From the
Department of Biochemistry, Cell and Developmental Biology, Emory University School of Medicine, Atlanta, Georgia 30322,
Department of Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University School of Medicine, Atlanta, Georgia 30322
Received for publication, January 31, 2003
, and in revised form, March 18, 2003.
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ABSTRACT |
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INTRODUCTION |
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Protein cargoes that contain classical NLSs are recognized in the cytoplasm by a heterodimeric receptor composed of importin/karyopherin and importin/karyopherin
(3, 810). Importin
recognizes and binds the NLS, and importin
translocates the trimeric import complex through the nuclear pore (3). Delivery into the nucleus is dependent on the small GTPase Ran, which governs the interactions between the nuclear transport receptors and macromolecular cargoes and thus confers directionality to nucleocytoplasmic transport (3, 11). Once the cargo is delivered into the nucleus, the transport receptors are recycled to the cytoplasm (3).
Release of NLS-cargo into the nucleus is essential for both the function of the cargo and for recycling of the nuclear transport receptors to the cytoplasm (1115). Thus, once the NLS-cargo·importin ·importin
complex reaches the nuclear face of the pore, the complex must be disassembled to deliver the cargo to the nuclear interior and to recycle the import receptors in an NLS-free state. The best characterized signal for the dissociation of the import complex is RanGTP-dependent dissociation of importin
from the trimeric complex (1619). In the nucleus, RanGTP binds to importin
, which causes a conformational change in importin
that results in the release of importin
(20). RanGTP-triggered dissociation of importin
generates a transient NLS·importin
complex. The mechanism of NLS-cargo dissociation from importin
is not as well characterized, although recent studies suggest that the importin
export receptor, Cse1p, the nucleoporin, Nup2p, and the N-terminal auto-inhibitory domain of importin
could all play a role (19, 2123). Gilchrist et al. (19) demonstrated that Cse1p and Nup2p can facilitate release of NLS-cargo in vitro. Furthermore, our recent data support a model where the autoinhibitory domain of importin
couples the Ran-dependent dissociation of importin
with the subsequent release of NLS-cargo from importin
(23). Thus, these factors may cooperate or act sequentially to facilitate NLS-cargo release. It is not yet clear, however, how this occurs or how these factors functionally interact in vivo.
Domain analysis shows that importin has an N-terminal domain that binds importin
(the importin
binding domain; IBB), a central armadillo domain that constitutes the NLS binding pocket, and a C-terminal region that appears to be important for binding to the export receptor Cse1p (14, 18). The structure of mouse importin
revealed that the N-terminal IBB domain of importin
can form an intramolecular interaction with the NLS binding pocket of importin
(22). This observation suggested that the IBB, in addition to mediating binding to importin
, could also regulate cargo binding through an auto-inhibitory mechanism (22). This may occur through competition between the IBB and NLS-cargo for binding to the NLS binding pocket of importin
, which could facilitate the release of cargo into the nucleus. Additionally, the IBB could prevent NLS-cargo rebinding once it has been released. Thus, the IBB appears to act as a regulatory switch between the cytoplasmic form of importin
, which has a high affinity for NLS-cargo because of binding to importin
, and the nuclear form, which has a low affinity for NLS-cargo.
Importin structural studies suggest that monopartite NLSs and the auto-inhibitory sequence bind to the NLS binding pocket of importin
in a similar manner (22, 24, 25). Indeed, a conserved cluster of basic amino acids, which resembles a classical basic NLS, serves as the auto-inhibitory sequence (yeast importin
residues 54KRR56) (22, 23). In vitro analysis of the energetic landscape of NLS sequences binding to importin
revealed details of the specific interactions required for NLS binding to the pocket of importin
(26). In particular, Hodel et al. (26) defined the requirements for specific amino acid residues within an NLS that are critical for high affinity interaction with importin
. We hypothesize that the auto-inhibitory sequence depends on similar energetic molecular interactions with the NLS binding pocket. This would be consistent with its role in NLS-cargo release through a direct competition mechanism.
The present study further characterizes the auto-inhibitory sequence within the N-terminal domain of Saccharomyces cerevisiae importin . We have utilized site-directed mutagenesis, in vitro binding assays, and in vivo analyses to investigate the intramolecular interaction between the N-terminal IBB domain and the NLS binding pocket of importin
. Through these analyses we demonstrate that the auto-inhibitory sequence binds to the NLS binding pocket through energetic interactions that are analogous to those for a monopartite NLS. We present data in support of our hypothesis that the auto-inhibitory function of the IBB domain is responsible for essential in vivo functions. Our experiments demonstrate that the severity of the in vivo phenotypes are directly correlated to the reduction of auto-inhibition measured in vitro, suggesting that the in vivo phenotypes are directly related to the loss of auto-inhibitory function. Furthermore, we present data in support of functional overlap between the N-terminal domain of importin
, Cse1p, and Nup2p in vivo. We propose that the auto-inhibitory N-terminal domain of importin
and Cse1p function together in NLS-cargo release, whereas Nup2p functions through a different mechanism in this essential process.
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EXPERIMENTAL PROCEDURES |
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Generation of Importin MutantsAmino 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 (Invitrogen). DNA containing the mutations was subcloned from the bacterial expression vector into the yeast expression plasmid for in vivo studies. For all constructs generated, the presence of each desired mutation and the absence of any other mutations was confirmed by DNA sequencing.
Expression and Purification of Recombinant ProteinsAssays were performed with purified recombinant S. cerevisiae proteins Srp1p (importin ) and Rsl1p (importin
). Full-length His6-importin
(residues 1542), His6-SV40 (SPKKKRKVEAS)-NLS-GFP, and His6-Myc (PAA-KRVKLD)-NLS-GFP were expressed in the Escherichia coli strain BL21 (DE3) and purified by nickel affinity chromatography essentially as described (26, 29). Importin
was expressed and purified as described previously (29).
Fluorescence Anisotropy AssayFluorescence 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 Myc-NLS-GFP to importin were measured essentially as described previously (26, 29). Briefly, SV40-NLS-GFP or Myc-NLS-GFP was diluted in phosphate-buffered saline 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 increasing amounts of wild-type or mutant importin
proteins were 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 are calculated as detailed at www.biochem.emory.edu/Hodel/Research/BindingCurves/fitting_curves.htm. Binding energies were calculated using
G = RT ln Kd, where RT =0.59 kcal/mol.
In Vivo Functional AnalysisThe in vivo function of each of the importin variants was tested using a plasmid shuffle technique (30). Plasmids encoding each of the importin
mutant proteins were individually transformed into SRP1 deletion cells (ACY324) containing the URA3 SRP1 maintenance plasmid, pAC876 (23). Single transformants were grown in liquid culture to saturation, serially diluted (1:10), and spotted on minimal medium plates lacking leucine as a control or on fluoroorotic acid (5-FOA) plates. The drug 5-FOA eliminates the URA3 plasmid-encoded wild-type importin
(pAC876) (30). Plates were incubated at the indicated temperatures for 35 days.
Immunoblot AnalysisImmunoblot analysis was performed by standard methods as described (31). 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 either monoclonal anti-myc antibody (1:2000 dilution; Oncogene) or polyclonal anti-GFP antibody (1:10,000 dilution) (32).
MicroscopyDirect fluorescence microscopy was used to localize GFP fusion proteins in live cells. For all experiments, cells were stained with DAPI (1 µg/ml) 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 (Chroma Technology) using an Olympus BX60 epifluorescence microscope equipped with a Photometrics Quantix digital camera.
Integration into the Yeast GenomeThe importin -GFP mutants were integrated at the endogenous importin
locus of wild-type (ACY192) cells using a standard integration strategy (23). The integration created a duplication at the endogenous importin
locus such that both endogenous importin
and importin
-GFP were each expressed from SRP1 promoters. TUB1-GFP was integrated at the URA locus as described previously (33).
In contrast to the importin -GFP integrations, the A55-importin
mutant replaced the wild-type copy of importin
. To integrate A55-importin
, the A55 mutation was subcloned into the SRP1 open reading frame cloned in the LEU2 integrating plasmid, pRS305 (34), to create srp155-pRS305 (pAC1128). A55-importin
was then integrated at the endogenous SRP1 locus by linearization of srp155-pRS305 and transformation into the wild-type diploid ACY247. Transformants that grew on plates lacking leucine were selected, and the presence of the A55-importin
mutation was confirmed by PCR and sequencing. The heterozygous diploid was subsequently sporulated, and tetrads were dissected to generate the haploid A55-importin
strain, srp155 (ACY642). This integration strategy is designed to make A55-importin
the only copy of importin
expressed in the haploid strain.
Yeast Genetic AnalysesSynthetic interactions between A55-importin (srp155) and other nuclear transport factors were tested by creating double and triple mutant strains. These strains were made by crossing the various haploid mutant strains (srp155, cse11 (35),
nup2 (Research Genetics)). The srp155 strain was covered by a SRP1 URA3 plasmid (pAC876) and mated to each of the mutants to be tested. The resultant heterozygous diploids were sporulated and dissected to generate the appropriate double and triple mutant haploid strains. For suppression analysis, high copy plasmids (2µ) expressing various nuclear transport factors were transformed into srp155 (ACY642) covered by a SRP1 URA3 plasmid (pAC876). Genetic interactions (synthetic growth defects/lethality and suppression) were assessed by growing single colonies in liquid culture to saturation, serially diluting (1:10) and spotting on minimal medium plates as a control or on 5-FOA plates. Plates were incubated at the indicated temperatures for 36 days.
FACS AnalysisCells were prepared for FACS analysis by staining with propidium iodide (36). Briefly, cells were ethanol-fixed at 4 °C, washed, and resuspended in 1 ml of 50 mM sodium citrate, pH 7.0. Cells were then treated with 0.08 mg/ml RNase A for1hat50 °C, followed by 0.25 mg/ml proteinase K for 1 h at 50 °C, before incubation in 8 µg/ml propidium iodide. Each sample was analyzed with a FACScalibur cytometer.
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RESULTS |
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Auto-inhibitory Function of Importin MutantsThe A54 and A55 mutations are in residues that correspond to NLS residues that are critical for a high affinity interaction with the NLS binding pocket (26). Thus, we would predict that the auto-inhibitory domains of the A54 and A55 proteins should have weaker binding to the NLS binding pocket and should therefore have a decrease in auto-inhibitory function when compared with wild-type importin
. To examine both NLS binding and auto-inhibitory function for each importin
variant, we used a quantitative, fluorescence anisotropy, solution binding assay (23, 26, 29). Full-length wild-type importin
binds weakly to a classical SV40-NLS because of the N-terminal auto-inhibitory IBB domain. However, when the same experiment is carried out in the presence of a stoichiometric amount of importin
, the N-terminal auto-inhibition of full-length importin
is relieved, and much tighter binding (
30-fold) to the SV40-NLS is observed. In contrast, our previous work demonstrates that the A3-importin
mutant (54KRR56
AAA) has decreased auto-inhibitory function without impacting importin
binding (23). Due to this decreased auto-inhibition, the A3 protein binds to SV40-NLS-GFP
7-fold more tightly than full-length wild-type importin
(23). This assay actually examines three aspects of importin
function: binding to NLS-cargo, auto-inhibitory function (measured by the ability of full-length importin
to bind NLS-cargo in the absence of importin
), and binding to importin
(based on the relief of auto-inhibition).
To examine NLS binding and the auto-inhibitory function for each importin protein that contains a single amino acid change, we performed the fluorescence anisotropy assay using a monopartite SV40-NLS-cargo. The assay was carried out with each importin
protein in the presence and absence of importin
(Fig. 2). Typical curves for binding of SV40-NLS-GFP to the mutant importin
proteins in the absence of importin
are shown in Fig. 2A. As described under "Experimental Procedures," these curves are used to calculate Kd values for the interaction between NLS-cargo and importin
(see Table II). The Kd values can be used to determine the change in free energy (
G) to compare the wild-type and mutant proteins.
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Thus, to assess the impact of each amino acid change within the auto-inhibitory sequence, we compare the Kd values and calculate the change in free energy, G, for the binding of each importin
protein to NLS-cargo. As demonstrated previously (23), 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
. As a control, A3-importin
, which has decreased auto-inhibitory function, binds to SV40-NLS-GFP more tightly (Kd
73 nM) than full-length wild-type importin
(Kd
500 nM). A54-importin
binds to SV40-NLS-GFP with a similar affinity (Kd
83 nM) to A3-importin
suggesting that K54 is the most critical residue in the 54KRR56 autoinhibitory sequence. A55-importin
binds to SV40-NLS-GFP more tightly (Kd
240 nM) than wild-type importin
, an
2-fold increase in affinity. A56-importin
binds to SV40-NLS-GFP with a similar affinity (Kd
1300 nM) to wild-type importin
. To compare each of the importin
proteins, the change in free energy for the binding of each importin
protein to NLS-cargo in the absence (
) and presence (
) of importin
(Fig. 2B) was calculated. In the presence of importin
, the affinity of each of the mutant importin
proteins is similar to wild-type importin
suggesting that each mutant retains normal binding to importin
(Table II). These binding results also demonstrate that each of the mutant importin
/
complexes binds NLS-cargo with approximately the same affinity as the wild-type importin
/
complex.
Correlation of Importin Function with NLS BindingPrevious structural studies suggested that the auto-inhibitory sequence, KRR, resembles an NLS and binds to the NLS binding site in a similar way to a monopartite NLS (sites P2-P4) (22, 24). Alignment of NLS sequences with the auto-inhibitory sequence shows that residue K54 of importin
binds to importin
at the same position (P2) as the essential lysine of a monopartite NLS (Fig. 2C) (26). Mutation of K54 to A54 significantly decreases the auto-inhibitory function of importin
(Fig. 2A). The decrease in auto-inhibition can be expressed as a change in free energy (
G) for each importin
mutant when compared with wild-type protein (Fig. 2C). As shown in Fig. 2C, residues critical for auto-inhibitory function correlate with residues that are critical for an NLS binding to the NLS binding pocket of importin
, where mutation of the P2 binding residue of the NLS to alanine causes the largest decrease in binding affinity (26). The NLS residue that binds importin
in position 3 (P3) has an intermediate contribution, and the position 4 (P4) residue has a fairly weak contribution to the NLS binding energy (Fig. 2C). The in vitro auto-inhibitory behavior of A55-importin
and A56-importin
correlates with the energetic contributions of the corresponding residues within the SV40 and Myc monopartite NLSs (Fig. 2C). Thus, the auto-inhibition of each of the importin
mutants correlates with the energetic contribution of each residue to a functional NLS.
In Vivo Function of Importin MutantsOur in vitro experiments demonstrate that variants of importin
with point mutations within the 54KRR56 auto-inhibitory sequence exhibit a range of auto-inhibitory functions (Fig. 2 and Table II). To test the effect of different levels of auto-inhibition on importin
function 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 a plasmid borne wild-type copy of SRP1 (Fig. 3A). 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 complement
SRP1 cells whereas neither a vector alone nor the auto-inhibitory defective importin
(A3) can functionally replace SRP1 (Fig. 3A, compare the control and 5-FOA plates). Results shown in Fig. 3A indicate that cells expressing A54-importin
have a pronounced cold-sensitive phenotype (no growth at 16 °C) and grow extremely slowly even at 30 °C. Cells expressing A55-importin
grow more slowly than wild-type at 30 °C and are cold-sensitive at 16 °C. Cells expressing A56-importin
grow similar to cells expressing wild-type importin
at all temperatures.
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To confirm that each mutant protein is expressed at a similar level to wild-type importin , we analyzed their expression using a C-terminal triple myc tag. 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. 3B, compare lanes 36 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 the A54 and A55 mutations perturb the function of the importin
proteins.
Localization of the Mutant Importin ProteinsWe have found previously (23) that mutations that decrease the autoinhibitory function of importin
result in its accumulation within the nucleus. This accumulation is presumably because of the persistence of an NLS-cargo·importin
complex within the nucleus. To further examine the importin
variants, we analyzed the localization of each protein using C-terminal GFP-tagged fusion proteins. These importin
-GFP fusion proteins were expressed from the endogenous importin
promoter. We integrated each importin
-GFP fusion protein at the endogenous importin
locus and visualized them as described previously (23). Wild-type importin
localizes to the nuclear rim and the cytoplasm in wild-type cells when visualized in this manner (Fig. 3C, panel E). However, A3-importin
-GFP accumulates within the nucleus (Fig. 3C, panel A). The localization of A54-importin
-GFP (Fig. 3C, panel B) is similar to that of A3-importin
-GFP. A55-importin
-GFP also localizes to the nucleus but shows some cytoplasmic signal, suggesting that its localization is intermediate between A54-importin
and wild-type importin
(Fig. 3C, panel C). The localization of A56-importin
-GFP is similar to that of wild-type importin
-GFP (Fig. 3C, compare panel D to panel E).
Analysis of R54-Importin Even conservative mutation of the P2 lysine to arginine in SV40 NLS results in an
94-fold decrease in binding to the NLS binding pocket (26). To further test the hypothesis that the auto-inhibitory sequence interacts with importin
in the same manner as an NLS, we mutated K54 to an arginine residue to create R54-importin
(Fig. 1) and assessed the impact of this conservative mutation. R54-importin
binds to SV40-NLS-GFP with
5.5-fold higher affinity than wild-type importin
(Table II) as assessed by fluorescence anisotropy. This demonstrates that R54-importin
is defective in auto-inhibition and provides additional evidence that a monopartite NLS and the autoinhibitory sequence bind to the NLS binding pocket of importin
with similar energetics. In the presence of importin
the binding of SV40-NLS-GFP to R54-importin
is similar to wild-type importin
(Table II).
To test the function of the R54-importin protein in vivo, we used the plasmid shuffle technique and the importin
-GFP localization assay (see "Experimental Procedures"). R54-importin
was transformed into yeast cells deleted for the endogenous importin
gene (SRP1), and plasmid shuffle was used to replace the plasmid-borne wild-type copy of SRP1 (Fig. 4A). Results show that cells expressing R54-importin
as the only copy of importin
grow slowly at 30 °C and are not viable at 16 °C. The R54-importin
protein was expressed at similar levels to wild-type importin
(data not shown). In vivo localization analysis shows that the R54-importin
-GFP protein accumulates within the nucleus similar to A54-importin
-GFP (Fig. 4B).
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In Trans Localization of the Auto-inhibitory SequenceThe IBB domain of importin , which contains the auto-inhibitory sequence, binds weakly to the NLS binding pocket in trans (Kd
14 µm) (23). This suggests that the auto-inhibitory sequence should not bind to the NLS binding pocket with sufficient affinity to act as an NLS and direct a heterologous protein into the nucleus when expressed in vivo. To test whether the autoinhibitory sequence can act like an NLS to direct a heterologous protein to the nucleus, we fused importin
residues 4960 (see Fig. 1) in-frame with GFP-GFP to create IBB4960-GFP-GFP. The localization of this fusion protein was compared both to GFP-GFP, which lacks any NLS, and to SV40-NLS-GFP-GFP, a positive control that contains a canonical monopartite NLS. As shown in Fig. 5, the control GFP-GFP protein is diffusely localized throughout the cell. In contrast, SV40-GFP-GFP accumulates in the nucleus. The IBB4960-GFP-GFP is localized throughout the cell similar to GFP-GFP. This demonstrates that the auto-inhibitory sequence cannot efficiently act as an NLS and direct a protein to the nucleus when expressed in trans.
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Genetic Analysis of NLS Release FactorsThrough analysis of the auto-inhibitory defective mutant of importin , A3, we suggested 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 (23). Cse1p and Nup2p have also been reported to affect NLS-cargo release from importin
(19, 21). It has been suggested that these factors may cooperate or act sequentially to facilitate NLS-cargo release and may therefore functionally overlap. As shown in Fig. 3A, the A55 mutation in importin
causes a conditional growth phenotype that correlates with its defect in auto-inhibitory function. This cold-sensitive phenotype can be exploited for genetic analysis. As a genetic test for functional overlap between the auto-inhibitory function of the IBB domain of importin
, Cse1p, and Nup2p, we tested for any exacerbated growth defects (synthetic lethality) in double or triple mutant cells.
The A55-importin allele (srp155) was integrated into the genome to avoid copy number effects (see "Experimental Procedures"). As CSE1 is essential, we used a well characterized cold-sensitive allele, cse11 (35). NUP2 is not essential (37), and therefore we utilized a complete deletion of the open reading frame,
nup2. If Cse1p and Nup2p are involved in NLS-cargo release in the nucleus, cells with combined mutations in the importin
auto-inhibitory function, CSE1 and/or NUP2, might be more growth-compromised than any of the single mutants. We therefore generated each of the double mutants (srp155 cse11, srp155
nup2, and cse11
nup2) and the triple mutant (srp155 cse11
nup2) maintained by a plasmid-borne wild-type importin
(SRP1) as described under "Experimental Procedures." As reported previously (21, 35, 38) and shown in Fig. 6A, cse11 cells are cold-sensitive at 16 °C, and
nup2 cells do not have any detectable growth defect. Results shown in Fig. 6A demonstrate that the srp155 cse11 double mutant is inviable (compare the control and 5-FOA plates). This synthetic lethal phenotype was observed at all temperatures tested. In addition, the srp155
nup2 and cse11
nup2 double mutants grow more slowly than either single mutant. As shown in Fig. 6A, these phenotypes are more pronounced at cold temperatures.
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As a second genetic test for functional overlap, we assessed high copy suppression of the srp155 cold-sensitive phenotype. Importin , Cse1p, and Nup2p were expressed from a high copy plasmid, and their ability to suppress the cold-sensitive phenotype of srp155 was examined as described under "Experimental Procedures." Controls demonstrate that a wild-type importin
plasmid can complement the cold-sensitive phenotype of srp155 cells, whereas a vector alone cannot (Fig. 6B, compare the control and 5-FOA plates). Results shown in Fig. 6B demonstrate that importin
cannot complement the cold-sensitive phenotype of the srp155 cells. This is consistent with our in vitro analyses, Fig. 2B, where we show that the mutant importin
proteins bind to importin
with the same affinity as wild-type importin
. Nup2p also does not suppress the srp155 cold-sensitive phenotype. In contrast, overexpression of Cse1p partially suppresses the cold-sensitive phenotype of srp155 cells.
srp155 Cells Accumulate in G2/M at 18 °CImportin is required for the execution of mitosis as cells with conditional mutations of importin
arrest with a G2/M phenotype (39). Therefore, to further characterize the srp155 cells, we assessed the morphology of cells grown at either 30 or 18 °C and compared them with wild-type cells grown under the same conditions. Microscopic analysis revealed that the srp155 cells have similar morphology to wild-type cells when grown at 30 °C. Interestingly, at 18 °C the srp155 cells were larger, and there was a greater proportion of large budded cells within the population as compared with wild-type cells (for example see Fig. 7A, compare panels G and H). To determine whether these large budded cells were arrested uniformly within the G2/M phase of the cell cycle, we examined the microtubules by integrating TUB1-GFP into wild-type and srp155 cells (see "Experimental Procedures"). The TUB1-GFP integrated wild-type and srp155 cells were grown to log phase at both 30 and 18 °C and compared by microscopic analysis (Fig. 7A). As shown in Fig. 7A, panel D, there is a variation in spindle length in the large budded srp155 cells; therefore these cells are not uniformly arrested at the same point within G2/M of the cell cycle. This was also confirmed by staining the DNA with DAPI (data not shown). These data suggest that the auto-inhibitory defective cells are not arrested but rather pass through G2/M phase more slowly than wild-type cells.
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The microscopic analysis of the srp155 cells suggests that they spend more time in the G2/M phase of the cell cycle, and thus we predict that a higher percentage of cells should have replicated (2N) DNA than wild-type cells. To examine the DNA content of the srp155 cells, we performed FACS analysis of log phase cultures grown at both 30 and 18 °C and compared them with wild-type cells grown under the same conditions. Cells were stained with propidium iodide, and the DNA content was analyzed by flow cytometry. The FACS profile of wild-type cells grown at either 30 or 18 °C are indistinguishable in the distribution of cells with 1N and 2N DNA content (data not shown). Cells expressing the A55-importin protein have a similar profile to wild-type cells at 30 °C (data not shown). As shown in Fig. 7B, the 2N DNA peak of srp155 cells grown at 18 °C is markedly broader than that of srp155 cells grown at 30 °C, which is consistent with an increased proportion of cells in G2/M and hence slower progression through this phase of the cell cycle.
This G2/M phenotype of srp155 cells provides another assay to characterize the genetic interaction between the auto-inhibitory function of importin and Cse1p. As shown in Fig. 6B, expression of Cse1p from a high copy plasmid suppresses the cold-sensitive phenotype of srp155 cells. Therefore, we tested whether Cse1p expressed from a high copy plasmid could also suppress the G2/M phenotype of srp155 cells. Controls demonstrate that a wild-type importin
plasmid can suppress the srp155 G2/M phenotype whereas a vector alone cannot (Fig. 7B). Overexpression of Cse1p also suppresses the G2/M cell cycle phenotype of the srp155 cells.
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DISCUSSION |
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Our previous analysis of auto-inhibitory function utilized a variant of importin , A3, where all of the basic amino acids within the 54KRR56 sequence were mutated to alanine (23). In the current study, we show that amino acid K54 is the most important residue within the auto-inhibitory sequence. Indeed, the in vitro and in vivo phenotypes of the K54 protein are similar to those of the A3 protein. Even conservative mutation of K54 to arginine significantly decreases auto-inhibition. Mutation of residue R55 has an intermediate effect on auto-inhibition, whereas mutation of R56 appears to have no affect on importin
function. This graduated effect on auto-inhibitory function within the 54KRR56 sequence is consistent with the molecular interactions revealed by structural studies and is analogous to the energetic landscape of a monopartite NLS binding to the NLS binding pocket of importin
(22, 25, 26). Furthermore, we show that the severity of the in vivo phenotype is directly correlated to the reduction of in vitro autoinhibition, suggesting that the in vivo phenotypes are directly related to the loss of auto-inhibitory function.
The ability of the IBB domain to regulate NLS-cargo binding to the NLS binding pocket is presumably dependent on the energy the IBB gains from the intramolecular interaction versus an intermolecular interaction. Indeed, our data support a model where the energy for the binding of the auto-inhibitory sequence to the NLS binding pocket of importin is obtained from the in cis intramolecular interaction. We have shown previously (23) that in trans the IBB binds with low affinity (µM) to the NLS binding pocket of importin
. Consistent with this previous result, we demonstrate that the auto-inhibitory sequence is not able to target a heterologous protein into the nucleus of yeast cells when expressed in trans, suggesting that the binding affinity for importin
is not sufficient for nuclear localization. Although the auto-inhibitory sequence can bind to the NLS binding pocket like an NLS, it would not be energetically favorable for it to interact with importin
with an affinity comparable with a functional NLS. However, the autoinhibitory sequence can compete with an NLS for binding to the NLS binding pocket because of the physical tethering of the IBB to the NLS binding pocket domain of importin
, which significantly increases its local concentration. This requirement for an in cis interaction between the IBB domain and the NLS binding pocket of importin
also presumably prevents the N-terminal domains of adjacent importin
proteins from interacting with NLS-cargo pockets intermolecularly and forming dimers.
Although RanGTP is the major determinant of import complex dissociation (16, 17, 19), the N-terminal auto-inhibitory domain of importin , Cse1p, and Nup2p also play a role in the release of NLS-cargo into the nucleus (19, 2123, 25, 29). The trimeric import complex is disassembled by the RanGTP-triggered dissociation of importin
to release a dimeric NLS-cargo·importin
complex into the nucleus. The importin
auto-inhibitory function is essential to efficiently dissociate this dimeric intermediate (23). In this study we use genetic analyses of the auto-inhibitory domain of importin
, CSE1 and NUP2, to provide data in support of a model where these factors have overlapping functions in vivo that facilitate release of NLS-cargo from importin
. These in vivo data are consistent with a recent study by Gilchrist et al. (19) where they demonstrated that each of these interactions is able to increase the dissociation rate of the dimeric NLS-cargo/importin
intermediate in vitro.
Our genetic data suggest that Cse1p and Nup2p have distinct functions in NLS-cargo release. This conclusion is supported by two lines of genetic evidence. First, cse11 and srp155 are synthetically lethal, but nup2 and srp155 show only a modest synthetic interaction. Second, the cold-sensitive phenotype of srp155 cells is suppressed by Cse1p but not by Nup2p. Independent biochemical experiments support the idea that Cse1p and Nup2p act through distinct mechanisms in NLS-cargo release.2 As shown here and suggested previously (40), Cse1p and Nup2p demonstrate only minor genetic interactions. Thus, although the evidence suggests that both Cse1p and Nup2p participate in NLS-cargo release and/or importin
recycling, these proteins appear to function through distinct mechanisms.
A previous study (41) also demonstrated a genetic interaction between importin and Nup2p. This study utilized the srp131 allele of importin
(42). It should be noted that the srp131 allele has not been functionally characterized, and therefore it is not known what step within the importin
transport cycle is affected. In contrast, importin
binding, NLS binding, and auto-inhibitory function have all been analyzed for the srp155 mutant. Analysis of the genetic interactions of srp155 can therefore be interpreted in terms of the autoinhibitory function of importin
. In srp131, serine residue 116 is substituted with a phenylalanine residue (42). This residue is outside of the NLS binding pocket and is not within the IBB domain of importin
(24, 42). In the future it may be interesting to determine whether the srp131 mutant is specifically defective in a particular step in the importin
transport cycle so that it is possible to more definitively interpret studies with srp131 cells.
The ability of Cse1p to suppress the cold-sensitive phenotypes of srp155 cells suggests an intimate relationship between NLS-cargo dissociation and recycling of importin to the cytoplasm. There is also evidence that NLS-cargo and Cse1p cannot bind to importin
simultaneously (13, 14). This presumably prevents the recycling of importin
that is still bound to NLS-cargo in the nucleus and thus futile cycles of nuclear transport. It is not known how importin
and Cse1p interact, although we have shown that an auto-inhibitory defective mutant of importin
still interacts with Cse1p (23). This suggests that high copy suppression of the srp155 cold-sensitive phenotype by CSE1 is not because of a change in the binding affinity of the two proteins but rather an overlap of function between the two proteins. Future studies of the importin
-Cse1p interaction should allow us to dissect the mechanism of NLS-cargo release and importin
recycling.
Mutant srp155 cells accumulate in the G2/M phase of the cell cycle. This observation is consistent with a previous report (39) where the conditional srp131 mutation causes mitotic cell cycle defects. Loeb et al. (39) suggested that the importin -dependent transport of cell cycle regulators into the nucleus is critical for cell cycle progression. Indeed, the slow growth phenotype of srp155 cells could be because of the inefficient release of NLS-cargoes within the nucleus, in particular, specific cargoes required for mitosis. For example, a recent study identified a critical cargo, TPX2, of the importin
/
complex whose release from importin
is essential for mitotic progression in Xenopus (15). This highlights the importance of understanding how cargoes are efficiently dissociated from importin
within the nucleus to mediate their cellular function. Future studies of the A55-importin
protein may allow us to identify these mitotic cargoes.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM-58728 (to A. H. C.) and by National Science Foundation Grant MCB-9874558 (to A. E. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This 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, Emory University School of Medicine, 1510 Clifton Rd., NE, Atlanta, GA 30322. Tel.: 404-727-4546; Fax: 404-727-3954; E-mail: acorbe2{at}emory.edu.
1 The abbreviations used are: NLS, nuclear localization signal; DAPI, 4', 6-diamidino-2-phenylindole dihydrochloride; DIC, differential interference contrast; FACS, fluorescence-activated cell sorter; 5-FOA, 5-fluoroorotic acid; GFP, green fluorescent protein; IBB, importin binding; WT, wild-type.
2 D. Gilchrist and M. Rexach, personal communication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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