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INTRODUCTION |
In eukaryotic cells, the physical separation of nuclear DNA from
cytoplasmic protein synthesis necessitates the bidirectional transport
of a diverse set of macromolecules between the nucleus and the
cytoplasm. Proteins destined for the nucleus carry a nuclear localization signal (NLS)1
(1), whereas substrates to be exported from the nucleus harbor nuclear
export signals (NESs) (2, 3). The signals are recognized by soluble,
mobile transporters that mediate transport through the nuclear pore
complexes (NPCs) embedded in the nuclear envelope (4, 5). These
transporters are collectively termed karyopherins (kaps), but specific
members of the family have different names (see below). The first kaps
to be identified were shown to be responsible for importing basic (1)
(now termed classical) cNLS-bearing proteins into the nucleus. In this
case, the cNLS is recognized in the cytoplasm by a heterodimer of kap
(6) (also termed importin (7)) and kap 
(6) (also termed importin (8)). The subunit binds to the cNLS, whereas the subunit is responsible for docking the complex to the NPC (6, 9-12).
It is proposed that a series of docking and release steps facilitates
movement of the //NLS-cargo from the cytoplasmic filaments
through the NPC (13).
Other macromolecules transported through the NPC follow a similar fate
but are specifically recognized by different kap family members. The
Saccharomyces cerevisiae genome sequence suggests that the
yeast kap family may contain as many as 14 members, identified by their
predicted primary structural similarity to yeast kap , Kap95p (5,
14, 15). Although many members have been shown to be bona
fide transporters, others remain to be characterized. The first
kap -related proteins shown to be transport factors were a mammalian
protein termed karyopherin 2 or transportin (kap 2/Trn) (16, 17)
and its yeast orthologue, Kap104p (18). Both proteins import
mRNA-binding proteins into the nucleus. Kap 2/Trn recognizes a
38-amino acid residue sequence termed M9 within hnRNP A1 and mediates
its nuclear import (16). Similarly, Kap104p transports the nuclear
RNA-binding proteins Nab2p and Nab4p/Hrp1p (18). Nab4p/Hrp1p is the
yeast protein most similar in sequence to mammalian hnRNP A1 (19).
In addition to the kaps, the small GTPase Ran and its interacting
protein, p10/NTF2, play a critical role in providing directionality to
the import and export processes (20, 21). In the nucleus, Ran is
believed to be maintained in its GTP-bound state by the nuclear-restricted GTP exchange factor RCC1 (22). In contrast, the
localization of the Ran GTPase-activating protein to the cytoplasm and
the cytoplasmic filaments of the NPC (23, 24) is thought to ensure that
cytoplasmic Ran is maintained in its GDP-bound form. This separation of
the two pools of Ran is thought to play a role in vectorial transport
across the NPC, acting as a molecular switch in the binding and release
steps that occur during transport through the NPC (4, 20, 21). For
example, the formation of an import complex containing kaps / and
the cNLS is stable in the presence of Ran-GDP, but Ran-GTP triggers its
disassembly (13, 25). In contrast, the formation of an export complex, for example, between an NES-containing protein and its export factor,
Crm1/Exportin 1, is stabilized by Ran-GTP in a trimeric complex (26).
Presumably, as this complex reaches the cytoplasm and GTP hydrolysis on
Ran is catalyzed by Ran-GTPase-activating protein, the complex
disassembles. This mechanism is thought to ensure that cargoes are
loaded and released from their appropriate carriers in the correct
locations. Exactly how energy is utilized to drive translocation is not
clearly understood, but because GTP hydrolysis on Ran is not required
for (at least) a single round of either import or export (27-30), the
energy input may, in part, come from maintaining this gradient of Ran,
thereby indirectly driving translocation.
Although the mechanisms of mRNA export are yet to be elucidated, it
is likely that proteins bound to the mRNA act as adaptors for
export factors. The human immunodeficiency virus protein, Rev is an
excellent example of such an adaptor. It binds specifically to
incompletely spliced viral transcripts (31-34) and, in turn, binds to
cellular Crm1/Exportin 1 through its NES, thereby mediating export of
the viral mRNA (26, 35, 36). Because several proteins bind to
mRNA and are also exported from the nucleus, these shuttling proteins are candidate adaptors mediating mRNA export (3, 37, 38).
The best characterized such protein is hnRNP A1. hnRNP A1 is one of the
most abundant hnRNP proteins in mammalian cells and is essential for
efficient mRNA export (38, 39). Interestingly, the 38-amino acid
residue M9 sequence, which is recognized by Kap 2/Trn (16, 17), is
sufficient to confer shuttling activity upon a passenger protein (40).
A direct role, however, for M9 or Kap 2/Trn in mRNA export has
not been shown.
In yeast, the Kap104p substrates Nab2p and Nab4p/Hrp1p (18) also bind
mRNA in the nucleus and are essential for efficient mRNA
processing and export (19, 41-43). To further understand the function
of Kap104p in nuclear import, and potentially mRNA export, we have
undertaken a molecular approach to characterizing the interactions
between Kap104p and Nab2p and Nab4p/Hrp1p. We provide evidence for a
simple cycle in which, upon import, the Nabs are released from Kap104p
by the concerted action of Ran-GTP and newly transcribed RNA. We
suggest the Nabs are then exported with the mRNA, at which point,
Kap104p rebinds to RNA-binding domains of Nab2p and Nab4p/Hrp1p,
facilitating their release from the mRNA.
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EXPERIMENTAL PROCEDURES |
Plasmids and Strains
Yeast strains were derived from DF5 (Mat a/Mat
ura3-52/ura3-52 his3
200/his3200 trp1-1/trp1-1 leu2-3,
112/leu2-3, 112 lys2-801/lys2-801) or W303 (Mat a/Mat ade2-1/ade2-1 ura3-1/ura3-1 his3-11, 15/his3-11, 15 trp1-1/trp1-1 leu2-3, 112/leu2-3, 112 can1-100/can1-100)
(44). KAP104 strains are derivatives of DF5 cells and have
been described (18).
KAP104 and NAB2 open reading frames were
amplified from yeast genomic DNA by polymerase chain reaction, cloned
into the BamHI site of pGEX-2TK (Amersham Pharmacia
Biotech), and termed Nab2-pGEX2TK. Likewise, NAB4/HRP1 was
cloned into the BamHI site of pGEX-4T1 and termed
Nab4-pGEX4T1. Kap95p-GST (13) was a gift from M. Rexach, Stanford
University. Full-length Nab2p tagged with green fluorescent protein
(GFP) was a gift from C. Guthrie (University of California at San
Francisco, CA).
Nab2p and Nab4p Deletion Constructs
Nab2p deletion constructs 8-188, 200-264, and
306-499 were constructed by polymerase chain reaction.
Oligonucleotides containing SacI restriction sites were
designed to amplify NAB2 and pGEX2TK while omitting the
desired site of insertion. The fragments were purified, digested with
SacI, and ligated. Recombinant plasmids were sequenced to
confirm deletions. Nab4p fragments encoding amino acid residues
492-534, 392-534, and 241-400 were amplified from yeast genomic DNA
(Promega, Madison, WI) using polymerase chain reaction and
oligonucleotides containing restriction sites for cloning as described
below. Fragments were cloned into the BamHI and
EcoRI sites in pGEX-2TK or into the HindIII and
EcoRI sites of NES-GFP2-NLS (pKW430) or p12-GFP2-NLS
(pKW431) (36). PKW431 was a gift from K. Weis (University of California
at San Francisco, CA). In each case, the cNLS was removed and replaced with NAB2 or NAB4/HRP1 coding sequence upstream
of, and in-frame with, a mutant (p12; nes) or wild type NES followed by
two GFP coding sequences (2×GFP). The carboxyl-terminal deletion of
Nab4p was constructed by digesting full-length Nab4-pGEX4T1 with
NdeI and SmaI. T4 DNA polymerase was then used to
create blunt ends, and the sample was ligated.
Recombinant Protein Preparation
GST fusion proteins were expressed separately in
Escherichia coli (BL21 DE3 pLYS-S) (Novagen, Madison, WI).
Overnight cultures were diluted and grown for 3-4 h at 37 °C.
Expression of the chimeras was induced by addition of
isopropyl-1-thio--D-galactopyranoside to a final
concentration of 2 mM, and the cells were incubated at
37 °C for an additional 2 h. Cells were harvested and washed with water, and pellets were rapidly frozen with dry ice ethanol. Pellets were then resuspended in 30 ml of transport buffer (20 mM HEPES-KOH, pH 7.4, 110 mM KOAc, 2 mM MgCl2, 1 µM CaCl2,
1 µM ZnCl2, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Tween 20) containing 300 µl of
solution P (0.4 mg/ml pepstatin A, 18 mg/ml phenylmethylsulfonyl fluoride (18)) and immediately lysed by sonication. The resulting lysate was clarified by centrifugation at 2500 × g for
15 min and then 17,000 × g for 20 min at 4 °C. The
lysates were frozen with dry ice ethanol and stored at 
80 °C.
Ran Preparation
Yeast Ran (Gsp1) (45) was a gift from M. Floer and G. Blobel
(The Rockefeller University, New York, NY). Ran was loaded with three
forms of guanine nucleotide, GTP, GTP
S (guanylyl imidodiphosphate, nonhydrolyzable form of GTP), and GDP as described (13). A reaction mixture containing 30 mM EDTA, 4 mM
dithiothreitol, 1.2 mM guanine nucleotide in binding buffer
(20 mM HEPES-KOH, pH 6.8, 2 mM
MgOAc2, 150 mM KOAc) was incubated with equal
volume of Ran (0.8 µg/µl) at room temperature for 90 min. Magnesium
acetate was added to a final concentration of 30 mM, and
samples were incubated at 4 °C for 15 min and stored at
80 °C.
In Vitro Solution Binding Assay
Protein Purification and Binding--
Protein lysates were
thawed in ice water and incubated with 20 µl of glutathione (GT)
resin (Amersham Pharmacia Biotech) preequilibrated in transport buffer
for 1 h at 4 °C on a platform rocker. Beads were pelleted for
5 s and washed with transport buffer four times. To remove
proteins from the resin, the GST chimeras were cleaved with 0.3 National Institutes of Health units of thrombin (Sigma) and incubated
at room temperature for 15 min. Thrombin was deactivated after the
incubation time by the addition of 1 unit of hirudin (Sigma). Cleaved
proteins were collected by removing GT-bound GST-containing proteins by
centrifugation as above. The supernatant was transferred to a new tube
and cleared of residual GST-containing proteins by addition of 10 µl
of fresh resin, followed by a 30-min incubation at 4 °C and centrifugation.
For assays involving interactions between two or more proteins, the
initial binding steps were done as above, and cleaved proteins were
incubated with GST fusions, prebound to the GT resin, for 1 h at
4 °C. Samples were centrifuged, and the supernatant precipitated
with trichloroacetic acid. The bound fractions were washed three times
prior to cleavage from the resin as described above and analyzed by
SDS-PAGE and Coomassie Blue staining.
Single-stranded DNA (ssDNA) Binding--
Recombinant proteins
were purified and cleaved as above. ssDNA-cellulose (Amersham Pharmacia
Biotech) was prepared according to the manufacturer's instructions and
stored at 4 °C. Before use, 100 µl of slurry was washed twice with
transport buffer. ~7.5 µg of cleaved proteins was incubated in 500 µl with ssDNA (35 µl of 50% slurry) for 1 h at 4 °C.
Samples were centrifuged briefly and washed three times with transport
buffer. Aliquots of the ssDNA-protein complex were made and incubated
with additional protein samples prepared as described above for 1 h at 4 °C. Samples were centrifuged, and the unbound fractions were
collected and precipitated with trichloroacetic acid. Bound fractions
were boiled with SDS sample buffer, and samples were analyzed by
SDS-PAGE and Coomassie Blue staining.
Ran Binding--
Protein complexes were formed as above and
separated into aliquots. Three µg of Ran-GTPS, Ran-GTP, Ran-GDP,
or buffer alone was added, and samples were incubated for 30 min at
room temperature. Samples were centrifuged for 3 s, and unbound
fractions were collected. The beads were washed, and bound proteins
were cleaved from the resin with thrombin. Bound and unbound fractions
were analyzed by SDS-PAGE and Coomassie Blue staining.
GFP Analysis
Wild type or mutant cells containing GFP-tagged proteins were
grown in selective media, and cells were visualized directly by
fluorescent microscopy using a Zeiss Axioskop 2. Images were captured
using a Spot camera (Diagnostic Instruments Inc., Sterling Heights,
MI). In the case of temperature-sensitive cells, cultures were grown
overnight at 23 °C, shifted to 37 °C for the indicated time, and
examined for GFP distribution. Sample temperatures were maintained on
the microscope using a heated stage (Minitube, Minitube Canada,
Woodstock, ON, Canada).
The shuttling assay was performed as described by (36, 46) with the
following modifications: kap104-16 cells containing Nab2p-GFP were grown at 23 °C overnight and then shifted to 37 °C
or left at 23 °C in the presence or absence of 0.1 mg/ml
cycloheximide for 4.5 h. Nab2p-GFP was visualized by fluorescent microscopy.
Ran-GTP/RNA Release
Kap104p-GST was expressed and purified in E. coli as
described above. Immobilized Kap104p-GST was incubated for 1 h
with recombinant Nab4p/Hrp1p at 4 °C. Complexes were then incubated
with 3 µg of Ran-GTP, 10 µg of total yeast RNA, or 3 µg of
Ran-GTP + 10 µg of total yeast RNA for 30 min at room temperature.
Unbound fractions were collected, and bound fractions were washed and
cleaved with thrombin. Samples were analyzed by SDS-PAGE and Coomassie
Blue staining.
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RESULTS |
Identification of an rg-NLS in Nab2p and
Nab4p/Hrp1p--
Immunopurification studies showed that Kap104p
isolated from the yeast cytoplasm copurified with its two import
substrates, Nab2p and Nab4p/Hrp1p (18). However, it is unclear from
these results whether this represented a trimeric complex or a pair of
two member complexes containing Kap104p and Nab2p or Nab4p/Hrp1p. To
examine this, GST chimeras of Kap104p, Nab2p, and Nab4p/Hrp1p were
expressed in E. coli. Nab2p-GST and Nab4p/Hrp1p-GST were immobilized on GT-Sepharose and incubated with recombinant Kap104p. In
both cases, Kap104p bound directly to the fusion proteins in vitro (Fig. 1A). The
relative amounts of protein, as detected by Coomassie Blue staining,
suggests that under these conditions, Kap104p binds to Nab2p and
Nab4p/Hrp1p with similar capacity. Under the same conditions, we were
unable to detect the formation of a trimeric complex (data not
shown). Thus, the cytoplasmic interactions previously observed
were likely two independent, cytoplasmic, Kap104p-substrate import
complexes.
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Fig. 1.
Nab2p and Nab4p/Hrp1p bind Kap104p through a
conserved RG-rich domain. A, GST fusions of Nab2p and
Nab4p/Hrp1p (Nab4p) were expressed in E. coli and
immobilized on glutathione-Sepharose 4B beads to yield ~0.3 µg of
protein per µl of packed beads. Recombinant Kap104p (10 ng/µl) was
added and incubated at 4 °C for 1 h. After washing, bound
fractions were released from GT resin by cleavage with thrombin and
analyzed by SDS-PAGE and Coomassie Blue staining. As a control, GST
alone was bound to GT to yield ~1.5 µg of protein per µl of
packed beads, incubated with 10 ng/µl of recombinant Kap104p, and
treated as above. No detectable Kap104p bound to GST alone.
B, deletion constructs of Nab2p and of Nab4p/Hrp1p were
expressed as GST fusions and bound to GT-Sepharose. The line
diagram represents the regions of each protein expressed.
Structural domains of Nab2p and Nab4p/Hrp1p are indicated and described
in the text. Purified recombinant Kap104p was added (10 ng/µl), and
assayed for its ability to bind each chimera by SDS-PAGE of bound and
unbound fractions. Coomassie Blue staining of the Kap104p in each
fraction is shown on the right. C, the rg-NLS of
Nab2p was compared with full-length Nab4p/Hrp1p using MegAlign
(Lipman-Pearson: ktuple, 2; gap penalty, 4; gap length penalty,
12).
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To identify the NLS recognized by Kap104p, we first examined the
protein sequence of Nab2p. Nab2p contains two distinct functional RNA-binding motifs: amino acid residues 200-249 are rich in glycine and arginine residues and contain a sequence that fits the consensus RGG box sequence, and amino acid residues 249-474 contain a CCCH zinc
finger domain. Nab2p also contains a Gln-rich region between amino acid
residues 101 and 172 (38, 43, 47). We expressed GST chimeras harboring
deletions of these regions in E. coli and tested their
ability to bind Kap104p using in vitro binding assays (Fig.
1B). Nab2p lacking the Gln-rich region (8-188; Fig.
1B) or the CCCH zinc finger domain (306-499; Fig.
1B) maintained their ability to bind Kap104p. In contrast,
Nab2p lacking the RGG box, within amino acid residues 201-264, failed
to bind Kap104p (201-264; Fig. 1B), suggesting that the
RGG motif of Nab2p is necessary for Kap104p binding. To test whether
this domain is sufficient for binding Kap104p, the RGG motif (amino
acid residues 200-249) fused to GST was assayed for binding to
Kap104p. As shown in Fig. 1B (200-249) the RGG box alone
was able to bind directly to Kap104p. These results are consistent with
recent studies mapping the Nab2p NLS. Both studies used two hybrid
analysis to map Kap104p-interacting domain to amino acid residues
198-252 (48) and 181-251 (49), respectively. Our results map the
Kap104p-interacting domain of Nab2p to amino acid residues 200-249,
supporting these findings and extending them to show direct binding of
this domain to Kap104p.
Similarly, we assayed Nab4p/Hrp1p to identify the region responsible
for binding to Kap104p. Nab4p/Hrp1p contains two RNA recognition motifs
between amino acid residues 161 and 291 and an RG-rich carboxyl
terminus (19, 38, 50, 51). Deletion of the carboxyl terminus
(321-534; Fig. 1B) of Nab4p/Hrp1p abolished its ability
to interact with Kap104p. Amino acid residues 241-400 of Nab4p/Hrp1p
fused to GST, containing a highly basic region (pI 10.01) between amino
acid residues 281-350, and a short stretch (amino acid residues
329-350; pI 12.1) rich in Gly, Arg, and Asn residues, failed to bind
to Kap104p in the solution binding assay (data not shown). In contrast,
the carboxyl-terminal fragments consisting of amino acid residues
392-534 (pI 9.11) or amino acid residues 492-534 (pI 11.44) of
Nab4p/Hrp1p fused to GST were sufficient to support Kap104p binding in
the solution binding assay (Fig. 1B). The extreme carboxyl
terminus (amino acid residues 506-534) is similar to the RGG box of
Nab2p but does not fit the RGG box RNA binding consensus sequence (38,
47). Fig. 1C shows the amino acid residue sequence alignment
between the Nab2p RGG box and Nab4p/Hrp1p. In Nab4p/Hrp1p, the domain
is 37 amino acid residues long, highly basic, and lacking hydrophobic
residues. In Nab2p, the RG-rich 41 amino acid domain is similarly basic
and contains only 6 hydrophobic amino acid residues.
To test whether these RG-rich motifs in Nab2p and Nab4p/Hrp1p could
serve as functional NLSs, the protein sequences were tagged with a
tandem repeat of two GFPs (2×GFPs) and visualized in living cells
(Fig. 2A). Although 2×GFP
alone localized to the cytoplasm (data not shown) each RG-rich
Kap104p-interacting domain of Nab2p and Nab4p/Hrp1p targeted the
chimeric GFP to the nucleus, as did the cNLS of SV40 large T antigen
attached to the same GFP (36). The results demonstrate that these
RG-rich Kap104p interacting domains act as functional NLSs in
vivo. To distinguish these NLSs from the basic cNLSs we term them
rg-NLSs.
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Fig. 2.
Kap104p-interacting domains are functional
Kap104p-dependent NLSs. Amino acid residues 200-249
of Nab2p (Nab2p rg-NLS), residues 492-534 of Nab4p/Hrp1p
(Nab4p rg-NLS), and the cNLS of SV40 large T antigen
(cNLS) were expressed as 2×GFP chimeras in wild type
(WT) W303 haploid cells (A). Cells were observed
using differential interference contrast (right column), and
GFP was detected by fluorescent microscopy (left column). In
each case, GFP was localized to the nucleus. The NLS-GFP chimeras were
also expressed in kap104-16 ts cells (B).
kap104-16 cells were maintained at 23 °C or grown at
23 °C and shifted to 37 °C for 4.5 h prior to examination of
the GFP chimeras by fluorescent microscopy. Note the cytoplasmic
localization of the rg-NLSs after the temperature shift.
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In an attempt to determine whether the in vitro binding data
reflect cellular Kap104p-mediated import of the rg-NLSs, we expressed each fusion in kap104 ts cells (Fig. 2B). In
these cells, at 23 °C both rg-NLS- and cNLS-containing chimeras were
imported into nuclei. After shifting the cells to 37 °C, the
rg-NLS-containing chimeras were mislocalized to the cytoplasm, whereas
the cNLS-GFP chimera remained nuclear. Similar results were observed
when KAP104 was placed under control of a GAL
promoter, and its expression repressed by growth of cells on
glucose-containing medium (data not shown). Interestingly, like a cNLS
(36), the rg-NLS can be overcome by attaching a functional NES to the
chimera (Fig. 3). This suggests that the
nuclear localization of these chimeras is not simply a result of
binding to sites within the nucleus. Taken together, these data
demonstrate that Kap104p mediates the import of Nab2p and Nab4p/Hrp1p
through its interaction with the rg-NLS domains of each protein.
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Fig. 3.
NES-rg-NLS chimeras are exported from nuclei
in vivo. rg-NLS regions of Nab2p and Nab4p were
cloned upstream of a mutant (mutant nes) or a functional
nuclear export signal (wild type NES) followed by two GFP
tags. GFP chimeras were visualized in wild type cells grown at
30 °C.
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Kap104p Is Able to Displace Nab2p and Nab4p/Hrp1p from
Single-stranded DNA--
Nab2p and Nab4p/Hrp1p have been shown to bind
mRNA using UV cross-linking studies and are believed to play a role
in the export of mature polyadenylated mRNAs (19, 41-43). The
interaction of Nab2p with RNA is likely mediated by both its RGG domain
and the CCCH motif, as each is sufficient to bind to ssDNA, but when
both are present, the interaction is strengthened (43). Similarly, Nab4p/Hrp1p RNA binding is likely mediated by both the RNA recognition motifs and the RG-rich carboxyl terminus. Both the amino-terminal fragment (321-534; Fig. 1B) containing RNA recognition
motifs 1 and 2 and the carboxyl-terminal fragment (392-534) lacking
the RNA recognition motifs but containing the RG-rich domain bind to
ssDNA in vitro (data not shown). Because both the
RGG-RNA-interacting domain of Nab2p and the RG-rich domain of
Nab4p/Hrp1p (at least partially) overlap with the Kap104p-interacting
domains, we wished to determine whether Kap104p could bind to Nab2p or
Nab4p/Hrp1p while complexed with RNA. The formation of such a complex
would support a possible role for Kap104p in mRNA export. Nab2p and Nab4p/Hrp1p were immobilized on ssDNA-cellulose and challenged with
Kap104p. As shown in Fig. 4, Kap104p did
not bind to preformed complexes and remained in the unbound fractions.
Interestingly, however, Kap104p also removed Nab2p and Nab4p/Hrp1p from
the ssDNA, in a concentration-dependent manner, shifting them
to the unbound fractions. As a control, Kap95p was added in similar
amounts but had no effect on the Nab-ssDNA complexes. These results
demonstrate that binding of Kap104p to the rg-NLS of both Nab2p and
Nab4p/Hrp1p destabilizes the interaction of these proteins with ssDNA.
The relatively large amounts of Kap104p needed to produce complete displacement from ssDNA are most likely due to the presence of other
RNA-binding motifs contributing to the interaction. Nevertheless these
data suggest that the binding Kap104p to the rg-NLS of newly synthesized Nab2p and Nab4p/Hrp1p in the cytoplasm may have a role in
preventing nonproductive cytoplasmic Nab-RNA interactions.
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Fig. 4.
Nab2p and Nab4p/Hrp1p are displaced from
ssDNA-cellulose by Kap104p but not Kap95p. Nab2p (upper
panel) or Nab4p/Hrp1p (lower panel) bound to ssDNA
(~1 µg of protein/5 µl of packed resin) was incubated with
Kap104p (4 and 16 ng/µl), Kap95p (14 and 10 ng/µl), or buffer alone
for 1 h at 4 °C. Polypeptides in bound (B) and
released (R) fractions were analyzed by SDS-PAGE and
Coomassie Blue staining. The addition of Kap104p caused the release of
Nab2p and Nab4p/Hrp1p from ssDNA, as detected by the presence of Nab2p
and Nab4p/Hrp1p in the unbound fractions. In contrast, Kap95p or buffer
alone did not cause any detectable disruption of the Nab-ssDNA
association.
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In addition, if Nab2p and Nab4p/Hrp1p exit the nucleus with newly
synthesized mRNA, the observations reported above may suggest a
role for Kap104p in promoting cytoplasmic disassembly of the mRNP. In
support of this model, Nab4p/Hrp1p, like its closest human homologue,
hnRNP A1, has recently been shown to exit the nucleus, presumably with
newly synthesized mRNA (42, 52). We therefore investigated whether
Nab2p also exits the nucleus. The distribution of a functional
Nab2p-GFP chimera2 was
monitored in kap104 ts cells under conditions where new
protein synthesis was inhibited. As expected, and as observed
previously for endogenous Nab2p (18), the fusion was nuclear at the
permissive temperature and, upon shift to the nonpermissive
temperature, accumulated in the cytoplasm (Fig.
5). Protein synthesis was inhibited by
the addition of cycloheximide to the culture, and upon temperature shift, Nab2p redistribution was still observed, indicating that the
Nab2p-GFP was exported from the nucleus to the cytoplasm. Nab2p-GFP did
not change its distribution in wild type cells under the conditions
tested, and cycloheximide treatment alone did not affect the
localization of Nab2p-GFP. Importantly, Kap104p is rapidly turned over
at the restrictive temperature in kap104-16 cells, and
mRNA export is ongoing (18). This suggests that Kap104p is not
required for export of mRNA and Nab2p from the nucleus but is
specific for the import leg of the Nab2p (and Nab4p/Hrp1p) cycle.
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Fig. 5.
Nab2p shuttles between the nucleus and
cytoplasm. Nab2p-GFP was expressed in wild type (WT)
and kap104 ts cells (kap104-16). Cells were
grown at 23 °C and either maintained at 23 °C or shifted to
37 °C for 4.5 h in the presence (+CHX) or absence of
0.1 mg of cycloheximide per ml. Nab2p-GFP localized to the nucleus at
23 °C; however, upon a shift to 37 °C, the GFP signal accumulated
in the cytoplasm, regardless of the presence of cycloheximide.
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The Role of Ran-GTP on Kap104p-mediated Transport--
Ran-GTP is
a believed to be a key regulator of nuclear transport (20, 21). Its
presence in the nucleus is thought to disrupt import-bound
substrate/kap complexes while stabilizing export-bound transporter/substrate complexes. As described in the Introduction, this
differential effect of Ran-GTP and GDP provides directionality to
import and export. To investigate the role Ran-GTP and Ran-GDP in
Kap104p-mediated transport, we first tested whether Ran-GTP or -GDP
bound to Kap104p. Kap104p-GST was immobilized on GT-Sepharose, and
Ran-GTP, Ran-GDP, or buffer alone was added. As shown in Fig. 6, Ran-GTP bound preferentially to
immobilized Kap104p, consistent with its role in nuclear import.
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Fig. 6.
Kap104p binds Ran-GTP. E. coli-expressed Kap104p-GST was immobilized on GT-Sepharose (~5
µg/10 µl of packed resin) and incubated with purified Ran-GTPS,
Ran-GTP, Ran-GDP (3 µg/25 µl), or buffer alone for 30 min at room
temperature. Bound and unbound fractions were analyzed by SDS-PAGE and
Coomassie Blue staining. Both Ran-GTPS and Ran-GTP bound to Kap104p,
whereas Ran-GDP did not.
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The binding of Ran-GTP to other -kaps, including Kap95p (13),
Kap121p (53, 54), Kap123p (54, 55), and kap 2/Trn (56, 57), has been
shown to disrupt their interaction with import substrates. To
investigate whether Ran-GTP had a similar disruptive effect on the
Kap104p-Nab complexes, Nab2p- and Nab4p/Hrp1p-Kap104p complexes were
formed on GT-Sepharose and challenged with Ran-GTP, -GDP, or -GTPS
(Fig. 7A). Addition of Ran-GTP
to this complex stimulated a moderate release of Kap104p from both
Nab2p and Nab4p/Hrp1p, as shown by a shift of some of the bound Kap104p
to the unbound fraction. Ran-GDP did not, however, stimulate Kap104p
release over what was observed with buffer alone. Quantitation of these results revealed that Kap104p release was stimulated 3-fold by the
presence of Ran-GTP, demonstrating that Ran must be in its GTP-bound
form to disrupt the Kap104p-substrate complexes. Furthermore, GTP
hydrolysis is not required for Kap104p release from its substrate, as
Ran-GTPS was also able to dissociate Kap104p from Nab2p and Nab4p/Hrp1p with similar efficiency as Ran-GTP. These results are
similar to those found by Rexach and Blobel (13) for the disruption of
the Kap95p/Kap60p/cNLS, by Ran-GTP. However, in our hands, the Ran-GTP
stimulated release of the Kap104p-Nab complex is far less complete than
that observed for Kap95p/Kap60p (data not shown).
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Fig. 7.
Ran-GTP and RNA act cooperatively to remove
Nabs from Kap104p. A, recombinant Kap104p (6 ng/µl)
was incubated with immobilized Nab2p-GST (~1.5 µg/10 µl of packed
resin) (left panel) and Nab4p/Hrp1p-GST (~2 µg/10 µl
of packed resin) (right panel) for 1 h at 4 °C.
Complexes were incubated with Ran-GTPS, Ran-GTP, Ran-GDP, or buffer,
as indicated, for 30 min at room temperature. Polypeptides that
remained bound to the column and those that were released were analyzed
by SDS-PAGE and Coomassie Blue staining. Kap104p release was
quantitated using Image-Quant software, revealing that Ran-GTPS and
Ran-GTP caused a 3-fold increase in the displacement of Kap104p from
its substrate over that observed with Ran-GDP or buffer alone.
B, immobilized Kap104p-GST (~1 µg/10 µl of packed
resin) was incubated with cleaved Nab4p (6 ng/µl). Complexes were
then incubated with 1) Ran-GTP, 2) Ran-GTP + total yeast RNA, or 3)
total yeast RNA. Released fractions and bound fractions (after cleavage
from the resin with thrombin) were analyzed by SDS-PAGE and Coomassie
Blue staining. The results of quantitation of Nab2p/Hrp1p in the
released fractions in three separate experiments are represented
graphically below the gel. Ran-GTP-mediated release was normalized to
1.0.
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Because Kap104p stimulates release of Nabs from single-stranded DNA
(Fig. 4), we investigated whether the Ran-GTP-dependent disruption of the Kap104p-Nab complex was stimulated by the addition of
RNA (Fig. 7B). In this case, we bound Kap104p to
GT-Sepharose and formed a complex with Nab4p/Hrp1p. Under these
conditions, addition of Ran-GTP or RNA alone stimulated a weak release
of Nab4p/Hrp1p from the column. Strikingly, Ran-GTP and RNA had a cooperative effect in releasing the substrate. Quantitation of the
release under these conditions revealed that RNA and Ran-GTP alone
released approximately similar amounts of Nab4p/Hrp1p, but together,
their effect on substrate release was cooperative, stimulating substrate release greater than 7-fold.
| |
DISCUSSION |
Here, we show that Nab2p and Nab4p/Hrp1p both interact directly
and independently, through a similar RG-rich domain, with the
karyopherin Kap104p. Because this domain is both necessary and
sufficient for Kap104p binding and acts as a
KAP104-dependent nuclear import signal in yeast,
we term this domain an rg-NLS to distinguish it from cNLSs recognized
by Kap60p/Kap95p (kap and ). The Kap104p-dependent
pathway is considered to be homologous to the mammalian Kap 2/Trn
pathway (14, 15, 48). Kap104p shares the most sequence similarity to
Kap 2/Trn; likewise, Nab4p/Hrp1p is the most similar to mammalian
hnRNP A1, the prototype import substrate of Kap 2/Trn. Both the
rg-NLS defined here (see also Refs. 48 and 49) and the Kap
2/Trn-interacting (M9) domain of hnRNP A1 are rich in glycines.
Recently, however, a consensus Kap 2/Trn-interacting motif has been
defined, and this motif shows no similarity to the rg-NLSs. This
suggests that the NLSs recognized by Kap 2/Trn and Kap104p are
different and offers an explanation for why Nab2p cannot be imported by
Kap 2/Trn in vitro and for our observations that
overexpression of mammalian Kap 2/Trn does not complement
kap104 cells.3
The rg-NLS is similar to the NLS in Npl3p (58), which is recognized by
the yeast kap family member Mtr10p (58, 59). Because the deletion
of either MTR10 (58, 59) or KAP104 (18) is not
lethal, it seems reasonable that in the absence of one, the other may
substitute for the loss. In support of this, these two transport
pathways genetically interact, as cells carrying a deletion of both
MTR10 and KAP104 are not viable (data not shown). It is also likely that both Mtr10p and Kap104p import other proteins in
addition to the three substrates already defined. Of course, several
other nuclear proteins in the yeast proteome contain RG-rich domains,
thereby making them candidate substrates; however, whether these are
functional NLSs in the context of these proteins remains to be shown.
Newly transcribed mRNA is bound by proteins in the nucleus, and the
resulting heteroribonuclear protein particles are processed and
transported out of the nucleus. Studies in metazoan cells demonstrate
that whereas some hnRNP proteins are retained in the nucleus, others,
such as hnRNP A1, accompany the mRNA into the cytoplasm (3, 37, 38,
60-62). Because of their ability to exit and reenter the nucleus,
these proteins are collectively called shuttling proteins. Previously,
we showed that Nab2p and Nab4p/Hrp1p are imported into the nucleus by
Kap104p and proposed that both Nab2p and Nab4p/Hrp1p exit the nucleus
with RNA (18). This has been shown to be true for Nab4p/Hrp1p (42, 52),
and here we demonstrate that Nab2p also exits the nucleus, presumably bound to mRNA. We propose that in the cytoplasm, Kap104p binds the
rg-NLS, and this destabilizes the Nab interaction with mRNA. This
idea is supported by our in vitro binding studies. Addition of Kap104p to Nab2p and Nab4p/Hrp1p bound to ssDNA causes their release
from the column. The rg-NLS of Nab2p overlaps with the RGG box.
Furthermore, our data suggest the RG-rich carboxyl terminus of
Nab4p/Hrp1p contributes to RNA binding. Thus, because both rg-NLSs in
Nab2p and Nab4p/Hrp1p reside within these RNA-binding domains, binding
Kap104p to this region could lead to displacement of the RNA thereby
contributing to mRNP disassembly. However, other factors may also
mediate the release of the Nabs from the mRNA in the cytoplasm
in vivo. In the cytoplasmic environment of a low Ran-GTP
concentration, the overlap of the rg-NLS with the RNA binding activity
may drive the reaction to a new round of import, by either direct
displacement of the RNA or simply by preventing the rebinding of the
Nab to mRNA.
Our results also suggest that upon import of the Nab-Kap104p complexes,
a combination of Ran-GTP and newly transcribed RNA act cooperatively to
dissociate Nab2p and Nab4p/Hrp1p from Kap104p. This is similar to the
release of Npl3p from Mtr10p (58). Imposing a role for RNA, in
combination with nuclear Ran-GTP, in the release of Nab2p and
Nab4p/Hrp1p from Kap104p may (as proposed for Npl3p (58)) allow the
local unloading of the cargo at the site of transcription. Such a
mechanism would prevent the premature release of mRNA-binding
proteins until they are required to bind RNA. Similarly, several cNLSs
appear to overlap with DNA- and RNA-binding domains (63). Thus, this
may be a general mechanism invoked to facilitate intranuclear protein sorting.
Although Nab2p and Nab4p/Hrp1p are required for efficient mRNA
export, and they interact directly with both RNA and Kap104p, our
results strongly support a model in which Kap104p is specific for the
import of the Nabs, and not in mRNA export. Similarly, although
hnRNP A1 is required for efficient mRNA export in metazoan cells,
kap 2/Trn is not likely involved in this process (39, 56, 64) but is
specific for the import of (among other proteins (56)) hnRNP A1 (16,
17). In both cases, cells have evolved a system of maintaining separate
directionality to import and export. Ran-GTP in the nucleus
destabilizes import-substrate/kap complexes; however, as demonstrated
here, this may not be sufficient for completely unloading the cargo.
Masking of the NLS by competition with overlapping RNA or DNA binidng
sites, or by assembly into a multicomponent complex, may also
contribute to local release. Interestingly, the M9 domain of hnRNP A1
is not accessible to kap 2/Trn in the context of the nuclear hnRNP
(56). Because the M9 domain does not bind RNA (40), this masking is
likely due to other proteins in the nuclear hnRNP. It would be
interesting to determine whether proteins in yeast nuclear hnRNPs also
mask NLSs and whether they, as in mammalian cells, differ from
cytoplasmic hnRNPs. Thus, upon export, some hnRNP proteins may be shed,
allowing access of the NLSs to karyopherins in the cytoplasm and lead
to another round of import.
TOP
ABSTRACT
INTRODUCTION
Medical Research Council of Canada and Heritage Scholar. To whom
correspondence should be addressed: Medical Sciences Bldg., Rm. 5-14, Dept. of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-6062; Fax: 780-492-0450; E-mail: john.aitchison@ ualberta.ca.
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