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Molecular and Cellular Biology, February 1999, p. 1547-1557, Vol. 19, No. 2
Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School, and The Dana-Farber
Cancer Institute, Boston, Massachusetts 02115
Received 12 August 1998/Returned for modification 12 October
1998/Accepted 28 October 1998
Proteins to be transported into the nucleus are recognized by
members of the importin-karyopherin nuclear transport receptor family.
After docking at the nuclear pore complex (NPC), the cargo-receptor complex moves through the aqueous pore channel. Once cargo is released,
the importin then moves back through the channel for new rounds of
transport. Thus, importin and exportin, another member of this family
involved in export, are thought to continuously shuttle between the
nuclear interior and the cytoplasm. In order to understand how nuclear
transporters traverse the NPC, we constructed functional protein
fusions between several members of the yeast importin family, including
Pse1p, Sxm1p, Xpo1p, and Kap95p, and the green fluorescent protein
(GFP). Complexes containing nuclear transporters were isolated by using
highly specific anti-GFP antibodies. Pse1-GFP was studied in the most
detail. Pse1-GFP is in a complex with importin- Macromolecules move between the
nucleus and the cytoplasm via aqueous channels spanning the nuclear
envelope, termed nuclear pore complexes (NPCs). Transported molecules
include proteins that move from the cytoplasm into the nucleus, RNAs
that move outward to the cytoplasm, and proteins that shuttle back and
forth. Thus, the processes of macromolecular import and export are
intimately connected.
In general, transport in or out of the nucleus begins with recognition
of the transported cargo by its cognate nuclear transport "receptor". Proteins destined for the nuclear interior contain nuclear localization sequences (NLSs). The best characterized NLSs are
from simian virus 40 T antigen and nucleoplasmin (44). Proteins containing these so-called "classical" NLSs are recognized in the cytoplasm by a heterodimeric receptor termed importin (or karyopherin) (29, 65). The NLS is bound by the smaller
importin- Although many NLS-containing proteins use importin- The exit of proteins (and at least some RNA/protein complexes) from the
nucleus appears to occur in a manner reciprocal to protein import, as
illustrated by the human immunodeficiency virus Rev protein. Once
inside the nucleus, Rev binds to Rev response element-containing RNAs
and moves out of the nucleus (19). Rev and other similarly
exported proteins contain a short stretch of leucine-rich amino acids,
now termed the nuclear export signal (NES), that mediates their nuclear
export (19, 26). The phenomenon of NES-dependent export led
to the identification of export "receptors," e.g., mammalian
exportin and yeast Xpo1p/Crm1p, that bind NESs (22, 25, 61,
77). Exportins are also members of the importin- A general model is that cargoes move into or out of the nucleus
complexed with their receptor. Once the cargo-receptor complex has
reached its proper destination (i.e., the nucleoplasm or cytoplasm), the cargo dissociates and the transport receptors recycle for new
rounds of transport. In support of this view, some importin- In addition to the The NPC is a complex array of proteins spanning the double membrane
bilayer of the nuclear envelope. The complete composition of the
mammalian NPC is not known but, due in part to the sequencing of the
yeast genome, the composition of the simpler yeast NPC is almost
completely known (17). However, how macromolecules pass
through the NPC channel remains a mystery. Blot overlay experiments have shown interactions between importins and various nucleoporins (Nups) in yeast and mammalian cells. Many Nups contain repeats of FXFG
and/or GLFG. These repeats have been suggested to function as
Taken together, macromolecular transport in or out of the nucleus
begins with binding of the transported cargo to the correct importin or
exportin. After docking at the NPC, the cargo-receptor complex somehow
moves through the nuclear pore channel. Thus, loading and unloading of
cargoes and the interactions of cargo-receptor complexes with the NPC
are the defining events of nuclear transport.
To understand how the cargo-receptor complexes move through the NPC, we
have undertaken studies with the yeast Saccharomyces cerevisiae. The S. cerevisiae genome encodes at least
14 proteins with some similarity to importin- Construction of yeast strains expressing GFP-tagged
importins.
To replace PSE1 with an engineered gene
encoding Pse1-GFP, we subcloned a ClaI-KpnI
fragment containing the PSE1 open reading frame (ORF) fused
in frame to the S65T V163A mutant of GFP and the NUF2 3' UTR
(42) from pPS1069 (74) into the nonreplicating plasmids pRS304 and pRS306 (76). The resulting plasmids,
pPS1538 (TRP1) and pPS1539 (URA3), were
linearized with NsiI or BstEII, and the linear
DNA was transformed into PSY580 [(MATa ura3-52 leu2 Antibodies.
To raise the anti-GFP polyclonal antibody,
recombinant GFP was purified from an Escherichia
coli-overproducing strain and injected into rabbits. Briefly,
pJK83 a plasmid for the expression of 6×His-tagged GFP (S65T; V163A
mutant) was transformed into XL1-Blue bacteria. To generate pJK83, the
ORF of GFP was amplified by PCR with BamHI-linked
oligonucleotides. The resulting PCR product was digested with
BamHI and ligated into the bacterial expression vector pQE9
(Qiagen). 6×His-GFP was prepared by using Ni-NTA agarose resin
(Qiagen) as directed by the manufacturer. The protein was injected into
New Zealand White rabbits, and anti-GFP antibody was affinity purified
from raw serum with GFP covalently attached to NHS-derivatized
Sepharose (Pharmacia). GFP-specific antibodies were eluted with first
100 mM glycine (pH 2.5) and then 100 mM triethylamine (pH 11.5) and
neutralized. Eluted antibodies were dialyzed against phosphate-buffered
saline (PBS) and stabilized with 10 mg of bovine serum albumin (BSA)
per ml and 0.05%. NaN3, with a final antibody
concentration of 1 mg/ml.
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Interactions between a Nuclear Transporter and a
Subset of Nuclear Pore Complex Proteins Depend on Ran GTPase
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
and -
(Srp1p and
Kap95p in yeast cells) that is sensitive to the nucleotide-bound state
of the Ran GTPase. In addition, Pse1p associates with the nucleoporins
Nsp1p, Nup159p, and Nup116p, while Sxm1p, Xpo1p, and Kap95p show
different patterns of interaction with nucleoporins. Association of
Pse1p with nucleoporins also depends on the nucleotide-bound state of
Ran; when Ran is in the GTP-bound state, the nucleoporin association is
lost. A mutant form of Pse1p that does not bind Ran also fails to
interact with nucleoporins. These data indicate that transport
receptors such as Pse1p interact in a Ran-dependent manner with certain nucleoporins. These nucleoporins may represent major docking sites for
Pse1p as it moves in or out of the nucleus via the NPC.
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
subunit, which interacts with the larger importin-
subunit for docking at the NPC and subsequent passage into the nucleus
(11, 18, 31, 32, 36, 56, 81). In some cases, importin- appears to bind and transport cargoes without importin- (34, 40).
/ to enter the
nucleus, others do not contain the classical NLS and do not interact
with importin-/. Instead, they interact with different import
receptors that are members of a family of proteins related to
importin-. For example, the mRNA-binding protein hnRNPA1 contains a
novel NLS that binds to transportin for its nuclear import (10, 24, 64). Transportin is one of several importin--like proteins that have no corresponding -like partner, bind cargo directly, and
dock at and move through the NPC (reviewed in reference
84).
family.
Related export receptors for tRNAs have recently been identified
(4, 33a, 50).
proteins have been shown to cycle between the nucleus and the cytoplasm
(38, 48, 77). In doing so, proteins not only interact
with their respective cargoes but also with proteins of the NPC
(75).
proteins, the GTPase Ran and its regulators are
central to the movement of macromolecules through the NPC. Ran is found
in both the nucleus and the cytoplasm, whereas the Ran
GTPase-activating protein (GAP) functions in the cytoplasm (7, 14,
35, 54) and the Ran GTP exchange factor (GEF) in the nucleus
(3, 9, 60). This asymmetric distribution of the RanGAP and
GEF with respect to the nuclear envelope has led to models of how
molecules move in a vectorial manner between the nucleus and the
cytoplasm (e.g., references 30, 45, and 53). According to one model, cytoplasmic RanGAP
means that the Ran-GDP concentration would be high in the cytoplasm.
The nuclear location of Rcc1 (the Ran GEF) would cause the
concentration of Ran-GTP to be high in the nucleus. These distinct
nucleotide-bound states of Ran are proposed to promote the binding
and/or release of cargo from its particular carrier in the proper
compartment and thus allow recycling of the receptors (12, 22, 30,
39, 49, 67). Recent reports suggest that importin- can move
through the NPC without binding cargo (46). However, the
precise mode of Ran action remains controversial.
-binding sites, and solution binding assays have provided some
support for this hypothesis (2, 37, 56, 67, 69). However,
these in vitro binding experiments with recombinant proteins may not
adequately reproduce the specificity of the -Nup interaction. Complexes containing importins and nucleoporins isolated from Xenopus cells indicate more-specific interactions
(75) with certain nucleoporins on both the nucleoplasmic and
cytoplasmic NPC surface.
(59). These
proteins and their metazoan relatives compose the importin-
superfamily. The cell thus appears to have evolved an array of related
carrier proteins to deliver diverse cargoes into and out of the
nucleus. The relatedness of the carriers may reflect interactions with common transport factors such as Ran, whereas their diversity may
reflect their ability to distinguish cargoes and nucleoporins. In order
to address how importin-s move through the NPC, we have studied the
interactions between several yeast importin- family members and
nucleoporins by creating yeast strains where the only functional
version of a particular nuclear transport receptor is fused to green
fluorescent protein (GFP). Using highly specific anti-GFP antibodies,
we have isolated and characterized complexes containing members of the
importin family. In doing so, we have found that different
importin-s associate with distinct nucleoporins. Further, by
analyzing one family member, Pse1p, in detail in mutants that affect
the nucleotide-bound state of Ran, we show that interactions with
distinct nucleoporins are Ran dependent.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
1 trp163 GAL+) and mutant strains
rna1-1 and prp20-1. The prp20-1
(PSY713 [45]) and rna1-1 (PSY714
[14]) strains result from backcrosses with FY86
(PSY581) and FY23 (PSY580). FY86 and FY23 are isogenic haploid S288c
strains that are auxotrophic for different amino acids with opposite
mating types (82). Similarly, to replace SXM1, we
subcloned a SalI-KpnI fragment containing the
SXM1 gene fused to GFP from pPS1117 (74) into
pRS304, and the resulting plasmid pPS1541 was linearized with
NsiI and transformed into PSY580. For XPO1, an
ApaI-BamHI fragment containing
XPO1-GFP (pKW470 [77]) was subcloned into
pRS304, linearized with XcmI, and transformed into PSY580.
To replace the KAP95 gene, we generated pJK169, which contains a 3' fragment of the KAP95 gene amplified by PCR
with the primers GATATTGCCTATGAGCTCG and
GGctcgagCTAAGGATAATTGACGCTTC. The resulting PCR product was
digested with SacI and XhoI and ligated into the
similarly cut pPS967 (41). The resulting plasmid featured
the last 1,689 bp of the KAP95 ORF fused in frame to the
S65T V163A mutant GFP and the NUF2 3' UTR. pJK169 was
linearized with AgeI and transformed into an S288c-derived
diploid strain. Transformants were selected for Ura+
prototrophy and checked by fluorescence microscopy for expression of
Kap95p-GFP. The diploid strain was sporulated, and the functionality of
Kap95p-GFP was assessed by determining the viability of the Ura+ spores. Of 12 tetrads dissected, 11 had four viable
spores and, in all cases, the Ura+ phenotype segregated
2:2. All Ura+ spores exhibited nuclear rim signal by
fluorescence microscopy. Furthermore, immunoblot analysis with a
polyclonal antibody raised against the Kap95p protein indicated that,
in Ura+ cells, there was no expression of the unfused
Kap95p protein. Therefore, we conclude that the Kap95p-GFP protein is
functional. Because the Kap95p-GFP protein is functional, pJK169 was
transformed into the haploid strain PSY685 (MATa
ura3-52 his3200 trp163 leu21) to make PSY1227.
Immunoprecipitations.
Lysates for immunoprecipitations were
usually prepared from 50-ml cultures grown at 25°C in yeast
extract-peptone-dextrose (YEPD) medium to a cell density of
107 cells/ml. Wild-type cells transformed with pPS814 (a
gift from Jim Haseloff, Medical Research Council, Cambridge, United
Kingdom) encoding a fusion of GFP to -galactosidase were grown in
selective medium. Cells were washed in PBS (137 mM NaCl, 1.76 mM
KH2PO4, 5.4 mM Na2HPO4,
2.7 mM KCl; pH 7.2) and resuspended in 1 ml of lysis buffer (PBS plus 3 mM MgCl2, 1 mM EDTA, and 0.5% Triton X-100) with protease
inhibitors added (0.5 mM phenylmethylsulfonyl fluoride [PMSF]; 2.5 µg each of leupeptin, chymostatin, antipain, pepstatin A, and
aprotinin per ml [all from Sigma]). In some cases, radioimmunoprecipitation assay (RIPA) buffer (1% Nonidet P-40, 0.5%
sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS] in 150 mM
NaCl and 50 mM Tris-HCl [pH 8.0]) with protease inhibitors was used.
To lyse cells, a one-third volume of glass beads (425 to 600 µm in
diameter) was added, and the samples were cooled on ice and treated
three times mechanically for 1 min each cycle with a Mini-Bead Beater
(Advanced Laboratory Research, Inc., Franklin, Mass.) at maximum speed.
Lysates were clarified by centrifugation for 10 min at
12,000 × g at 4°C. To remove all cell debris, the supernatants were transferred into fresh tubes and centrifuged again.
Protein concentrations of the lysates were determined with a protein
assay kit (Bio-Rad). Equal amounts of protein corresponding to
approximately 25 ml of culture were incubated with 7.5 µl of anti-GFP-beads for 30 min at 4°C. The beads were washed three times
in lysis buffer and two times in lysis buffer without Triton X-100.
Bound proteins were eluted with SDS buffer (50 mM Tris-HCl [pH 6.8],
100 mM dithiothreitol [DTT], 2% SDS, 0.1% bromophenol blue, 10%
glycerol) and separated on an 8 or 12% gel by SDS-polyacrylamide gel
electrophoresis (PAGE), followed by transfer to nitrocellulose membrane
(Protan; Schleicher & Schuell). Membranes were temporarily stained with
Ponceau-S (Sigma) to confirm equal loading. Silver-staining analyses of
the corresponding samples revealed the GFP fusion to be the major
component, with additional bands present at lower levels, some of which
changed in a Ran-dependent manner (data not shown). To generate
anti-GFP-beads, we incubated 200 µl of protein G-Sepharose (50%
slurry; Pharmacia) with 100 µl of affinity-purified anti-GFP
antibodies (1 mg/ml) in PBS with 10 mg of BSA per ml for 15 min at
25°C. The beads were washed three times with PBS and then resuspended
in PBS plus 0.05% NaN3 to a final volume of 200 µl.
Immunoblots. To detect proteins, membranes were incubated for 1 h at 25°C with antibodies diluted in PBS-2.5% milk powder-0.05% Tween, followed by horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch Laboratories) and detection with enhanced chemiluminescence (Amersham Corp.). To analyze GFP-tagged proteins, we diluted the affinity-purified antibodies as follows: GFP-specific antibodies, 1:5,000; Pse1p-specific antibodies, 1:4,000; Kap95p-specific antibodies, 1:500; Srp1p-specific antibodies, 1:5,000; Nup159p-specific antibodies, 1:10,000; Nsp1p-specific antibodies, 1:1,000; Nup116p-specific antibodies, 1:1,000; and Pom152p-specific MAb 118C3, 1:5. To detect Ran/Gsp1p we used a 1:1,000 dilution of the Ran/Gsp1p-specific antibodies in PBS, 2.5% milk powder, and 0.01% Tween.
Immunofluorescence. Cells were spheroplasted with 300 µg of Zymolase (100T; ICN) in solution P (0.1 M K2HPO4-KH2PO4 [pH 6.5], 1.2 M sorbitol) plus 25 mM DTT, washed in solution P, placed on slides coated with 0.3% polylysine, and permeabilized by the addition of 0.5% Nonidet P-40 in solution P for 5 min. Cells were blocked for 1 h with 5 mg of BSA per ml in 0.1 M Tris (pH 9.0), 150 mM NaCl, and 0.3% Triton X-100, followed by overnight incubation with a 1:2,000 dilution of MAb 414 (Babco) and a 1:100 dilution anti-Nop1p antibodies. Cells were washed two times with 0.1 M Tris (pH 9.0)-150 mM NaCl (Buffer A) and two times with 0.1 M Tris (pH 9.5)-100 mM NaCl-50 mM MgCl (Buffer B) and incubated for 1 h with mouse-specific Texas Red-conjugated secondary antibodies (Jackson Immunoresearch Laboratories) in a 1:1,000 dilution. Samples were washed two times in Buffer A and two times in Buffer B, and chromatin was stained with 1 µg of 4'6-diamidino-2-phenylindole (DAPI) for 5 min in Buffer B, followed by two washes with Buffer B.
GFP microscopy. A Nikon Optiphot-2 epifluorescence microscope and a ×100 Plan APO 1.4 NA DIC objective were used to observe the yeast cells. A Chroma filter number 41018 (excitation HQ470/40, dichroic Q495LP, emission HQ 500LP; Chroma Technology Corp., Brattleboro, Vt.), together with a Princeton Instruments Micromax camera equipped with a Kodak KAF 1400 chip (1,317 × 1,035, 6.8 × 6.8 µm pixels; Princeton Instruments), operated by Metamorph Imaging software (Universal Imaging Corp., West Chester, Pa.), was used to capture the GFP images. The final figures were produced by using Adobe Photoshop without further manipulation.
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RESULTS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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The main focus of the following experiments is the dynamic
behavior of the importin--like protein Pse1p. Pse1p is an essential protein implicated in the import of ribosomal proteins and certain transcription factors into the nucleus and perhaps also in the export
of mRNAs from the nucleus (40, 43a, 69, 72, 74). Moreover,
its function is at least in part redundant with that of the
nonessential Kap123p, as evidenced by the synthetic lethality occurring
between pse1-1ts and kap123
mutants (74).
In order to study the dynamics of Pse1p and some of its relatives, we have employed the following general strategy. Gene fusions were created that encode the entire protein of interest fused to a bright derivative of GFP (43) so that their distribution in cells can be observed directly by fluorescence microscopy without perturbations introduced by cell fixation. These were then introduced into the yeast genome so as to completely replace their wild-type counterparts, making the GFP fusion protein the only functional version in the cell. One of the strengths of this approach is that the distributions and interactions of the proteins are being studied at their normal levels and are not impacted by possible artifacts of overexpression from multicopy plasmids or competition from endogenous wild-type protein. Moreover, since the proteins remain fully functional, as evidenced by their ability to substitute for the wild-type version, the GFP moiety does not appear to alter their functions.
Replacement of PSE1 by a chimeric gene encoding a functional Pse1-GFP fusion protein is described in Materials and Methods. An immunoblot with anti-Pse1p antibodies confirms that the only version of Pse1p present in these strains is the GFP fusion. Pse1p is present in wild-type cells as a protein of the predicted molecular size of 121 kDa (Fig. 1A, lane 1). However, in lysates from cells where the endogenous PSE1 is replaced by PSE1-GFP, the anti-Pse1p antibody recognizes only a protein of 150 kDa (Fig. 1A, lane 2), which corresponds to the predicted molecular size of Pse1p plus GFP. This is confirmed by immunoblots probed with anti-GFP antibodies. No cross-reacting protein is present in the wild-type cells (Fig. 1A, lane 3), whereas the 150-kDa Pse1-GFP is observed in the strain containing the corresponding gene fusion as the only version of PSE1 (Fig. 1A, lane 4). Confirmation that Pse1-GFP is completely functional is illustrated in Fig. 1B; cells containing Pse1-GFP grow in a manner identical to the wild-type PSE1 cells at both 25 and 37°C.
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In order to compare the behavior of Pse1p to other importin--like
proteins, strains were similarly constructed that contained only the
GFP-tagged versions of SXM1, XPO1/CRM1, and
KAP95. In each case, we confirmed that fusion proteins of
the correct size were made and were functional in that their presence
resulted in normal cell growth at all temperatures (data not shown).
Fluorescence microscopy of cells grown at 25°C revealed a similar
localization of Pse1-GFP, Sxm1-GFP, and Kap95-GFP. The three
importin-s are located at the nuclear envelope (Fig.
2A, first three panels), with some
protein present in the cytoplasm and the nucleus as well. This
distribution is consistent with the idea that these proteins may move
between the nuclear interior and the cytoplasm, with a rate-limiting
step occurring at the NPC. Sxm1-GFP, compared to Pse1-GFP and
Kap95-GFP, shows more intranuclear accumulation. In contrast, Xpo1-GFP
at steady state is restricted to the nucleus and the nuclear envelope
(Fig. 2A, last panel). Unlike the other three proteins, Xpo1p is
proposed to function only in export and thus may spend less time in the
cytoplasm (77).
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Interaction of Pse1-GFP with other importins. In order to analyze interactions of Pse1p with other transport factors and the NPC, we isolated Pse1-GFP from cell lysates by using anti-GFP specific antibodies. Highly specific anti-GFP antibody was raised and affinity purified with recombinant GFP. The resulting antibodies were coupled to Sepharose beads for adsorption of the GFP fusion proteins from cell extracts.
Conditions were established for efficient solubilization of the GFP fusion proteins. In order to efficiently extract Pse1-GFP, we found that detergent must be present in the lysis buffer. A comparison of the amount of Pse1-GFP present in lysates prepared by glass bead lysis by using buffer without detergents, buffer with 0.5% Triton X-100, or RIPA buffer revealed only a minor fraction of the total Pse1-GFP extracted when no detergent was present in the buffer (data not shown). In contrast, the amount of Pse1-GFP in lysates prepared with 0.5% Triton X-100 represented a significant fraction of the total and did not increase when the more stringent RIPA buffer was used. Therefore, all experiments were carried out by lysing cells in buffer containing 0.5% Triton X-100. Pse1-GFP, Sxm1-GFP, Xpo1-GFP, and Kap95-GFP were isolated intact from cell lysates by immunoprecipitation with GFP-specific antibodies coupled to protein G-Sepharose. All tested GFP-tagged proteins were efficiently precipitated, including a negative control of LacZ-GFP (Fig. 3A, top panels). The bound fractions analyzed represent approximately 25-fold more than that analyzed for total lysates.
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should bind importin-. Therefore, as a
control for our experimental conditions, we probed the
immunoprecipitates for the presence of importin- by immunoblotting
with affinity-purified antibody, raised against Srp1p, the yeast
homologue of importin-. As predicted, a significant portion of the
total Srp1p remained associated with Kap95-GFP (Fig. 3A, lane 9). No
Srp1p associated with Sxm1-GFP, Xpo1-GFP, or LacZ-GFP. However, a
reproducibly smaller amount of Srp1p was always observed in association
with Pse1-GFP (Fig. 3A, lane 6). Moreover, when the same precipitates were probed with affinity-purified antibody against Kap95p, it was also
present in association with Pse1-GFP. These results suggest that Pse1p
may bind directly to Srp1p/Kap95p or be part of larger complex that
perhaps also contains NPC binding partners, a topic which will be
addressed below. The observation that other importin-s do not
display a similar interaction suggests that this association with
Srp1p-Kap95p is specific to Pse1p.
Ran regulators affect the behavior of Pse1p. In yeast cells, the two major Ran/Gsp1p regulators are encoded by PRP20 and RNA1. Prp20p corresponds to the nuclear-localized GTP exchange factor for Gsp1p (20). Temperature-sensitive prp20-1 mutants result in loss of function of Prp20p, a block in nuclear transport, and presumably a decrease in the ratio of nuclear Ran-GTP to Ran-GDP (1, 23). Rna1p corresponds to the cytoplasmically located GAP for Gsp1p (5, 8, 14). The use of temperature-sensitive rna1-1 mutants results in loss of function of Rna1p, a block in nuclear transport and presumably a corresponding increase in the ratio of Ran-GTP to Ran-GDP in the cell (14, 35). In order to directly examine the effect on the distribution of Pse1-GFP of the nucleotide-bound state of Gsp1p in cells, we replaced the genomic PSE1 gene with PSE1-GFP in rna1-1 and prp20-1 strains. Both strains remained temperature sensitive after the gene replacement (data not shown).
The intracellular distribution of Pse1-GFP in the mutant cells was examined by fluorescence microscopy at 25°C and following a shift to the nonpermissive temperature of 37°C. In rna1-1 cells, some of the nuclear envelope-associated GFP fluorescence was lost, with a corresponding increase in the cytoplasmic fluorescence (Fig. 2B, middle panel), suggesting less association of Pse1-GFP with the nuclear envelope. In contrast, in prp20-1 cells most of the Pse1-GFP fluorescence was concentrated at the nuclear rim (Fig. 2B, right panel), a finding consistent with an increase in the association of Pse1-GFP with the NPC. The total amount of Pse1-GFP was not altered in any of the mutant backgrounds when compared to the wild-type cells (data not shown). The apparent high concentration of Pse1-GFP at the nuclear envelope in prp20-1 cells suggests that the nucleotide-bound state of Ran/Gsp1p plays a role in Pse1p's docking at or release from the NPC as it moves in or out of the nucleus. The association of Pse1-GFP with the Srp1p/Kap95p complex is disrupted in rna1-1 cells. Lysates were prepared from rna1-1 cells at either permissive or nonpermissive temperature, and Pse1p-interacting proteins were assessed by immunoprecipitation with anti-GFP antibodies as described above. In both cases, the amount of coprecipitating Srp1p/Kap95p was significantly decreased (Fig. 3B, lanes 2 and 5) compared to extracts prepared from wild-type cells (Fig. 3B, lanes 1 and 4). On the other hand, little effect on the interaction of Pse1-GFP with importins was observed for extracts prepared from prp20-1 cells at either temperature (Fig. 3B, lanes 3 and 6). If anything, the amount of bound Kap95p/Srp1p increased slightly in prp20-1 cells. Moreover, under any conditions that generated a high concentration of Ran/Gsp1p-GTP, a decrease in the level of binding of Srp1p/Kap95p to Pse1-GFP was observed. That this effect is mediated by the nucleotide-bound state of Ran is supported by the following observations (data not shown). First, the addition of the nonhydrolyzable GTP-analogue GMP-PNP to extracts decreased the amount of Srp1p associated with Pse1-GFP. Second, the overexpression of a mutant Ran/Gsp1p stabilized in the GDP-bound form (T26N) as opposed to overexpression of mutant Ran/Gsp1p stabilized in the GTP-bound form (G21V) (71, 83) also increased the amount of Srp1p associated with Pse1-GFP. Ran-GTP is known to dissociate the importin-/ complex (12, 30, 67). Dissociation could also release Srp1p/Kap95p from Pse1p or perhaps from a common binding
site shared with Pse1p at the NPC.
Interactions between importins and proteins of the NPC.
To
examine interactions between members of the importin- family and
proteins of the NPC, we probed equal amounts of the immunoprecipitated Pse1-GFP, Sxm1-GFP, Xpo1-GFP, and Kap95-GFP complexes with antisera specific for various nucleoporins. Both Kap95-GFP and Pse1-GFP complexes contained significant amounts of the nucleoporin Nsp1p, whereas little Nsp1p was present in association with Sxm1-GFP or
Xpo1-GFP (Fig. 4A). As we have previously
reported, we consistently observe enrichment of a
higher-molecular-weight protein (which we designate Nsp1p*) that reacts
with the affinity-purified anti-Nsp1p antibody in association with
Kap95p but not with Pse1p (80). The nature of this
presumably modified form of Nsp1p is not known, but treatment with
phosphatase does not affect its mobility on the SDS gel (data not
shown).
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complexes contain only minor
amounts of Nup159p/Rat7p (Fig. 4A, lanes 3 to 5).
The immunoprecipitates were also probed with antibodies to a number of
other nucleoporins which were not found to be present in the complex.
These include antibodies to Pom152p (Fig. 4A), Nup145p, Gle2p, Nup82p,
and Nup1p (data not shown).
Consistent with the alterations in localization of Pse1-GFP at the
nuclear envelope in rna1-1 and prp20-1 mutants,
the interactions of Pse1p with nucleoporins are also affected in these
mutants. The amount of Nsp1p, Nup116p, and Nup159p associated with
Pse1-GFP is drastically reduced when immunoprecipitates are prepared
from rna1-1 cells (Fig. 4B, lanes 2 and 5). In contrast,
Pse1-GFP still displays interactions with Nsp1p, Nup116p, and Nup159p
in extracts from prp20-1 cells (Fig. 4B, lanes 3 and 6),
suggesting that the Pse1p-NPC complex is stable when the concentration
of Ran-GDP is high but not when Ran-GTP predominates. Interestingly,
the amount of Nup159p associated with Pse1-GFP did decrease somewhat in
the prp20-1 strain compared to the wild type (Fig. 4B, lanes 3 and 6).
Mutations in PSE1 affect the localization of the mutant
protein.
We have previously described temperature-sensitive
alleles of PSE1 (74). Sequence analysis of the
PCR-generated pse1-1 reveals 11 mutations corresponding to
10 amino acid changes and a G-to-A mutation 11 bp upstream of the ATG
(see Fig. 5A). Five of the ten amino acid changes are located in the
N-terminal region of Pse1p within the Ran-binding domain, as defined by
homology among importin-s (27).
|
Mutant Pse1-1p does not interact with Ran-GTP. No Ran/Gsp1p was detectable in a complex with mutant Pse1-1-GFP, whereas wild-type Pse1-GFP could be isolated complexed to Ran/Gsp1p. Equal amounts of cell lysate from either wild-type or pse1-1 cells (grown at 25°C) were adsorbed to anti-GFP Sepharose and analyzed for the presence of Ran/Gsp1p. In the presence of the GTP analog GMP-PNP, no Ran/Gsp1p was bound by Pse1-1-GFP, whereas binding to wild-type Pse1-GFP was observed (Fig. 6A, lanes 3 and 4). This result was also seen for wild-type and mutant Pse1-GFP proteins when cells were shifted to 37°C (data not shown). As we observed previously, mutant Pse1-1p is produced at lower levels than wild-type Pse1p, and this is confirmed by the total amount of GFP fusion associated with the Sepharose beads (Fig. 6A, top panels). Thus, in order to ensure that we did not miss the presence of Ran/Gsp1p-GTP associated with Pse1-GFP, 10 times more pse1-1 lysate was also tested. Even under these conditions, no Ran/Gsp1p was present in the Pse1-1 immunoisolated complexes (Fig. 6B).
|
Mutant Pse1-1p fails to interact with importins and some proteins of the NPC. The ability of Pse1-1-GFP to interact with nucleoporins and importins compared to wild-type Pse1-GFP was examined at both 25 and 37°C. Compared to the wild-type Pse1-GFP, very low amounts of Srp1p/Kap95p were present in the immunoprecipitated Pse1-1-GFP complex (Fig. 7, compare lanes 1 and 2 to lanes 3 and 4) at either temperature. Similarly, the amount of copurifying Nup116p and Nup159p was significantly less for Pse1-1. At 25°C, some Nsp1p remained associated with the mutant Pse1-1-GFP, but at 37°C most Nsp1p was absent from the Pse1-1-GFP (Fig. 7, lanes 3 and 4).
|
| |
DISCUSSION |
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|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
Elucidating how nuclear transporters such as Pse1p and other
members of the importin- superfamily move through the aqueous channel of the NPC provides the key to understanding the vectorial nature of nuclear transport. The asymmetric distribution of the major
regulators of the Ran GTPase plays a central role in the transport
process. The levels of Ran-GDP would be high in the cytoplasm due to
the localization of the RanGAP (Rna1p) in the cytoplasm and at the NPC
(14, 35), perhaps on its cytoplasmic face. Conversely, the
levels of Ran-GTP would be high in the nucleus because of the
nuclear-localized exchange factor, Rcc1 (or Prp20p in yeast cells)
(60). Additional regulators reside in both compartments and
further effect the nucleotide-bound state of Ran (e.g., references 15, 52, 55, 63, 73, and 79).
To understand further how macromolecules are transported in and out of
the nucleus, we have studied in detail the protein interactions of an
essential member of the importin- family, Pse1p, in the yeast
S. cerevisiae. We find that under conditions where the Ran
nucleotide exchange factor, Prp20p, is inactive, Pse1p associates with
the importin-/ heterodimer and with certain nucleoporins. When
the RanGAP, Rna1p, is inactive, these interactions are lost and less
Pse1p appears to be localized at the nuclear envelope. Taken together,
we suggest that these interactions reflect major NPC binding sites for
certain nuclear transport receptors as they pass through the nuclear
pore channel.
There are several possible explanations for how certain cargoes together with their transport receptors move between the nucleus and the cytoplasm. By one model, cargo loading would allow the receptor-cargo complex to move in the proper direction through the NPC (22, 32, 36, 45, 49). Once through, cargo unloading would result in a change in confirmation, resulting in recycling of the transport receptor for further rounds of transport (6, 12, 67). Cargo loading and unloading would be effected by the different nucleotide-bound states of Ran in the nucleus versus the cytoplasm. This model is supported by the observations that Ran-GTP dissociates cargo from importins, which would be expected to occur on the nucleoplasmic side of the pore (21, 30, 67). Furthermore, Ran-GTP stimulates binding of NES-bearing cargoes to exportins, which also would occur in the nucleus prior to their movement out (4, 22, 67). Additional experiments have also indicated the presence of Ran-GDP in complex with importin and cargo as it docks on the cytoplasmic side of the NPC during the first step of import (13).
A second possibility is that the nuclear transport receptors simply
move continuously in both directions through the NPC independent of
cargo and/or Ran. Support for this possibility comes from observations that importin- mutants that do not bind Ran can still enter the nucleus (28, 46, 58). However, this may not accurately
reflect the state of importin in the cell, where it may always be
exposed to some form of Ran. Thus, in any model, the interaction of
transport receptors with the NPC, as well as loading and unloading of
cargoes, are the defining events of nuclear transport.
Several features of the yeast system have allowed us to develop a powerful approach to simultaneously assess the dynamics and the protein-protein interactions of transporters such as Pse1p under physiological conditions. First, we have taken advantage of the ability of yeast to efficiently express functional GFP fused to a number of proteins. We also substituted the GFP fusions for the normal copy of a particular gene, thus creating yeast strains where the only version of the protein of interest is the GFP fusion expressed at its wild-type levels. Finally, we have generated a highly specific polyclonal anti-GFP antibody that, when coupled to Sepharose, efficiently precipitates GFP fusion proteins from yeast cells. We can use these tools to observe the location of a particular protein without artifacts introduced by fixation or overexpression, and we can employ GFP as an effective affinity tag.
Using this approach, we have documented interactions between Pse1p and the NPC. We found that Pse1p localizes at the nuclear envelope and interacts with three nucleoporins, Nup159p, Nup116p and Nsp1p, under conditions where Ran-GTP should be low, e.g., in the prp20-1 mutant (1). When Ran-GTP is high, e.g., in an rna1-1 (14) mutant or in the presence of GMP-PNP, Pse1p is located in the cytoplasm and no longer binds to the three nucleoporins. These data are summarized in Fig. 8. Moreover, a mutant Pse1p that can no longer bind Ran does not efficiently localize at the NPC or bind to nucleoporins but does appear to be able to enter the nucleus.
|
One interpretation of these data is that Nup159, Nup116, and Nsp1p are part of a major docking site for Pse1p as it moves into the nucleus. Thus, when Ran-GDP is high in prp20-1, this might favor the NPC docked form. When Ran-GTP is high, this would prevent docking and Pse1p would remain cytoplasmic. This interpretation is supported by the observations that at least a portion of Nsp1p and Nup159p are located on the cytoplasmic side of the NPC (47, 70).
Another possibility is that the different nucleoporin interactions represent distinct steps as Pse1p moves in or out of the nucleus. For instance, the association with Nsp1p may be the import docking step favored by Ran-GDP. Conversely, the binding to Nup159p might reflect movement of Pse1p out of the nucleus, which might require interaction of Pse1p with Ran-GTP once it has entered the nucleus. Thus, in prp20-1 where Ran-GTP would be lower, the interaction of Pse1p with Nup159p is slightly lower compared to wild-type cells. These ideas are supported by the fact that nsp1 mutants have a block in nuclear import (57, 70), whereas nup159/rat7 mutants have a block in nuclear export (at least of export of mRNAs (16)). However, our data do not address which nucleoporins are in direct contact with Pse1p as opposed to part of a larger complex.
We also created strains expressing only functional Sxm1-GFP, Xpo1-GFP,
and Kap95-GFP. Interestingly, we find that the pattern of nucleoporin
interactions differs for different importin- family members. All
three importin-s bind Nup159p, but to a much lesser extent than that
observed for Pse1p. Only marginal binding to Nup116p is observed for
all three as well. Sxm1p and Xpo1p also show only a marginal level of
Nsp1p binding. However, as we previously reported, Kap95p binds very
strongly to Nsp1p and to a higher-migrating form of Nsp1p
(80). These differences may indicate that not all
importin-s take the same route through the NPC. If this is the case,
differential interactions between importin-s and Nups may contribute
to their vectorial movement.
Others have assessed interactions between some yeast importin-s and
Nups. For example, Kap95p was shown to bind Nup1p, Nsp1p, Nup145p,
Nup100p, Nup159p, Nup116p, and Nup57p in blot overlay experiments
(37, 47, 67, 69). Similarly, Sxm1p bound Nsp1p, Nup1p, and
Nup159p (68). However, it is difficult to ascertain the
biological significance of these interactions as they were carried out
with isolated proteins displayed on blots and did not take into account
the distribution of the various Nups on the NPC in vivo. Moreover, they
do not display the Ran-dependent binding that we have observed. In a
more physiological study with Xenopus oocytes, Imp was
found to interact with three nucleoporins in a Ran-dependent manner
(75). Two of these, TPR and Nup153, reside on the nuclear
surface of the NPC (62, 66, 78). It is proposed that these
interactions reflect a terminal import step prior to release of cargo
from Imp (75). Imp also interacted with Nup358, which
lies on the cytoplasmic fibers extending from the NPC (56, 85,
86). This may reflect an initial docking step. It is not yet
clear if yeast NPCs contain such elaborate cytoplasmic fibers.
In addition to nucleoporin interactions, we detected an association of
Srp1p/Kap95 with Pse1p and not with other importin-s. This
interaction was also dissipated by Ran-GTP. There are at least two
explanations for these observations. One is that there is a direct
interaction between Pse1p and Srp1p/Kap95p. The second is that the
interaction is indirect and reflects a common binding site for the two
nuclear transporters at the NPC. The interaction of both Kap95p and
Pse1p with Nsp1p suggests that this may be the site of their interaction.
In sum, we have developed a strategy for simultaneously assessing the in vivo dynamics and biochemical interactions of a nuclear transport receptor. In doing so, we have been able to document Ran-dependent interactions between Psep1p and the NPC. These data further our understanding of how macromolecules may transit between the nuclear interior and the cytoplasm.
| |
ACKNOWLEDGMENTS |
|---|
We especially thank Laura Davis, Michael Rout, Susan Wente, Dirk Gorlich, Chuck Cole, and Ed Hurt for providing us with numerous antibodies. We thank the members of the Silver laboratory and Anita Corbett for comments on the manuscript.
This work was supported by grants from the National Institutes of Health to P.A.S. and by Postdoctoral Fellowships from the Deutsche Forschungsgemeinschaft and the Japanese Science Foundation to M.S. and T.T., respectively. M.D. was supported by an NIH Training Grant in Biophysics to Harvard University, and J.K. was supported by an NIH Training Grant to The Dana Farber Cancer Institute.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: The Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Phone: (617) 632-5102. Fax: (617) 632-5103. E-mail: pfas.harvard.silver{at}edu.
Present address: ZMBH, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany.
Present address: Ludwig Institute for Cancer Research, University
of California at San Diego, La Jolla, Calif.
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Top
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Discussion
References
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