell (14, 15), perhaps to function as a "disaggregase" separating Kap60p from NLS-cargoes. The KaRF activity of Cse1p and
Nup2p on Kap60p·NLS-cargo complexes may not be required in cases
where the Kap60p·NLS-cargo complex dissociates rapidly on its own.
Nup1p and Nup2p accelerate the disassembly of import complexes by
increasing 16-19-fold the rate of dissociation of NLS-cargo from
Kap60p·Kap95p heterodimers (from koff = 1.6 × 104 s1 to 2.5 × 103 s1) (Fig. 8 and Table II). This
reaction is orders of magnitude slower than the Nup2p- or
Cse1p·Gsp1p-GTP-mediated dissociation of NLS-cargo from Kap60p
monomers (Table II), but it is unique in that it uses the
Kap60p·Kap95p heterodimer rather than Kap60p monomers as a substrate,
and it does not require Gsp1p-GTP action. This mechanism may be most
important to yeast when levels of nuclear Gsp1p-GTP are low due to
starvation or stress. Because Kap95p and Kap60p bind independently to
Nup1pN (Fig. 2C) and Nup2p (12, 16), we cannot
distinguish whether Nup1pN and Nup2p trigger an allosteric change in
Kap60p structure, Kap95p structure, or both, that results in release of
NLS-cargo from Kap60p. Interestingly, the average time needed for
Nup1pN to trigger NLS-cargo release from Kap95p·Kap60p
(t1/2 = 276 s) is longer than the average time
a Kap95p·Kap60p·NLS-cargo complex spends bound to Nup1pN
(t1/2 21 s), which implies that a
Kap95p·Kap60p· NLS-cargo complex can interact with Nup1pN
without releasing the NLS-cargo. This explains the presence of
NLS-cargo bound to Kap60p·Kap95p·Nup1pN complexes in
Fig. 2C.
The dissociation of Kap·cargo complexes from Nup1pN (Table I) and
other FG Nups is fast. Among all of the Kap·Nup interactions tested
the slowest dissociation was the dissociation of Kap95p·Kap60p heterodimers from Nup1pN (t1/2 21 s)
(Fig. 4E and Table I). Even in that case the dissociation occurs orders of magnitude faster than the slow intrinsic rate of
dissociation of NLS-cargo from Kap60p·Kap95p (t1/2 = 73 min), of NLS-cargo from Kap60p (t1/2 = 6-7
min), of Kap60p from Kap95p (t1/2 = 36 min), and of
Kap95p from Kap60p·NLS-cargo (t1/2 = 73 min)
(Table I). This fast spontaneous dissociation of Kap95p·Kap60p from
nucleoporins may occur rapidly enough to support a stochastic nuclear
import mechanism. In such mechanism, karyopherins translocate across
the NPC via facilitated diffusion involving a series of fast
association and dissociation events with multiple FG Nups (17) (Fig.
1). Possibly, Gsp1p-GTP or additional proteins assist this stochastic
mechanism by accelerating the rate of dissociation of karyopherins from
nucleoporins. However, our current assay cannot measure dissociation
rates on the time scale necessary to test this hypothesis.
The association rate (kon) of Kap95p·Kap60p
with Nup1p was calculated using the equation kon = koff/KD, the measured affinity of Kap95p·Kap60p toward Nup1pN (KD 51 pM), and the measured rate of dissociation of the
complex (koff 3.3 × 102
s1) (Table I). We find that Kap95p·Kap60p binds
Nup1pN at a very fast rate (kon 6.5 × 108 M1s1). This
rate
