From the
Department of Physiology, McGill University, Montreal, Quebec H3G 1Y6,
Canada and the University of Saarland, 66421
Homburg/Saar, Germany
Received for publication, February 14, 2003 , and in revised form, April 23, 2003.
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ABSTRACT |
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INTRODUCTION |
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In higher eukaryotes, translocation of Ran across the nuclear envelope requires Ntf2, a small protein that specifically binds to Ran-GDP (6, 8, 9). Ntf2 transports Ran-GDP into the nucleus where RCC1 converts it to Ran-GTP, thereby dissociating the Ran-Ntf2 interaction. The nucleoporin p62 is likely to play an important role for the translocation of Ran across the NPC, as both Ran and Ntf2 bind to p62. In particular, the FXF repeats present in the N-terminal domain of p62 are involved in these binding reactions (10). Similarly, the yeast nucleoporin Nsp1p, a functional homolog of p62, binds to Ran and Ntf2 (10). As well, the repeat domain of Nsp1p interacts with Gsp1p in vitro (11), indicating that the interactions Ran-p62 and Nsp1p-Gsp1p have been conserved and Gsp1p accumulates in yeast nuclei by the same mechanism as in metazoan cells (6, 8, 9). This idea is further supported by our recent results, which show that mutations in the yeast NTF2 gene interfere with Gsp1p nuclear accumulation (7).
In addition to soluble factors, nuclear trafficking requires nucleoporins. With about 30 different nucleoporins, the NPC overall organization in yeast is less complex but similar to higher eukaryotes (3, 12). Various yeast nucleoporins have been shown to be involved in nuclear transport of proteins, RNA, or both types of macromolecules. For instance, Nup49p and Nsp1p play a role in protein import as well as mRNA export (1318). For RAT2/NUP120, NUP82, and NUP159, only mutations affecting the nuclear export of mRNA have been reported so far (1923). Recent studies on nuclear export of 60 S ribosomal subunits showed that yeast strains carrying mutations in NSP1, NUP49, or NIC96 accumulate 60 S subunits, whereas NUP84 or NUP85 deletion mutants were not impaired (24). Moreover, mutations in NSP1 and NUP159 interfere with nuclear export of the precursor of signal recognition particle (25). With respect to nucleocytoplasmic transport, the nucleoporin Nsp1p is particularly interesting as it assembles into biochemically distinct protein complexes (12, 18, 19, 2527). Nsp1p is associated with Nup49p, Nup57p, and Nic96p both on the cytoplasmic and nuclear side of the NPC (12, 16, 19, 26, 28). In contrast, the Nup82p-Nsp1p-Nup159p subcomplex is found on the cytoplasmic side only (12, 19, 20, 22, 23, 27, 28), where it interacts with Nup116p-Gle2p, an association likely to be required for mRNA export (29, 30). Despite the pivotal role of Nsp1p in nucleocytoplasmic trafficking, only its C-terminal domain, which does not contain FXF repeats, is essential for viability. Furthermore, mutant nsp1-ts18 fails to incorporate the Nsp1p-Nup57p-Nup49p complex into NPCs. Thus, NPCs in nsp1-ts18 are lacking the repeats provided by this core complex. Although impaired in growth, the nsp1-ts18 mutant is viable (18). These observations are consistent with the idea that members of the Nsp1p complex perform a function that is redundant with other nucleoporins.
Previous in vitro analyses revealed that the middle FG repeat
domain of Nsp1p directly interacts with Gsp1p and also with Ran, the mammalian
homolog of Gsp1p. By contrast, binding to the essential C-terminal domain was
not observed (10,
11). Various mutations located
in subdomains coil 14 of the essential C-terminal part of Nsp1p show
distinct defects in nucleocytoplasmic trafficking. As such, strain
nsp1-5 (coil 2) fails to accumulate Mat2 and Pho2p in nuclei
at the non-permissive temperature. Likewise, nsp1-5 and
nsp1-ala6 (coil 2) are impaired in export of the 60 S ribosomal
subunit (24,
31). Furthermore, mutant L640
S (coil 1, Ref. 32) shows mRNA
export defects (18), whereas
L697P (coil 2), ts18 (coils 3 and 4), and W644C (coil 1) are deficient in
nuclear import of GFP-Npl3p and classical nuclear import under nonpermissive
conditions (this contribution and see Ref.
18).2
Together with Nsp1p and other nucleoporins Nic96p forms an NPC subcomplex (28). Nic96p is organized into three domains, with heptad repeats in the N-terminal portion and stretches of uncharged amino acid residues in the central domain. Deletion of Nic96p heptad repeats and point mutations in the central portion, such as nic96-1, impair nuclear protein trafficking (16). By contrast, RNA export defects have not been observed in these mutants (16). Like nsp1p, mutant nic96p can alter the export of 40 S and 60 S ribosomal subunits from the nucleus (24, 33). Likewise, Nic96p is required for classical nuclear protein import (16).
Synthetic lethal screens with mutant nsp1 alleles identified NUP84 and NUP85 as components genetically interacting with NSP1 (34). Nup84p, Nup85p, and Rat2p/Nup120p are part of the same NPC subcomplex consisting of several distinct proteins (34, 35), and cells carrying a deletion of NUP84, NUP85/RAT9, or RAT2/NUP120 show defects in poly(A)+ mRNA export (21, 3437). Moreover, disruption of the NUP84 or the NUP85 gene causes abnormalities of the NPC and nuclear envelope organization (34, 35). In addition, mutant alleles of RAT2/NUP120 or deletions of nucleoporin genes like NUP133 induce clustering of NPCs (13, 21, 36, 38). However, cluster formation does not correlate with transport defects, and both mutants show clustering even under permissive conditions (13, 21). Mutations in Nup133p are synthetic lethal with nup85, rat2/nup120, and nsp1 (see Ref. 39 and references therein), and two-hybrid screens have identified Nup84p as a component that interacts with Nup133p (40). Furthermore, Nup133p associates with the Nup84p complex in vitro and in vivo (41, 42).
Previous studies have revealed that several nucleoporins, in particular Nsp1p, participate in nuclear trafficking. This prompted us to determine their role in the nuclear accumulation of Gsp1p, a GTPase implicated in various nuclear transport reactions. Although the interaction of Gsp1p/Ran and Ntf2 with FXF repeats of nucleoporins is well established, other components of the NPC involved in Gsp1p nuclear transport have yet to be defined. For instance, zinc finger-containing nucleoporins that bind Ran-GDP in mammalian cells have not been found in Saccharomyces cerevisiae (reviewed in Ref. 4). We have now identified several non-repeat yeast nucleoporins that play a role in the nuclear accumulation of Gsp1p, thereby regulating trafficking across the NPC.
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EXPERIMENTAL PROCEDURES |
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PlasmidsTo monitor classical nuclear transport, the gene encoding SV40-GFP (43) was transferred into a centromeric plasmid carrying the URA3 marker. Expression of SV40-GFP is controlled by the ADH1 promoter. Nuclear accumulation of SV40-GFP requires classical nuclear import to be constitutively active as described previously (7). A centromeric plasmid carrying the GFP-GSP1 gene and a LEU2 marker was kindly provided by D. Lau and E. Hurt (Heidelberg, Germany). To allow expression in mutant strains that are Leu+, we have transferred the ADH1 promoter and the GFP-GSP1 coding sequence into a centromeric plasmid containing the URA3 gene. To generate NTF2-GFP, the complete coding sequence of NTF2 was fused in-frame to GFP, and the correctness of the construct was verified by DNA sequencing. For regulated gene expression in yeast, NTF2-GFP was cloned into centromeric plasmids containing the GAL1 promoter and the URA3 or LEU2 marker. Expression of NTF2-GFP was induced by overnight growth at room temperature in selective medium supplemented with 2% galactose.
Generation and Affinity Purification of AntibodiesHis6-tagged Gsp1p was affinity-purified to raise antibodies in mice (7). Polyclonal antibodies were generated against purified His6-tagged Rna1p essentially as described (7). For immunofluorescence studies antibodies were preadsorbed to the His6-tag and affinity-purified with immobilized His6-Rna1p.
Fluorescence MicroscopyFixing of yeast cells, generation of sphero-plasts, and incubation with various antibodies have been described previously (7). Affinity-purified secondary antibodies (Jackson ImmunoResearch, West Grove, PA; Molecular Probes, Eugene, OR) were diluted 1:1000 (Cy3-coupled anti-mouse IgG) or 1:250 (TRITC-labeled anti-rabbit IgG). Cells were incubated with secondary antibodies for 60 min at room temperature and washed three times in PBS/bovine serum albumin. DNA was stained with 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI), and slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA). To visualize GFP-containing reporter proteins, cells were fixed for 10 min in 3.7% formaldehyde, collected by centrifugation, and resuspended in 0.1 M potassium phosphate, pH 6.5, 1.1 M sorbitol. Cells were immobilized on polylysine-coated multiwell slides and stained with 1 µg/ml DAPI in PBS/bovine serum albumin for 2 min at room temperature. Slides were then mounted as described above. Cells were inspected with a Nikon Optiphot at x1,000 magnification and photographed with TMAX 400 films. Negatives were scanned and processed with Adobe Photoshop 5.5.
Western BlottingWestern blot analysis was carried out essentially as described (44). In brief, equal amounts of protein from unstressed and stressed cells were separated by SDS-PAGE and blotted to nitrocellulose. Filters were blocked with 5% skimmed milk in PBS, 0.1% Tween 20 and incubated with primary antibodies in PBS/Tween 20/milk overnight at 4 °C. To detect anti-Gsp1p antibodies, filters were incubated with horseradish peroxidase-coupled secondary antibodies in PBS/Tween 20/milk for 1 h at room temperature. Filters were washed, and immunoreactive material was visualized with an ECL system (PerkinElmer Life Sciences).
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RESULTS |
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The Nucleoporin Mutants Nup133, rat2-1, and
nup85
Mislocalize Gsp1p upon Heat StressWhen the
clustering strain nup133
was incubated for 3 h at 37 °C,
the non-permissive temperature, cytoplasmic levels of Gsp1p increased as
compared with cells kept at room temperature
(Table II). Moreover, after 6 h
at 37 °C the Gsp1p concentration gradient had collapsed in most cells
(Fig. 2D and
Table II). Likewise, in the
clustering mutant rat2-1 the Gsp1p nucleocytoplasmic gradient
dissipated at the restrictive temperature
(Fig. 2H). Double
immunofluorescence labeling with antibodies against nucleoporins and Gsp1p
demonstrated that the GTPase associated with NPC clusters in both mutants
under permissive conditions (data not shown). To characterize further the
defect of nup133
and rat2-1 in maintaining a
nucleocytoplasmic gradient of Gsp1p at 37 °C, we compared levels of the
GTPase in control and heat-treated cells by Western blot analysis. When equal
amounts of protein from unstressed and heat-shocked cells were analyzed in
parallel, Gsp1p levels were reduced in nup133
, but no drastic
changes were observed for rat2-1
(Table II and
Fig. 3).
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Additional members of the Nup84p-Nup133p NPC module were studied for Gsp1p
distribution. As such, 6 h of heat exposure abolished the gradient formation
in nup85 cells but did not alter the Gsp1p concentration
gradient in nup84 cells, which carry a complete
disruption of the NUP84 gene (Fig.
2 and Table II). Although the deletion of NUP85 prevented Gsp1p gradient formation at
elevated temperatures, concentrations of the GTPase were similar to unstressed
cells (Fig. 3).
Taken together, our results demonstrate that three members of the Nup84p-Nup133p NPC subcomplex which in vitro are essential for its assembly are involved in concentrating Gsp1p in nuclei of stressed cells.
The Nucleocytoplasmic Gradient of Gsp1p Collapses in the mRNA Transport Mutant mtr7-1 under Restrictive ConditionsYeast cells carrying the mtr7-1 mutation, also called acc1-7-1, fail to synthesize very long chain fatty acids under non-permissive conditions. At the restrictive temperature NPCs appear as "spots," and increased cytoplasmic concentrations of nucleoporins are detected (data not shown and see Ref. 45). Furthermore, the integrity of the nuclear envelope and nuclear export of mRNAs are defective at 37 °C (45). Thus, the mtr7-1 allele has a more general effect on nuclear envelope organization that could also alter nucleocytoplasmic trafficking and retention of Gsp1p. In line with this idea, we found elevated cytoplasmic concentrations of the GTPase upon shift to 37 °C for 3 h (Table II). After 6 h at 37 °C, Gsp1p was no longer accumulated in nuclei. Furthermore, the total concentration of the GTPase was reduced (Fig. 3).
Mutations in the Essential C-terminal Domain of NSP1 Have Only Minor Effects on the Distribution of Gsp1pWe have analyzed the role of several NSP1 mutations (see Introduction) in establishing a Gsp1p concentration gradient. Despite pronounced consequences for nuclear transport, mutations in the essential C-terminal domain of Nsp1p had less severe effects on Gsp1p distribution and concentration (Table III). For instance, at room temperature nsp1-5 displayed a poor nucleocytoplasmic Gsp1p gradient, with elevated cytoplasmic levels of Gsp1p (Fig. 4B). Upon exposure to heat, however, Gsp1p gradients improved, and the GTPase became restricted to nuclei (Fig. 4D and Table II). One possible interpretation of these results is the preferential degradation of Gsp1p in the cytoplasm in response to heat stress. Indeed, when equal amounts of protein from unstressed and stressed cells were analyzed by Western blotting, the concentration of Gsp1p was found to be slightly decreased at 37 °C (Fig. 3 and Table II).
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Improved nucleocytoplasmic Gsp1p gradients at the non-permissive temperature were also detected in other strains mutated in different coils of the C-terminal Nsp1p domain, i.e. mutants nsp1-ala6, L640S, ts18, or W644C. By contrast, Gsp1p protein levels did not change noticeably at 37 °C in these strains (Table II). Taken together, mutations in different coils of the C-terminal segment, which have strong defects in nuclear trafficking, did not drastically alter the concentration gradient of Gsp1p or levels of the GTPase.
Deletion of the C-terminal NIC96 Domain Changes the Distribution of
Gsp1pWe tested the distribution of Gsp1p in different nic96p
mutants (see Introduction), with nic96N missing
residues 2863, nic96-1 carrying the mutations L260P and P332L,
and nic96
C lacking residues 532839 of the
C-terminal domain. All of the mutants mislocalized Gsp1p after prolonged heat
treatment, but nic96
C showed the most severe effect
(Fig. 4L). By
contrast, levels of Gsp1p were not drastically altered in any of the
nic96 mutants under restrictive conditions
(Fig. 3 and
Table II). In summary, our
results show that the C-terminal domain of Nic96p plays a critical role in
generating or maintaining a high concentration of Gsp1p in nuclei.
Effect of Mutant Nucleoporins on Classical Nuclear Protein
ImportTo determine whether classical nuclear protein import is
altered in nucleoporin mutants under the conditions used for our experiments,
we have introduced the fluorescent reporter protein SV40-GFP. To concentrate
SV40-GFP in nuclei, classical nuclear protein import has to be constitutively
active, and the inhibition of import can be monitored by the appearance of
SV40-GFP in the cytoplasm (7).
At room temperature, all strains accumulated SV40-GFP in nuclei, although some
cytoplasmic localization was detected for several of the mutant strains
(summarized in Table III). Upon
exposure to 37 °C, wild type cells adapted, and the reporter protein was
concentrated in nuclei when cells were inspected after 3 and 6 h of heat
treatment (Table III). By
contrast, cells carrying the mutations nup133, rat2-1,
mtr7-1, nsp1-, nsp1-ala6, nsp1-ts18, nic96
C,
nup84, and nup85
failed to accumulate
SV40-GFP in nuclei after heat exposure. However, this is not a general defect
in nucleoporin mutants. For instance, rat7-1, a mutant impaired in
mRNA export (20), did not show
a defect in classical nuclear import when incubated for 6 h at 37 °C
(Table III and Supplemental
Material Fig. 1). Based on the
Gsp1p distribution upon incubation at 37 °C (see above), we have assigned
the classical transport mutants to two different groups: group A, cells for
which the Gsp1p gradient collapsed in response to heat stress (this includes
nup133
, rat2-1, mtr7-1, nic96
C, and
nup85
); group B, several nsp1 mutants and
nup84 display an intact Gsp1p gradient after 6 h at
37 °C. Defects in classical nuclear protein transport for members of group
A can be explained by the failure of cells to build a nucleocytoplasmic
gradient of Gsp1p. By contrast, different mechanisms of import inhibition
operate for mutants in group B. In the following, we have further
characterized members of group A.
Import of Gsp1p in Mutant Nucleoporin StrainsWith a
molecular mass of 25-kDa, Gsp1p is small enough to diffuse in and out of
the nucleus. Once diffused through the NPC, retention could concentrate Gsp1p
in nuclei. However, transport of Gsp1p across the nuclear envelope could also
result in nuclear accumulation of the GTPase. In the second scenario, elevated
levels of Gsp1p in the cytoplasm will indicate a defect in Gsp1p nuclear
import. To determine whether Gsp1p nuclear import plays a role in mutants of
group A, we have monitored the nuclear accumulation of GFP-Gsp1p, a protein of
52 kDa, which is too large to diffuse efficiently across NPCs. To this
end, cells synthesizing the reporter protein were incubated at room
temperature or 37 °C (Fig.
5 and Table III),
and the fusion protein was subsequently localized by fluorescence microscopy.
In wild type cells, GFP-Gsp1p accumulated in nuclei of unstressed and stressed
cells, and the same was observed for several of the mutant strains such as
nsp1-5 (Fig. 5). By
contrast, strains nup133
, rat2-1,
nic96
C, and mtr7-1 showed increased amounts of
GFP-Gsp1p in the cytoplasm upon exposure to heat. Interestingly,
nup133
and mtr7-1 also had elevated levels of
GFP-Gsp1p in the cytoplasm at the permissive temperature
(Fig. 5 and
Table III), suggesting defects
in GFP-Gsp1p nuclear transport even at room temperature. Likewise, in mutant
nup85
cells GFP-Gsp1p was only slightly accumulated in nuclei
when cells were grown at room temperature. Exposure of nup85
to heat stress did not drastically change the GFP-Gsp1p distribution
(Fig. 5 and
Table III).
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An alternative model for Gsp1p mislocalization in mutant cells would predict that the failure to retain the GTPase in nuclei increased Gsp1p cytoplasmic concentrations. Augmented cytoplasmic GTPase levels would then simply reflect Gsp1p release from nuclei into the cytoplasm. This redistribution should be independent of de novo Gsp1p synthesis. We attempted to test this possibility by incubating cells at the non-permissive temperature in the presence of the protein synthesis inhibitor cycloheximide. Although incubation with cycloheximide for up to 3 h does not collapse the Gsp1p gradient (7), 6 h of exposure to cycloheximide abolished Gsp1p gradient formation, even in wild type cells (Fig. 1J). We were therefore unable to determine whether nuclear retention defects contribute to changes in the Gsp1p localization when cells were kept for 6 h at non-permissive conditions. Nevertheless, we have clearly shown that several of the mutants studied are deficient in Gsp1p nuclear import (see above).
Nuclear Envelope Association of Ntf2p-GFP Is Altered in Several
Nucleoporin MutantsA possible explanation for the elevated
cytoplasmic levels of GFP-Gsp1p under non-permissive conditions could be a
failure of Ntf2p, the nuclear carrier for Gsp1p, to properly interact with
NPCs. We have addressed this question with Ntf2p-GFP, a reporter protein that
concentrates at the nuclear rim in wild type cells at room temperature. A
similar distribution is also observed when wild type cells have been incubated
at 37 °C for 6 h (Fig.
6A). Although mutant strains nup133 and
rat2-1 show a strong association of Ntf2p-GFP with NPC clusters under
permissive conditions, exposure to heat significantly reduced Ntf2p-GFP
localization to clusters (Fig. 6,
A and B). In addition, mutant
nup85
cells displayed reduced binding of Ntf2p-GFP even at
room temperature. This supports the idea that Nup133p, Rat2p, and Nup85p are
required to promote binding of Ntf2p to NPCs of stressed cells. In contrast,
the association of Ntf2p-GFP with nuclear envelopes was only slightly
diminished by heat treatment in mtr7-1, several nsp1, and
nic96 mutants (Fig. 6, A
and B). Even though nup84
cells showed reduced binding of Ntf2p-GFP to the nuclear envelope under normal
and stress conditions, the difference to wild type cells or between control
and heat-treated nup84 cells was not statistically
significant.
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The Guanine Nucleotide Exchange Factor Prp20p Mislocalizes to the
Cytoplasm in nup85, mtr7-1, and
nic96
CCollapse of the Gsp1p gradient may also be
caused by the mislocalization of Gsp1p interacting factors, i.e. the
guanine nucleotide exchange factor Prp20p or the GTPase-activating protein
Rna1p. Under normal growth conditions Prp20p is concentrated in nuclei, where
it is essential to generate Gsp1p-GTP. By contrast, most of Rna1p resides in
the cytoplasm, although nuclear pools of Rna1p have been detected
(46). When analyzed by
immunofluorescence, Prp20p was concentrated in nuclei of wild type and mutant
cells at room temperature (Fig.
7). However, upon heat treatment a significant redistribution of
Prp20p is seen in nup85
, mtr7-1, and
nic96
C. Prp20p mislocalization was most prominent in
mtr7-1, and after 3 h of heat stress less than 35% of the cells had
Prp20p concentrated in nuclei. Moreover, elevated cytoplasmic levels of Prp20p
were detected even after 1 h of incubation at 37 °C. A difference in
Prp20p distribution was also detected for rat2-1, but the effect was
less significant for rat2-1 (p = 0.02) as compared with
nup85
, mtr7-1, and nic96
C
(p < 0.01).
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We have also determined the distribution of Rna1p in wild type and mutant cells under different growth conditions. As reported by others (46) most of Rna1p was cytoplasmic, and the association with nuclei became increased upon heat shock (data not shown). However, we did not detect drastic differences between wild type and mutant cells in the localization of Rna1p. In summary, our studies have identified several nucleoporin mutants in which a collapse of the Gsp1p concentration gradient under stress conditions can be attributed, at least in part, to a mislocalization of Prp20p.
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DISCUSSION |
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To identify novel components of the nuclear envelope that are involved in Gsp1p nuclear accumulation, we have analyzed yeast strains that carry a deletion or mutation in various nucleoporin genes or in MTR7. So far, components of the nuclear envelope other than FG repeat-containing nucleoporins have not been shown to participate in Gsp1p trafficking. Our results demonstrate for the first time that the non-repeat nucleoporins Nup133p, Nup120p/Rat2p, Nup85p, and Nic96p, as well as acetyl-CoA carboxylase regulate the distribution of the small GTPase.
To define the mechanisms that lead to the collapse of the Gsp1p gradient in
mutant strains, we have determined whether nuclear import of the fusion
protein GFP-Gsp1p is compromised under non-permissive conditions. GFP-Gsp1p is
too large to diffuse efficiently across the NPC, and elevated levels of this
reporter protein in the cytoplasm suggest that nuclear import is impaired in
nup133, rat2-1, nup85
,
nic96
C, and mtr7-1. Interestingly,
nup133
, rat2-1, nup85
, and mtr7-1
showed elevated cytoplasmic levels of GFP-Gsp1p even at room temperature,
suggesting that import is already affected under these conditions.
Nup84p, Nup85p, and Nup120p/Rat2p are present in the same NPC subcomplex,
called the Nup84p complex, which also contains Nup145p-C, Seh1p, and Sec13p
(35). Furthermore, Nup133p
associates with the Nup84p complex, indicating that the Nup84p-Nup133p unit
represents a building block of the NPC
(41,
42). To assemble the Nup84p
module Nup85p, Nup120p/Rat2p and Nup145p-C are required, whereas Nup84p and
Seh1p are dispensable (35).
Deletion of NUP84, NUP85, or NUP120/RAT2 changes
nuclear membrane and NPC organization
(34,
35). In particular, a complete
disruption of NUP84 in nup84 cells leads
to an altered distribution of NPCs
(34). Despite the effects of
the nup84 allele on nuclear envelope and NPC
assembly, Gsp1p gradients did not collapse in this mutant at the
non-permissive temperature. These results emphasize that the changes in Gsp1p
localization observed by us for several nucleoporin mutants cannot simply be
ascribed to altered nuclear organization. In contrast to
nup84 cells, Gsp1p gradients collapsed in strains
nup85, rat2-1, and nup133
under
restrictive conditions, which points to a specific role of Nup85p,
Nup120p/Rat2p, and Nup133p in nuclear accumulation of the GTPase. We have now
demonstrated that the defect in rat2-1 and nup133
cells can be attributed to the inefficient association of Ntf2p with NPCs upon
exposure to heat stress. However, Nup133p is not essential for binding Ntf2p
to the NPC under non-stress conditions, demonstrating the presence of
redundant binding sites. Nevertheless, failure of Ntf2p to interact with
mutant NPCs in stressed cells will ultimately dissipate the Gsp1p gradient, as
Ntf2p is required for Gsp1p nuclear import. In contrast to rat2-1 and
nup133
mutants, nup85
, missing another
component of the Nup84p complex, displayed reduced Ntf2p-GFP binding even at
room temperature. This is in line with the observation that GFP-Gsp1p in
nup85
cells already mislocalized at room temperature, similar
to what was detected at 37 °C. Furthermore, Prp20p, the guanine nucleotide
exchange factor for Gsp1p, redistributed in nup85
at elevated
temperature (see below). Thus we have identified multiple defects for this
mutant that will contribute to the collapse of the Gsp1p gradient (summarized
in Table IV). We have also
carried out experiments to further analyze the interaction between Ntf2p and
Nup133p. In line with the idea that Ntf2p forms complexes with this
nucleoporin, we were able to co-purify both components (not shown). However,
results for this co-purification were variable, most likely reflecting the
transient nature of this interaction.
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Even though nuclear export of the ribosomal 60 S subunit depends on the Gsp1p-GTPase cycle, members of the Nup84p complex did not show a major defect in this export reaction (24). These apparent differences can be explained by the distinct conditions used in our analyses. To study export of the 60 S ribosomal subunit, cells were exposed to 33 °C, followed by a 4-h shift to 20 °C. It is presently not known whether the Gsp1p concentration gradient dissipates at 33 °C. Furthermore, it is possible that a collapsed Gsp1p concentration gradient can be rebuilt when cells are returned to 20 °C.
Our data clearly demonstrate a deficiency of rat2-1 cells in the
formation of a Gsp1p gradient and of Ntf2p binding to the NPC under
non-permissive conditions. Moreover, a weak defect was also detected for the
nuclear concentration of Prp20p upon exposure to heat. However, it should be
noted that previous studies of rat2-1 did not reveal a nuclear
protein import defect (21). In
these analyses nuclear import was monitored with a reporter protein containing
the N-terminal 33 amino acid residues of yeast histone H2B fused to
-galactosidase (21).
Yeast histones H2A and H2B were recently shown to be imported into nuclei by
several members of the
-importin family, including Kap114p, Kap121p, and
Kap95p (49). These
-importins directly bind to N-terminal histone nuclear localization
signals (49). By contrast,
SV40-GFP, the reporter protein used by us, is expected to accumulate in nuclei
via the classical Srp1p/Kap95p import pathway. The specific requirements for
nuclear import may differ for the two substrates, thereby explaining the
distinct effects of the rat2-1 mutation on their nuclear
accumulation. Our results reveal the discrete functions of Rat2p in transport
of different cargoes. Although non-classical import mediated by the histone
H2B nuclear localization signal is not impaired at the non-permissive
temperature, a drastic effect is seen for classical nuclear protein import and
Gsp1p nuclear concentration. Our data are in accordance with the idea that
individual members of the Nup84p-Nup133p NPC module can selectively affect
specific aspects of nucleocytoplasmic trafficking.
Nup133p, Nup85p, Nup120p/Rat2p, and Nic96p are located on both the nuclear
and the cytoplasmic side of the NPC
(12). None of these
nucleoporins contains FXFG or GLFG repeats, and only Nic96p carries
heptad repeats implicated in coiled-coil interactions (reviewed in Ref.
39). In line with the latest
models for nuclear trafficking (reviewed in Refs.
47 and
50) several mechanisms, not
mutually exclusive, can be proposed for the Gsp1p gradient collapse and
failure to import GFP-Gsp1p in mutant nucleoporin strains. (a) The
organization of NPCs may be changed in a fashion that alters the binding or
translocation of Ntf2p-Gsp1p to the NPC. (b) Mislocalization of
Gsp1p-interacting factors could affect the Gsp1p gradient. (c)
Mutations might modulate the nuclear pore channel size, thereby preventing
passage of macromolecules across the NPC. (d) Nuclear retention of
the GTPase could be altered by an unknown mechanism. Because SV40-GFP, a
protein of 45 kDa molecular mass, was still able to exit the nucleus in
mutants that collapsed the Gsp1p gradient, a more general obstruction of the
channel seems unlikely. Moreover, we have demonstrated that nuclear
translocation of GFP-Gsp1p was changed in several of the nucleoporin mutants,
even at permissive conditions. Therefore, we favor the first two scenarios,
i.e. changes in the interaction of Ntf2p-Gsp1p with nucleoporins and
relocation of Gsp1p-interacting factors, and we have identified these defects
in several nucleoporin mutants. As a result, the initial binding of
Ntf2p-Gsp1p to NPCs or its subsequent translocation into the nucleus may be
prevented. In addition, the redistribution of Prp20p will affect the
Gsp1p-GTPase cycle, generating elevated levels of Gsp1p-GTP in the cytoplasm.
In either case, the consequence will be a collapse of the Gsp1p gradient.
Like several nucleoporins, MTR7 is also required for Gsp1p gradient formation. MTR7 encodes an acetyl-CoA carboxylase, an enzyme required for de novo synthesis of long chain fatty acids that were proposed to stabilize the NPC at the pore membrane interface (45). Under restrictive conditions, destabilization of NPCs is likely to affect all trafficking reactions, including the nuclear accumulation of Gsp1p, as observed by us. However, even at room temperature nuclear import of GFP-Gsp1p was impaired in mtr7-1 cells, demonstrating that transport already has been altered by changing the lipid composition of the nuclear envelope.
Factors other than Ntf2p, Prp20p, and Rna1p may also be affected and
contribute to the dissipation of the Gsp1p gradient in nucleoporin mutants
studied by us. As such, members of the -importin family have been
proposed to play a role in nuclear retention of Ran/Gsp1p
(6,
7). At this point it is not
clear how much individual nuclear carriers are involved in this process. As of
yet, it is therefore not possible to determine to which extent a potential
redistribution of
-importins in nucleoporin mutants affects the Gsp1p
gradient formation.
In addition to the identification of novel factors involved in Gsp1p nucleocytoplasmic distribution, our results help explain the classical and non-classical transport defects seen for several of the mutants used in this study. Many of the nuclear trafficking reactions require Gsp1p and presumably nucleocytoplasmic gradients of the GTPase. If mutant yeast strains fail to generate a Gsp1p gradient under non-permissive conditions, all nuclear trafficking reactions that rely on such a gradient will be prevented. Thus, the failure to concentrate Gsp1p in the nucleus because of altered binding of Ntf2p to the NPC or relocation of Prp20p may be a primary consequence of some of the nucleoporin mutations and contribute to the observed transport defects. Furthermore, reduced Gsp1p levels as observed for some of the mutations analyzed by us can be expected to alter nuclear transport as the GTPase may become the limiting factor for classical or non-classical pathways.
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FOOTNOTES |
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The on-line version of this article (available at
http://www.jbc.org)
contains Fig. 1.
Both authors contributed equally to this work.
¶ Supported by grants from National Science and Engineering Research Council of Canada, Canadian Institutes of Health Research, and the Heart and Stroke Foundation of Quebec. A Chercheur National of Fonds de la Recherche en Santé du Québec. To whom correspondence should be addressed: Dept. of Physiology, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-2949, Fax: 514-398-7452; E-mail: ursula.stochaj{at}mcgill.ca.
1 The abbreviations used are: NPC, nuclear pore complex; DAPI,
4',6-diamidino-2-phenylindole; Nup, nucleoporin; PBS, phosphate-buffered
saline; TRITC, tetramethylrhodamine isothiocyanate; GFP, green fluorescent
protein.
2 S. M. Bailer and E. C. Hurt, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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