From the § Department of Biochemistry, School of
Medicine and the Graduate Program in Biochemistry, Cell
and Developmental Biology, Emory University, Atlanta, Georgia 30322
Received for publication, October 25, 2002, and in revised form, December 10, 2002
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ABSTRACT |
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Proteins that contain a classical nuclear
localization signal (NLS) are recognized in the cytoplasm by a
heterodimeric import receptor composed of importin/karyopherin In eukaryotes, the nuclear envelope provides an essential barrier
that separates the nuclear genome from the intermediary metabolism,
signaling systems, and translation machinery of the cytoplasm.
Selective bi-directional transport of macromolecules across this
nuclear envelope regulates critical cellular processes such as gene
expression (1, 2). All nucleocytoplasmic transport of macromolecules
occurs through large proteinaceous structures, called nuclear pore
complexes (NPC),1 that
perforate the nuclear envelope (3, 4). These macromolecular cargoes are
specifically targeted to and transported through NPCs by a family of
soluble nuclear transport receptors (5, 6).
The small GTPase, Ran, governs the interactions between the nuclear
transport receptors and macromolecular cargoes (5, 7). Import receptors
bind cargo in the absence of RanGTP, whereas export receptors bind
cargo in a trimeric complex with RanGTP (5, 8). This mode of regulation
requires an asymmetric distribution of RanGTP, with more RanGTP in the
nucleus than in the cytoplasm. To achieve this asymmetry, the Ran
regulatory proteins are compartmentalized with the GTPase
activating protein (RanGAP), which generates
RanGDP, in the cytoplasm (9) and the guanine nucleotide exchange factor (RCC1), which generates RanGTP, in the nucleus (10).
The best-characterized nuclear import process occurs via receptor
recognition of a classical nuclear localization signal (NLS). This
classical NLS is typified by a cluster of basic amino acids (monopartite) or two clusters of basic amino acids separated by a
10-12 amino acid linker (bipartite) (11, 12). A heterodimeric import
receptor, composed of importins Dissociation of the NLS-cargo/importin The convergence of data from structural analyses and in
vitro binding studies provides insight into how importin To analyze the in vivo requirement for of the
auto-inhibitory activity of importin Strains, Plasmids, and Chemicals--
All chemicals were
obtained from Sigma or USBiological unless otherwise noted. All
DNA manipulations were performed according to standard methods (29),
and all media was prepared by standard procedures (30). All yeast
strains and plasmids used in this study are described in Table
I. A complete deletion of the
SRP1 open reading frame was created using a standard
PCR-based strategy (31) in the wild-type diploid ACY247 to create the
haploid Generation of Importin In Vivo Functional Analysis--
The in vivo function
of each of the importin Immunoblot Analysis--
Immunoblot analysis was performed
essentially as described previously (34). Briefly, cultures were grown
to log phase in appropriate media at 30 °C. Cells were harvested by
centrifugation and washed twice in water and once in PBSMT (100 mM KH2PO4, pH 7.0, 15 mM (NH4)2SO4, 75 mM KOH, 5 mM MgCl2, 0.5% Triton
X-100). Cells were subsequently lysed in PBSMT with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 3 µg/ml each of
aprotinin, leupeptin, chymostatin, and pepstatin) by glass bead lysis.
Equal amounts of total protein (generally 10 µg) were resolved by
SDS-PAGE and immunoblotted with monoclonal anti-myc antibody (1:2000
dilution; Oncogene), polyclonal anti-GFP antibody (1:10,000 dilution)
(35), anti-GST (1:5000; Santa Cruz Biotechnology), anti-His (1:5000; Santa Cruz Biotechnology), polyclonal anti-Cse1p (1:5000) (36), or
polyclonal anti-importin Expression and Purification of Recombinant Proteins--
Assays
were performed with purified recombinant S. cerevisiae
proteins Srp1p (importin Fluorescence Depolarization Assay--
Fluorescence anisotropy
measurements were carried out using an ISS PC1 fluorometer fitted with
polarization filters. The dissociation constants for the binding of
SV40 NLS-GFP and IBB-GFP to importin Solid Phase Binding Assay--
As previously described (26),
importin
GST-importin Microscopy--
Indirect immunofluorescence microscopy was
performed as described elsewhere (37) with the following conditions.
The myc antibody was used at 1:100 dilution, and the GFP antibody was used at 1:2000 for incubation with cells overnight at 4 °C. The Texas Red-labeled anti-mouse secondary antibody (Jackson
ImmunoResearch, 1:1000 dilution) was incubated with cells for 2 h
at room temperature. DNA was stained with DAPI (1 µg/ml). Samples
were viewed through a Texas Red-optimized filter (Chroma Technology)
using an Olympus BX60 epifluorescence microscope equipped with a
Photometrics Quantix digital camera.
Direct fluorescence microscopy was used to localize GFP fusion proteins
in live cells. For all experiments, cells were stained with DAPI to
visualize the DNA and confirm the location of the nucleus. The
localization of the fusion proteins was monitored by directly viewing
the GFP signal in living cells through a GFP-optimized filter as
described for indirect immunofluorescence microscopy.
Immunoprecipitation of Importin
Generation of Mutant Importin In Vivo Function of Importin
To confirm that each mutant protein is expressed at a similar level to
wild-type importin Identification of an Importin
To examine NLS binding and the auto-inhibitory function for
each importin
In contrast to the A1 and A2 mutants, the A3-importin
Our analysis of the auto-inhibition of A3-importin Interaction of Importin
For the competition binding assay, we first used fluorescence
anisotropy to measure the binding of wild-type IBB-GFP to importin Dissociation of the Importin Localization of A3-importin
To address this possibility, we re-examined the localization using an
assay that can more readily distinguish between importin Co-immunoprecipitation of Importin
Presumably, at least two major NLS-cargo/importin
Agarose-conjugated anti-myc antibody beads were used to
immunoprecipitate importin Interaction of A3-importin
As a complement to the in vitro binding experiments, we
examined importin
To examine whether the lack of A3-importin
When the ED mutations are combined with A3-importin This study demonstrates that the N-terminal IBB domain of importin
An in vivo analysis of the specific defect of A3-importin
The release of NLS-cargo from importin Although our data suggest that auto-inhibition is essential for
efficient NLS-cargo release from importin Although our study suggests that the essential role of importin We note that the absolute dissociation constants measured in our
studies differ from previously reported values (18, 24), particularly
in the binding of importin Although RanGTP binding to importin and
. The importin
subunit recognizes classical NLS sequences, and
the importin
subunit directs the complex to the nuclear pore.
Recent work shows that the N-terminal importin
binding (IBB) domain
of importin
regulates NLS-cargo binding in the absence of importin
in vitro. To analyze the in vivo functions
of the IBB domain, we created a series of mutants in the
Saccharomyces cerevisiae importin
protein. These
mutants dissect the two functions of the N-terminal IBB domain,
importin
binding and auto-inhibition. One of these importin
mutations, A3, decreases auto-inhibitory function without impacting
binding to importin
or the importin
export receptor, Cse1p. We
used this mutant to show that the auto-inhibitory function is essential
in vivo and to provide evidence that this
auto-inhibitory-defective importin
remains bound to NLS-cargo
within the nucleus. We propose a model where the auto-inhibitory
activity of importin
is required for NLS-cargo release and the
subsequent Cse1p-dependent recycling of importin
to the cytoplasm.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
(also known as karyopherin
and
), mediates the nuclear import of proteins that contain a
classical NLS (13-15). Over the last several years many studies have
led to a detailed model for the individual steps in the classic nuclear
transport cycle (5, 16): 1) importin
binds to the NLS-cargo to form
a trimeric import complex with importin
; 2) this NLS-cargo/importin
/importin
complex is targeted to the NPC by importin
; 3) the
complex then translocates into the nucleus where it encounters RanGTP;
4) upon binding RanGTP, importin
dissociates from
NLS-cargo/importin
; 5) NLS-cargo is released from importin
; and
6) once cargo is released, importin
is recycled to the cytoplasm by
its export receptor, Cse1p/CAS, in a trimeric complex with RanGTP.
Thus, the directionality and efficiency of nuclear import of NLS-cargo
is accomplished not only by the Ran GTPase cycle but also by an
additional series of protein-protein interactions, occurring in defined
locations, that result in changes in affinities of the transport
receptor for NLS-cargo.
/importin
import complex
is critical for delivery of NLS-cargo into the nucleus (7).
RanGTP-mediated dissociation of importin
from the trimeric import
complex (17) results in an NLS-cargo/importin
complex. Precisely
how NLS-cargo is then released from importin
is unknown; however, a
recent study shows that Cse1p and the nucleoporin, Nup2p, can
facilitate cargo release in vitro (18). The release of cargo
is important in the subsequent functions of both the cargo (7) and
importin
(19-21). For example, a recent study identified a
critical cargo, TPX2, of the importin
/
complex whose release
from importin
is essential for mitotic progression (22). Although
this study did not determine the mechanism of cargo release, it
highlighted the importance of understanding how cargoes are efficiently
dissociated from importin
within the nucleus to mediate their
cellular function. Furthermore, Cse1p, the export receptor for importin
, can only interact with importin
that is not bound to NLS-cargo
(19-21). Thus, for importin
to be recycled to the cytoplasm, it
must be dissociated from NLS-cargo within the nucleus. This ensures
that importin
is recycled to the cytoplasm only when it has
released NLS-cargo; and thereby provides a mechanism for assuring
uni-directional transport of NLS-cargo into the nucleus.
binds
to and regulates interactions with NLS-cargo (17, 23-26). Domain analysis has shown that importin
has an N-terminal domain that binds importin
(the importin
binding
domain or IBB), a central armadillo domain that constitutes the NLS
binding pocket, and a C-terminal region that appears to be important
for binding to its export receptor Cse1p/CAS (21, 27). Structural
studies have extended the knowledge obtained from this domain analysis. Conti et al. (25) solved the structure of truncated
Saccharomyces cerevisiae importin
(amino acid residues
89-530) in the presence of an NLS peptide revealing details of how the
central armadillo domain of importin
creates specific binding
pockets for NLS-cargo. More recently, the structure of full-length
mouse importin
, solved in the absence of NLS-cargo, showed that the
N-terminal IBB domain of importin
can form an intramolecular
interaction with the NLS-binding pocket of importin
(23). This
observation suggested that the IBB, in addition to mediating binding to
importin
(28), could have a second role as an auto-inhibitory
domain to regulate cargo binding (23). In support of this hypothesis, in vitro binding studies have shown that importin
lacking the proposed auto-inhibitory domain (
IBB-
), binds more
tightly to NLS-cargo than full-length importin
(26), and that this
auto-inhibition of full-length importin
binding to NLS-cargo is
relieved in the presence of importin
(17, 26). Further analysis of
the N-terminal IBB domain of importin
revealed a proposed internal NLS that could serve as an auto-inhibitory sequence to regulate NLS
binding through intramolecular competition for the NLS binding site
(23, 24). Taken together, these studies suggest a dual role for the
N-terminal domain of importin
in 1) binding to importin
and 2)
auto-inhibition of NLS-cargo binding.
, we created a series of
importin
mutants. These mutants dissect the two functions of the
N-terminal domain, importin
binding and auto-inhibition. One of
these mutants specifically decreases auto-inhibitory function without
impacting RanGTP-regulated binding to importin
. Despite normal
binding to both importin
and Cse1p, this importin
mutant is
unable to function in vivo. These experiments demonstrate
that the auto-inhibitory function of importin
is essential in
vivo. Our data support the hypothesis that the auto-inhibitory
mutant is deficient in NLS-cargo release from importin
and the
subsequent recycling of importin
to the cytoplasm. Thus, we propose
that the auto-inhibitory function of importin
is necessary for
efficient release of NLS-cargo into the nucleus via intramolecular
competition of the N-terminal IBB domain at the NLS-binding site.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SRP1 strain (ACY324) maintained by an SRP1
URA3 plasmid (pAC876). Importin
-GFP and A3-importin
-GFP
were integrated at the endogenous importin
locus of wild-type
(ACY192) and cse1-1 (ACY671) cells using a standard
integration strategy. This integration strategy creates a duplication
at the endogenous importin
locus such that both endogenous importin
and importin
-GFP are expressed from an SRP1
promoter.
Strains and plasmids used in this study
Mutants--
Amino acid substitutions
were introduced in the S. cerevisiae importin
(SRP1) coding region using PCR. For most mutants, mutagenesis was carried out on importin
in the bacterial expression vector pProEX-HTb (PerkinElmer Life Sciences). Mutations were subcloned
from the bacterial expression vector into the yeast expression plasmid
for in vivo studies. An 88-amino acid N-terminal deletion of
importin
,
IBB-
(pAC959), was created using the following
PCR-based strategy. An importin
plasmid (pAC876) was used as a
template to amplify the importin
promoter and
IBB-
open
reading frame starting at a PCR-introduced ATG start codon preceding
leucine residue 89. These products were further amplified by PCR to
form
IBB-
(amino acid residues 89-542) expressed from the
endogenous importin
promoter. The resulting PCR product was cloned
into the yeast centromeric (CEN) plasmid pRS315 (32). For
all constructs generated, the presence of each desired mutation and the
absence of any other mutations were confirmed by DNA sequencing.
mutants was tested using a plasmid shuffle
technique (33). Plasmids encoding each of the importin
mutant
proteins were individually transformed into SRP1 deletion
cells (ACY324) containing the URA3 SRP1 maintenance plasmid,
pAC876. Single transformants were grown in liquid culture to
saturation, serially diluted (1:10), and spotted on minimal media
plates lacking leucine as a control or on fluoro-orotic acid (5-FOA)
plates. The drug 5-FOA eliminates the URA3 plasmid-encoded wild-type importin
(pAC876) (33). Plates were incubated at 30 °C
for 3 days.
antibody (1:5000 dilution) (37).
), Kap95p (importin
), Cse1p, and Gsp1p
(Ran). Full-length His6-importin
(residues 1-542),
His6-
IBB-importin
(residues 89-530),
His6-IBB-GFP (importin
residues 1-88), His6-SV40 (SPKKKRKVEAS)-NLS-GFP, His6-Cse1p,
and His6-Ran were expressed in the Escherichia
coli strain BL21(DE3) and purified by nickel affinity
chromatography essentially as described previously (26, 38, 39).
Importin
was expressed and purified as described previously (26).
GST-importin
and GST-A3-importin
were expressed as described
elsewhere (40).
were measured essentially as
described previously (26, 38). Briefly, SV40 NLS-GFP or IBB-GFP was
diluted in PBS to the desired concentration (~20 nM) in a
total volume of 1 ml in a 1-cm quartz cuvette. Changes in the
anisotropy of the GFP fluorophore were monitored as aliquots of the
full-length wild-type or mutant importin
proteins were successively
added to the assay volume. Changes in anisotropy were used to calculate
the fraction of the GFP fluorophore bound, yielding a binding isotherm
for the reaction. The binding isotherm was then fit through nonlinear
regression to a simple binding equation to obtain dissociation
constants. All Kd values were calculated as detailed
at
http://www.biochem.emory.edu/Hodel/Research/BindingCurves/fitting_curves.htm. The dissociation constant for importin
binding to importin
was
measured using a competition assay (41) with IBB-GFP. The assay was
carried out as described above except changes in the anisotropy of
IBB-GFP were monitored in the presence of increasing amounts of
importin
. This yielded a dissociation constant for IBB-GFP binding
to importin
. To measure the binding of importin
to importin
, the binding of IBB-GFP was examined in the presence of three
different concentrations of full-length wild-type or mutant importin
. The Kd values for wild-type and mutant importin
proteins binding to importin
were determined by fitting the
resulting binding curves to an equation for the fraction of IBB-GFP
bound as a function of Kd for IBB,
Ki for the full-length importin
protein, the
total IBB-GFP concentration, the total importin
concentration, and
the total concentration of the full-length importin
protein.
Binding energies were calculated using
G = RT ln Kd, where RT =
0.59 kcal/mol.
was covalently coupled to epoxy-activated Sepharose beads
(Amersham Biosciences) at a concentration of 1 mg/ml. Approximately 0.2 mg/ml purified full-length wild-type or full-length mutant importin
or
IBB-
was incubated with 0.5 ml of importin
-coupled beads
in binding buffer (50 mM Tris-HCl, pH 7.8, 100 mM NaCl, 20 mM dithiothreitol) for 3 h at
4 °C. The beads were then washed five times with 1 ml of binding
buffer, and beads were either eluted with elution buffer (50 mM Tris-HCl, pH 7.8, 1 M NaCl) or incubated
with a RanGTP-RS as described previously (39). Briefly, recombinant Ran
was incubated with a RanGTP-RS (20 units of acetate kinase, 10 mM GTP, and 10 mM sodium acetyl phosphate) for
15 min on ice before addition to the beads at 4 °C for 2 h. The
supernatants from each reaction were subsequently saved as the eluant
fractions. The beads were washed twice with binding buffer before
elution with elution buffer. The unbound (2% of total unbound), bound (2% of total bound), and eluant (2% of total eluant) fractions were
resolved on a 10% SDS-PAGE gel and visualized by Coomassie Brilliant
Blue staining (42).
proteins were bound to
glutathione-Sepharose (Amersham Biosciences) and tested for interaction
with Cse1p essentially as described previously (40), with the following
modifications. The GST-importin
-conjugated beads were washed five
times with PBSMT before incubation with purified Cse1p (10 µg) and
Gsp1p (20 µg) in the presence or absence of the RanGTP-RS in a total volume of 500 µl. The bound (50% of total bound) fractions were resolved on a 8% SDS-PAGE gel and immunoblotted as described. For
quantification, immunoblots were analyzed using a fluorescence imager and Quantity One Software (Bio-Rad).
-myc--
Agarose-conjugated anti-myc antibody beads (9E-10 Santa
Cruz Biotechnology) were used to immunoprecipitate importin
-myc proteins. Wild-type cells (ACY192) expressing importin
-myc (pAC891) or A3-importin
-myc (pAC894) from the endogenous SRP1
promoter were transformed with galactose-inducible plasmids encoding
GFP (pAC1042) alone or a bipartite NLS
(KRTADGSEFESPKKKRKVE)-GFP (pAC1045) (38). Cells
were grown to early-log phase in synthetic media containing 2%
raffinose, induced with 2% galactose, and incubated at 30 °C for
3 h. Lysates were prepared as described for immunoblot analysis.
Six milligrams of total protein lysate was incubated with myc
antibody-coupled agarose beads (10 µl of volume of packed beads) for
5 h at 4 °C in the absence or presence of a RanGTP-RS. Beads
were washed twice in PBSMT and once in PBSM before elution with 30 µl
of sample buffer (125 mM Tris-HCl, pH 6.8, 250 mM dithiothreitol, 5% SDS, 0.25% bromphenol blue, 25% glycerol). The bound (50% of total bound) fractions were resolved on a
10% SDS-PAGE gel and immunoblotted as described. For quantification, immunoblots were analyzed using a fluorescence imager and
Quantity One Software (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Proteins--
Previous
structural and in vitro analyses identified an
auto-inhibitory sequence within the N-terminal IBB domain of importin
(23, 24, 26). The structural studies suggested that this auto-inhibitory domain interacts with the NLS binding pocket by mimicking an NLS sequence (23, 24). Alignment of the N-terminal domains
of importin
proteins from yeast, mouse, and human reveals three
clusters of conserved basic amino acids that could serve as
auto-inhibitory NLS sequences (Fig. 1).
To identify the amino acid residues that are required for the
auto-inhibitory function of importin
, we carried out an alanine
scan by individually substituting each of these basic clusters in yeast
importin
(Srp1p) to generate importin
mutants referred to as A1
(33RRRR36
AAAA), A2
(44RKAKR48
AAAAA), and A3
(54KRR56
AAA) (Fig. 1). The full-length
mouse importin
crystal structure showed that residues 44-54
(corresponding to yeast importin
residues 49-59) interact with the
larger NLS binding pocket (24). Although this suggests that our A3
mutant should target this intramolecular interaction, we completed a
broader, unbiased alanine scan and analyzed the function of each of the
mutant importin
proteins, A1, A2, and A3.
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Fig. 1.
Alignment of the N-terminal domains of
importin proteins. Amino acid sequence
alignment of yeast (sc) (residues 30-60), mouse
(mm), and human (hs) importin
proteins is
shown. The conserved clusters of basic residues that were mutated to
alanine to generate the A1, A2, and A3 mutants in S. cerevisiae importin
are indicated by the
boxes.
Mutants--
To test
the function of the mutant importin
proteins in vivo,
each mutant was transformed into yeast cells deleted for the endogenous
importin
gene (SRP1) and plasmid shuffle (see
"Experimental Procedures") was used to replace the functional
wild-type copy of SRP1 (Fig.
2A). This results in
SRP1 cells that express each of the mutant importin
proteins from their own promoter on a low copy centromeric plasmid as
the only copy of importin
. Controls demonstrate that a wild-type
importin
plasmid can rescue
SRP1 cells, whereas
neither a vector alone nor the N-terminally truncated importin
(
IBB-
) can functionally replace SRP1. Results shown in
Fig. 2A indicate that none of the importin
mutant
proteins, A1, A2, or A3, can function in vivo.
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Fig. 2.
Functional analysis of the importin
mutants in vivo.
A, the SRP1 deletion strain (ACY324) maintained
by a plasmid encoding wild-type importin
and expressing either
wild-type or mutant importin
proteins was spotted onto minimal
media (control) or 5-FOA plates as described under "Experimental
Procedures." All plates were incubated at 30 °C for 3 days.
B, levels of importin
expressed in wild-type cells
(ACY192) were examined by immunoblotting with an anti-myc antibody. Ten
micrograms of total yeast protein was loaded in each lane. The
positions of full-length importin
and
IBB-
are indicated by
the arrows.
, we analyzed their expression using a C-terminal
triple myc tag. Wild-type importin
-myc can functionally replace
endogenous importin
(data not shown). Immunoblotting of the
myc-tagged importin
proteins demonstrates that each of the mutant
proteins is expressed at approximately the same level as wild-type
importin
(Fig. 2B, compare lanes 3-6 with
lane 2). This suggests that none of the mutations in the
N-terminal IBB domain of importin
significantly affect the level of
the protein within the cell, but rather that these mutations perturb the function of the importin
proteins. Importin
undergoes a
series of defined protein-protein interactions during a nuclear transport cycle (16). Thus, to determine the basis for the loss of
function of each of the importin
mutants, we investigated the
interaction of the mutant importin
proteins with both NLS-cargo and
nuclear transport factors essential for importin
function.
Mutant with Decreased
Auto-inhibitory Function--
To examine both NLS binding and
auto-inhibitory function for each importin
protein, we used a
quantitative, fluorescence anisotropy, solution binding assay (26, 38).
Full-length wild-type importin
binds weakly to a classical SV40-NLS
due to the N-terminal auto-inhibitory IBB domain. However, when the
same experiment was carried out in the presence of a stoichiometric
amount of importin
, the N-terminal auto-inhibition of full-length
importin
was relieved and much tighter binding to the SV40-NLS was
observed. Thus, this assay actually examines three aspects of importin
function: binding to NLS-cargo (directly), auto-inhibitory function (directly, measured by the ability of full-length importin
to bind
NLS-cargo in the absence of importin
), and binding to
importin
(indirectly, based on the relief of auto-inhibition).
variant, we performed the fluorescence anisotropy assay using a monopartite SV40-NLS-cargo (26, 38). The assay was
carried out with each importin
protein in the presence and absence
of importin
(Fig. 3). Typical curves
for binding of SV40-NLS-GFP to wild-type importin
in the absence
(
) and presence (
) of importin
are shown in Fig.
3A. As described under "Experimental Procedures," these
curves were used to calculate Kd values for the
interaction between the NLS-cargo and importin
(Table II). The change in free energy
(
G) for the binding of each importin
protein to
NLS-cargo in the absence (
) and presence (
) of importin
is
shown in Fig. 3B. As previously demonstrated (24, 26),
wild-type importin
binds to SV40-NLS-GFP weakly
(Kd ~ 500 nM), but the affinity
increases ~30-fold (Kd ~ 18 nM) in
the presence of importin
. The A1-importin
and A2-importin
mutants show weak binding to SV40-NLS-GFP, comparable to wild-type importin
, suggesting that the N-terminal auto-inhibitory function is intact in these mutants. However, the binding to NLS is not enhanced
in the presence of importin
showing that there is no relief of
auto-inhibition. This observation implies that the A1-importin
and
A2-importin
mutants have decreased binding to importin
.
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Fig. 3.
Analysis of the auto-inhibitory function of
importin mutants. A, binding
of an NLS-GFP cargo to full-length importin
protein was measured by
anisotropy in the absence (
) or presence (
) of a stoichiometric
amount of importin
. The anisotropy is plotted versus the
concentration of importin
on a logarithmic scale. B,
binding of an NLS-GFP cargo to each full-length importin
protein
indicated was measured by fluorescence anisotropy in the absence (
)
or presence (
) of a stoichiometric amount of importin
. For each
importin
protein, the data were fit to an ideal binding curve to
yield a value for Kd. The calculated binding energy
(
G, kcal/mol) is shown on the left axis.
Standard deviations are indicated by error bars.
C, the N-terminal IBB domain of importin
(WT-IBB (
))
and A3-importin
(A3-IBB (
)) were fused to GFP, and their binding
to
IBB-
was measured by anisotropy. The anisotropy is plotted
versus the concentration of importin
on a linear
scale.
Binding of importin proteins to NLS-cargo
mutant binds
to SV40-NLS-GFP more tightly (Kd ~ 73 nM) than full-length wild-type importin
(Kd ~ 500 nM), an ~7-fold increase
in affinity. This suggests that A3-importin
has decreased auto-inhibitory function. When the assay is performed in the presence of importin
, the affinity of SV40-NLS-GFP for A3-importin
increases to approximately the same binding affinity
(Kd ~ 20 nM) as measured for wild-type
importin
(Kd ~ 18 nM), which
shows that the residual auto-inhibition can be relieved by importin
and suggests that the A3-importin
mutant retains normal binding to
importin
. In addition, these data show that the A3-importin
/
complex is able to bind NLS-cargo with approximately the same affinity
as the wild-type importin
/
complex. Similar results were
obtained when two different cargoes containing distinct NLS sequences
were used (data not shown).
suggests that
this mutant IBB domain has a decreased affinity for the NLS binding
pocket compared with a wild-type IBB domain. To directly examine this
interaction, we analyzed binding of the IBB domain to the NLS binding
pocket in trans. We compared the binding of wild-type and A3
IBB-GFP to
IBB-
using fluorescence anisotropy (Fig.
3C). Wild-type IBB-GFP binds to
IBB-
with a
Kd of ~14 ± 5 µM. Binding of
A3 IBB-GFP to
IBB-
was significantly less and was too weak to
accurately measure in this assay.
Proteins with Importin
--
The
interaction of importin
with importin
is essential for
targeting the import complex to nuclear pores (5). Therefore, to
evaluate the in vivo role of the auto-inhibitory function of importin
, it is critical to demonstrate that we have separated the
two functions of the N-terminal IBB domain of importin
(importin
binding and auto-inhibition). Although the auto-inhibitory binding assay suggests that A3-importin
retains wild-type binding to importin
, it does not measure this binding directly. To
quantitatively measure the binding of importin
to importin
we
utilized two assays. First, we used a competition assay where we
examined the ability of each full-length importin
protein to
compete with the IBB-GFP for binding to importin
. Second, we
performed a direct binding assay to measure binding of wild-type and A3
IBB-GFP to importin
.
.
This yielded a Kd of ~17 ± 5 nM
(Fig. 4). The competition experiment was
then carried out in the presence of three concentrations of each
importin
protein (wild-type, A1, A2, and A3) in competition with
IBB-GFP. This analysis yields equilibrium binding constants for the
interaction of wild-type and each of the mutant importin
proteins
with importin
(Table III).
Full-length importin
binds to importin
with a
Kd of ~0.6 µM. Both A1-importin
and A2-importin
show decreased affinity for importin
with decreases of ~15-fold and ~28-fold, respectively. In contrast, as
suggested by the NLS binding experiments, A3-importin
binds to
importin
with approximately the same affinity
(Kd ~ 0.4 µM) as wild-type importin
(Kd ~ 0.6 µM). To further confirm that the A3 mutation within the IBB domain does not affect the
interaction between importin
and importin
, we performed the
direct binding assay. This assay measures binding of wild-type and A3
IBB-GFP to importin
using fluorescence anisotropy. Binding curves
for IBB-GFP and A3 IBB-GFP are shown in Fig. 4. A3 IBB-GFP binds to
importin
with the same affinity (Kd ~ 20 ± 5 nM) as wild-type IBB-GFP (Kd ~ 17 ± 5 nM). These results indicate that the A3 mutant
of importin
has compromised auto-inhibitory function but binds to
importin
with wild-type affinity. Because this mutant dissects the
two functions of the IBB domain, we focused our subsequent experiments
on the A3 mutant to analyze the in vivo contribution of the
auto-inhibitory activity of importin
.
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Fig. 4.
Quantitative analysis of the interaction
between importin and A3-importin
. The N-terminal IBB domains of importin
(WT-IBB
(
)) and A3-importin
(A3-IBB (
)) were fused to GFP, and their
binding to importin
was measured by anisotropy. Data are plotted as
anisotropy versus the concentration of importin
on a
logarithmic scale.
Binding of importin proteins to importin
/Importin
Complex by
RanGTP--
To determine whether the A3-importin
can be
dissociated from importin
by RanGTP, we performed a bead
dissociation assay. We covalently attached purified recombinant
importin
to activated epoxy beads and then incubated the beads with
purified importin
to pre-form the importin
/
complex. We have
previously shown that full-length importin
specifically binds to
the importin
-coupled beads (26). Both wild-type and A3-importin
can bind to the importin
beads (Fig.
5A, lanes 2 and
4). Fig. 5B shows that both wild-type and
A3-importin
are released from importin
upon addition of an
RanGTP-regenerating system (RanGTP-RS) (39). Similar results were
obtained when the experiment was carried out using Ran loaded with 100 µM GTP
S in place of the RanGTP-RS to modulate the
nucleotide bound state of Ran (data not shown). RanGDP, which does not
bind with high affinity to importin
(43), did not dissociate any of
the pre-formed complexes (data not shown).
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Fig. 5.
Dissociation of the importin
/importin
complex by
RanGTP. A, recombinant importin
was bound to epoxy
beads and incubated with recombinant importin
to pre-form the
importin
/importin
complex as described under "Experimental
Procedures." Samples were resolved by SDS-PAGE and stained with
Coomassie Brilliant Blue. For each protein, U (unbound)
(lanes 1 and 3) and B (bound)
(lanes 2 and 4) fractions are shown.
B, the pre-formed importin
/importin
complexes were
incubated with a RanGTP-RS. The bound (B) (lanes
1 and 3) and eluant (E) (lanes 2 and 4) fractions were analyzed as described for
A. The positions of importin
, Ran, and importin
(which minimally leaches off the beads) are indicated by the
arrows.
--
Our in vitro
experiments demonstrated that the A3-importin
protein interacts
with importin
in a RanGTP-dependent manner that is
comparable to wild-type importin
. One prediction from these
experiments is that the A3 protein should be efficiently targeted to
the nucleus in vivo through a productive interaction with
importin
. Wild-type S. cerevisiae importin
protein
is localized throughout the cell with some accumulation at the nuclear rim and/or within the nucleus depending on the tag (37, 44). This
steady-state localization reflects a dynamic state where importin
enters the nucleus and is then exported by Cse1p (19, 20, 44). To
analyze the intracellular localization of importin
, we first
utilized C-terminal triple myc-tagged fusion proteins. These tagged
proteins were expressed from the importin
promoter on a centromeric
plasmid and localized by indirect immunofluorescence (Fig.
6A). Wild-type importin
-myc is localized throughout the cell but accumulates within the
nucleus (Fig. 6A, panel A). The A3-importin
-myc protein also accumulates within the nucleus (Fig.
6A, panel D). For comparison, the
localization of A2-importin
, which has compromised binding to
importin
, is also shown (Fig. 6A, panel G).
It is clear that, in contrast to wild-type and A3-importin
, the A2
protein does not accumulate within the nucleus. Although the
localization of wild-type and A3-importin
was very similar, we
consistently noticed less cytoplasmic signal for the A3 protein. This
raised the possibility that the A3 protein could have a more nuclear
steady-state localization than wild-type importin
.
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Fig. 6.
Localization of importin proteins. A, wild-type cells (ACY192) expressing
myc-tagged importin
plasmids were grown at 30 °C to log phase.
Cells were prepared for indirect immunofluorescence and stained with
anti-myc antibody to visualize importin
(panels A,
D, and G) and with DAPI to visualize DNA
(panels B, E, and H) as described
under "Experimental Procedures." Corresponding DIC images are shown
(panels C, F, and I). B,
integrated importin
-GFP or A3-importin
-GFP was viewed in
wild-type (ACY 192) (panels A and C) or cse1-1
(panels E and G) cells by direct fluorescence.
All cultures were grown at 30 °C. Corresponding DIC images are shown
(panels B, D, F, and H).
C, integrated A3-importin
-GFP cells were grown at
30 °C to log phase. Cells were prepared for indirect
immunofluorescence and stained with anti-GFP antibody to visualize
importin
(panel A) and with DAPI to visualize DNA
(panel B). The corresponding DIC image is shown (panel
C).
at the
nuclear rim and importin
within the nuclear interior (44). This
assay relies on visualization of an importin
-GFP fusion protein
that is expressed from the endogenous importin
promoter. We
integrated both wild-type and A3-importin
-GFP fusion proteins at
the endogenous importin
locus as described under "Experimental
Procedures." As previously reported (44), wild-type importin
accumulates at the nuclear rim in wild-type cells when visualized in
this manner (Fig. 6B, panel A). However, A3-importin
-GFP is localized within the nucleus (Fig. 6,
B (panel C) and C) suggesting that
this mutant protein is not efficiently recycled to the cytoplasm. The
localization of A3-importin
-GFP is similar to that of importin
-GFP in cse1-1 mutant cells where importin
is not
efficiently recycled to the cytoplasm (Fig. 6B, panel
E) (20, 36). A3-importin
-GFP also accumulates within the
nucleus of cse1-1 cells (Fig. 6B, panel
G).
and NLS-cargo--
Because
A3-importin
has decreased auto-inhibitory activity and a more
steady-state nuclear localization than wild-type importin
, it seems
likely that this mutant protein accumulates within the nucleus bound to
NLS-cargo. Thus, A3-importin
should bind to more NLS-cargo than
wild-type importin
in cell lysates. To test this prediction, we
performed a co-immunoprecipitation with cells expressing either
myc-tagged wild-type importin
or myc-tagged A3-importin
together with a bipartite NLS-GFP cargo. The bipartite NLS-GFP protein
is localized to the nucleus in vivo (data not shown).
complexes exist:
1) the trimeric import complex of NLS-cargo/importin
/importin
;
and 2) NLS-cargo/importin
, which may exist transiently after
importin
is released. Immunoprecipitation of importin
could
isolate both complexes; however, addition of RanGTP should dissociate
the trimeric complex, because RanGTP causes a conformational change in
importin
that results in importin
dissociation from importin
(17, 45). If A3-importin
is defective in NLS-cargo release from
importin
, it should bind more NLS-cargo in cell lysates than
wild-type importin
under the same conditions.
-myc from lysates (see "Experimental
Procedures"). The bound fractions were analyzed for
co-immunoprecipitation of the bipartite NLS-GFP reporter protein and
importin
. As a control, GFP alone was not co-immunoprecipitated
with either of the importin
proteins (data not shown). The
experiment was carried out in the presence of a RanGTP-RS to isolate
NLS-cargo/importin
complexes in the absence of importin
. The
RanGTP-RS dissociates importin
from the complex and decreases the
amount of NLS-GFP co-immunoprecipitated (compare lanes 1 and
2). This confirms that in the presence of RanGTP the primary
co-immunoprecipitated complex isolated is not the trimeric import
complex of NLS-cargo/importin
/importin
but a complex of
NLS-cargo/importin
. Thus, we compared the amount of NLS-GFP
co-immunoprecipitated with wild-type and A3-importin
under the same
conditions. Results of the experiment indicate that, in the presence of
RanGTP, ~3-fold more NLS-GFP co-immunoprecipitated with A3-importin
-myc than wild-type importin
-myc (Fig.
7A, compare lanes 2 and 3). Expression of the importin
-myc proteins and the
NLS-cargo protein was similar in each lysate (data not shown). Protein
bands were compared by fluorescence imaging under conditions
where none of the bands were saturated. For each sample the amount of
NLS-cargo present was calculated relative to the amount of importin
precipitated in that experiment.
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Fig. 7.
A3-importin is
defective in NLS-cargo release. A,
co-immunoprecipitation of importin
and NLS-cargo. Wild-type cells
(ACY192) expressing the bipartite NLS-GFP cargo protein (pAC1045) were
transformed with wild-type importin
-myc (pAC891) or A3-importin
-myc (pAC894). Cells were grown to early-log phase, and expression
of NLS-GFP was induced with galactose. Binding assays were carried out
as described under "Experimental Procedures." Wild-type importin
(lanes 1 and 2) and A3-importin
(lane 3) fractions are shown probed with anti-importin
antibody to detect importin
, anti-myc antibody to detect importin
, and anti-GFP antibody to detect the bound NLS-GFP reporter cargo.
Lanes are designated "
" or "+" to indicate whether binding
was carried out in the absence or presence of a RanGTP-RS.
B, in vitro interaction of importin
and
Cse1p. Wild-type and A3-importin
were expressed in E. coli as GST fusion proteins. The importin
GST fusion proteins
(~30 µg) were bound to glutathione beads and incubated with
purified His-tagged Cse1p (10 µg) and His-tagged Gsp1p (20 µg).
Lanes are designated "
" or "+" to indicate whether binding
was carried out in the absence or presence of a RanGTP-RS.
C, in vivo interaction of importin
and Cse1p.
Wild-type cells expressing importin
-GFP (panels A and
C), A3-importin
-GFP (panels E and
G), or A3-ED-importin
-GFP (panels I and
K) were transformed with either a 2µ plasmid expressing
Cse1p (pAC640) or a vector control (pAC8). Cells were grown to
early-log phase, and the GFP signal was viewed directly in living
cells. Corresponding DIC images are shown.
with Cse1p--
For importin
to
be functional in vivo, it must be efficiently recycled to
the cytoplasm by its export receptor, Cse1p/CAS. Formation of this
export complex requires both the direct interaction between importin
and Cse1p and the dissociation of NLS-cargo (19-21). To examine
the physical interaction of A3-importin
with Cse1p, we used a bead
binding assay. Both wild-type and A3-importin
were expressed in
E. coli as GST fusion proteins as described under
"Experimental Procedures." These proteins were bound to glutathione
beads and incubated with purified His6-tagged Cse1p in the
absence or presence of a RanGTP-RS. As shown in Fig. 7B, A3-importin
binds to Cse1p in a RanGTP-dependent
manner. Furthermore, in the presence of RanGTP, Cse1p binding to
A3-importin
is comparable to binding to wild-type importin
(Fig. 7B, compare lanes 2 and 3).
Protein bands were compared by immunoblotting and fluorescence imaging. Results shown are typical of three independent
experiments. In addition, both wild-type importin
and A3-importin
interacted with Cse1p to the same extent in a two-hybrid assay
(data not shown).
recycling by Cse1p in vivo. We took
advantage of the C-terminal GFP fusion protein, importin
-GFP,
expressed from the importin
promoter on a centromeric plasmid.
Previous work demonstrates that fusion of GFP to the C terminus of
wild-type importin
results in a protein that can function in
vivo when expressed as the only copy of importin
(44).
However, plasmid expression results in increased amounts of
importin
-GFP and thus a more nuclear localization than observed for
integrated importin
-GFP protein (as shown in Fig. 6B).
When expressed on a centromeric plasmid, both wild-type importin
-GFP and A3-importin
-GFP show primarily nuclear localization
(Fig. 7C, panels A and E).
Overexpression of Cse1p decreases the nuclear localization of wild-type
importin
-GFP presumably by facilitating export (44). Thus, cells
that overexpress Cse1p show a more diffuse (throughout the cell)
localization pattern for importin
-GFP. As previously reported, we
find that wild-type importin
-GFP is recycled to the cytoplasm and
diffusely localized throughout the cell when Cse1p is overexpressed on
a high copy plasmid (Fig. 7C, compare panels A
and C). This diffuse localization is observed in ~80% of
cells examined. In contrast, A3-importin
-GFP remains in the nucleus
even when Cse1p is overexpressed (Fig. 7C, compare panels E and G). There are two possible
explanations for why A3-importin
-GFP is not recycled to the
cytoplasm in cells that overexpress Cse1p. First, A3-importin
-GFP
may be bound to NLS-cargo and therefore unable to efficiently interact
with Cse1p. Several lines of experimentation have shown that importin
cannot interact simultaneously with NLS-cargo and Cse1p (19-21),
which presumably prevents recycling of importin
with NLS-cargo
still bound. Thus, overexpression of Cse1p should not cause
redistribution of a mutant importin
-GFP protein if it is still
bound to NLS-cargo. Second, the A3 mutation in importin
could
directly interfere with the physical interaction between importin
and Cse1p. However, our results shown in Fig. 7B demonstrate
that A3-importin
is still able to bind to Cse1p. This strongly
suggests that the lack of recycling of A3-importin
to the cytoplasm
by Cse1p is due to a tighter interaction between A3-importin
and
NLS-cargo in the nucleus.
recycling was in fact
due to bound NLS-cargo, we utilized a known importin
mutant,
ED-importin
(D203K/E402R), that has significantly reduced binding
to NLS-cargo (22). The interaction between S. cerevisiae ED-
IBB-
and SV40-NLS is too weak to detect in the anisotropy assay implying that the binding constant is at least in the millimolar range (data not shown). Because this mutant protein cannot bind to
NLS-cargo, it can be combined with the A3 mutation, to create an
A3-importin
mutant protein (A3-ED-importin
) that cannot bind
NLS-cargo. If this protein can be efficiently recycled to the
cytoplasm, it confirms that bound cargo prevents recycling of
A3-importin
. Alternatively, if the reason that A3-importin
is
not recycled to the cytoplasm is independent of binding to NLS-cargo,
then regardless of the ED mutation, A3-ED-importin
would not be
recycled and would remain in the nucleus.
to create
A3-ED-importin
-GFP, the steady localization of the double-mutant protein is within the nucleus (Fig. 7C, panel I)
indistinguishable from the localization of wild-type or A3-importin
-GFP. However, in contrast to the result obtained with A3-importin
-GFP, overexpression of Cse1p causes relocalization of
A3-ED-importin
-GFP to the cytoplasm in ~60% of cells (Fig.
7C, panel K). Immunoblotting demonstrates that
each of the importin
-GFP proteins is expressed at approximately the
same level as wild-type importin
-GFP and that cells transformed
with the CSE1 plasmid all have a similar increase in
expression of Cse1p (data not shown). Results of these experiments
suggest that A3-importin
accumulates in the nucleus bound to
NLS-cargo and thereby support our hypothesis that the auto-inhibitory
function of importin
is required for efficient NLS-cargo release in
the nucleus. In addition, these results show that A3-importin
can
interact with Cse1p in vivo when its ability to bind
NLS-cargo is abrogated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
has two essential functions in vivo. This domain is
known to bind importin
for targeting of the import complex to the nuclear pore (14, 15, 46). In addition, previous structural studies
demonstrated that the IBB domain contained an NLS-like sequence that
could compete directly with NLS binding to importin
through an
intramolecular interaction (23, 24). Investigations of the in
vitro behavior of importin
confirmed that the N-terminal domain inhibited binding of NLS-cargo to importin
(24, 26). This
auto-inhibitory function was relieved when importin
bound to
sequences of the N-terminal domain of importin
just upstream of the
NLS-like sequence (47). Here we create mutants of importin
that
dissect the two functions of the N-terminal domain, importin
binding and auto-inhibition. We demonstrate that mutations within the
NLS-like sequence in importin
(mutant A3) specifically decrease the
auto-inhibitory function of the N terminus without affecting binding to
importin
and provide evidence that this auto-inhibitory function is
required for efficient NLS-cargo release in the nucleus. Yeast cells
expressing this mutant of importin
as the sole copy of importin
are not viable, demonstrating that the NLS-like sequence mediates an
essential function of importin
in vivo.
shows that this mutant protein accumulates in the nucleus. This nuclear accumulation suggests that A3-importin
is not efficiently recycled from the nucleus to the cytoplasm by the export receptor, Cse1p. Our in vitro data showing that the A3 mutations do
not appear to have a direct impact on the binding of Cse1p to importin
suggest that this defect is due to impaired release of cargo in the
nucleus. Previous work has shown that the Cse1p/CAS association with
importin
does not depend on the N-terminal domain (40, 48). In
fact, the Cse1p/CAS binding site has been mapped to the C terminus of
human importin
(21). Our in vivo data support these
in vitro analyses, because a variant of the A3 mutant of importin
, where the NLS-binding function was destroyed (A3-ED), could be efficiently recycled to the cytoplasm by Cse1p. Taken together, these analyses suggest that the accumulation of A3-importin
in the nucleus is due to the persistence of an NLS-cargo/importin
complex in the nucleus, which inhibits the interaction between importin
and Cse1p. This strongly supports the hypothesis that an
essential function of the NLS-like sequence in the N-terminal domain of
importin
is the efficient release of NLS-cargo from importin
within the nucleus.
is necessary for both the
function of NLS-cargoes within the nucleus and for recycling of
importin
to the cytoplasm. The accumulation of A3-importin
in
the nucleus also demonstrates in vivo the intimate link
between cargo release and recycling of importin
. Because NLS-cargo
release is required for binding to Cse1p/CAS (19-21), it has been
proposed that Cse1p/CAS may facilitate release of NLS-cargo (7, 18). Unfortunately, the molecular details of the interaction of importin
with Cse1p/CAS have not yet been elucidated. Furthermore, it is not
known how Cse1p/CAS distinguishes between the NLS-cargo bound and free
importin
proteins. One possibility is that a conformational change
in importin
signals cargo release to Cse1p.
, it does not exclude the
possibility that additional factors may facilitate this dissociation. Specifically, a recent study demonstrated that in vitro both
Cse1p and the nucleoporin Nup2p facilitate dissociation of NLS-cargo from importin
(18). It is not yet clear how Nup2p may function in
cargo release, although the observation that Nup2p interacts with both
full-length and
IBB-
(44, 49) leads us to suspect that this
function is either independent of, or complementary to, the
auto-inhibitory function characterized in this study. Further work will
be necessary to determine how importin
, Cse1p, and Nup2p interact
as well as how Cse1p and Nup2p contribute to NLS-cargo delivery into
the nucleus in vivo.
auto-inhibition is within the nucleus for release of NLS-cargo from
importin
, we also present quantitative data that suggests a
secondary role in recognition/targeting in the cytoplasm. Indeed, a
cytoplasmic role would provide a mechanism by which formation of the
import complex is cooperative, leading from free importin
to a
trimeric import complex consisting of NLS-cargo, importin
, and
importin
(24). We have determined that full-length importin
binds to importin
with low affinity (Kd ~ 0.6 µM) in the absence of any NLS-cargo, presumably because the N-terminal domain forms an intramolecular interaction with the
NLS-binding pocket. In contrast, the IBB domain alone binds more
tightly to importin
(Kd ~ 17 nM).
This quantitative analysis of protein-protein interactions shows that,
in the cytoplasm, NLS-cargo, importin
, and importin
have a low
affinity for each other (micromolar Kd) unless they
are coincident, in which case they form a fairly tight trimeric complex
(nanomolar Kd). This cooperative complex formation
would presumably prevent nonproductive perpetual
Ran-dependent cycling of an importin
/importin
complex without NLS-cargo. Thus importin
is a dynamic adaptor
protein that has evolved to couple NLS-cargo import complex formation
in the cytoplasm and NLS-cargo import complex dissociation in the
nucleus through regulated protein-protein interactions.
to importin
. The values presented in
this study are unique in that they are taken from a measurement of
binding at equilibrium in solution. Previously reported values were
derived from assays that depended on the separation of bound ligand
from unbound (i.e. solid phase pull-down (18) or surface
plasmon resonance assays (24)). The dependence of these assays on the
kinetics of the binding reaction may play a role in the disparity in
the published values. Further experimentation will be needed to
determine the origin of the variance in measured binding constants.
is the primary signal that
initiates import complex disassembly, our data suggest that the IBB is
critical for relaying this signal to the NLS binding pocket on importin
. Thus, this study incorporates an NLS-cargo release step into the
classic nuclear transport pathway. A schematic is shown in Fig.
8: 1) NLS-cargo/importin
/importin
cooperatively associate to form the trimeric import complex in the
cytoplasm; 2) in the nucleus RanGTP mediates dissociation of importin
from the import complex to release an NLS-cargo/importin
complex; 3) the N-terminal domain of importin
competes with
NLS-cargo to bind the NLS-binding pocket; and 4) NLS-cargo is released
into the nucleus, and the auto-inhibitory N-terminal domain is bound to
the NLS-binding site. This renders importin
incapable of binding
nuclear NLS-containing proteins prior to export to the cytoplasm by
Cse1p. This model for protein import requires precise tuning of the
thermodynamic interactions between the various species for the reaction
to proceed efficiently in a single direction. In addition, it will be
interesting to determine what contribution other release factors, such
as Cse1p and Nup2p (18, 50), make to cargo release in
vivo.
View larger version (27K):
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Fig. 8.
A schematic depicting the proposed role of
the N-terminal auto-inhibitory function of importin in NLS-cargo delivery into the nucleus.
In summary, we present experimental evidence for the auto-inhibition of
importin in vivo and provide a mechanistic understanding of the essential nature of the auto-inhibitory function in NLS-cargo delivery into the nucleus as well as a potential role in NLS-cargo recognition/targeting in the cytoplasm. We propose that changes in the
binding affinities of the nuclear transport components underlie and
define the mechanism of regulation of nuclear transport. A complete
understanding of nuclear transport requires a quantitative model for
the nuclear transport process that correlates structural analyses,
in vitro interaction energies, and in vivo
functionality. Such analyses to determine the energetics of individual
mechanisms in the nuclear transport process will be required to fully
understand the rapid, selective, and highly regulated processes of
nuclear transport.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Maureen Powers for experimental suggestions and D. M. Green, A. Lange, and Dr. S. W. Leung for their critical review of the manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM-58728 (to A. H. C.) and by National Science Foundation Grant MCB-9874548 (to A. E. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, School of Medicine, Emory University, 1510 Clifton Rd., NE, Atlanta, GA 30322. Tel.: 404-727-4546; Fax: 404-727-3954; E-mail: acorbe2@emory.edu.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M210951200
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ABBREVIATIONS |
---|
The abbreviations used are:
NPC, nuclear
pore complex;
CEN, centromeric;
DIC, differential interference
contrast;
5-FOA, 5-fluoro-orotic acid;
GFP, green fluorescent protein;
GST, glutathione S-transferase;
IBB, importin binding;
NLS, nuclear localization signal;
RanGTP-RS, RanGTP regenerating
system;
PBS, phosphate-buffered saline;
DAPI, 4',6-diamidino-2-phenylindole;
GTP
S, guanosine
5'-3-O-(thio)triphosphate.
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REFERENCES |
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