Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021
Received for publication, November 29, 2000, and in revised form, March 9, 2001
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ABSTRACT |
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In Saccharomyces cerevisiae, Spo12p
is involved in mitosis and is essential for meiosis. We found that
Spo12p is imported into the nucleus by the karyopherin Kap121p. A
complex containing Spo12p and Kap121p was isolated from cytosol and was
also reconstituted with recombinant proteins, indicating that this
interaction is direct. Spo12p was mislocalized to the cytosol in
pse1-1, a temperature-sensitive strain harboring a
mutation of Kap121p, at the permissive temperature, confirming an
essential role for Kap121p in Spo12p import. Spo12p was also
mislocalized in a pse1-1/pse1-1 homozygous strain,
suggesting it is imported via the same pathway in diploid cells.
Furthermore, we found that pse1-1/pse1-1 shows a
sporulation defect similar to that of
spo12 Bidirectional exchange of macromolecules between the
nucleus and the cytoplasm occurs through a large complex embedded in the nuclear envelope, the nuclear pore complex
(NPC).1 In
Saccharomyces cerevisiae (herein referred to as yeast), the NPC is composed of about 30 proteins (nucleoporins), which provide docking sites for a family of soluble and structurally related transport factors, karyopherins (also known as
importins/exportins/transportins). In addition to docking at the NPC,
karyopherins bind cargo to be transported into and out of the nucleus
(see Refs. 1 and 2 for a review). They recognize specific sequence
elements in their cognate transport substrates that constitute the
signal for import (nuclear localization sequence, or NLS) or export
(nuclear export sequence, or NES). Kap Meiosis is one of the few differentiation pathways in yeast, and its
regulation necessarily depends on the expression control of subsets of
genes. Regulation of nuclear transport has been increasingly recognized
as an important mechanism for the control of gene expression (see Ref.
7 for a review). It is not known how most of the proteins involved in
meiosis are transported into the nucleus to participate in its
regulation. Spo12p was first identified as a protein whose absence
causes sporulating diploid cells to skip one meiotic division, leading
to the formation of two diploid spores (8). More recently, its
overexpression has been found to suppress a number of late mitotic
mutants (9, 10). Consistent with a role in exit from mitosis, cells in
G2/M are over-represented in a spo12 Spo12p has been shown to be nuclear in both haploid and diploid cells
(13). Here we report that in haploid cells Spo12p is imported into the
nucleus by Kap121p. Kap121p is a previously characterized karyopherin,
first implicated in ribosomal protein import (14) and mRNA export
(15) and later shown to be responsible for import of the transcription
factors Pho4p and Pdr1p (16, 17) (see "Discussion"). Evidence for
import of Spo12p by Kap121p includes binding to Kap121p both in
vivo and in vitro and mislocalization of Spo12p to the
cytosol in pse1-1, a temperature-sensitive strain of
Kap121p. We have also characterized the Spo12p NLS and identified residues within this region that are important for NLS function. Since
Spo12p has a role in both mitosis and meiosis, we sought to determine
whether the import pathway differs in diploid cells. In this case, we
found that Spo12p was mislocalized in a
pse1-1/pse1-1 homozygous strain, indicating
that it is imported using the same pathway in both haploid and diploid cells.
Yeast Strains and Plasmids--
The procedures for yeast
manipulation were as described (18). A diploid strain expressing
Spo12p-Protein A (PrA) was obtained by integrative transformation of
DF5 (19) with the coding sequence of four and a half IgG binding
repeats of Staphylococcus aureus protein A immediately
upstream of the SPO12 stop codon as described (20). Haploid
strains were obtained by sporulation and tetrad dissection. Proper
integration was assessed by PCR and Western blot analysis.
kap142
The entire coding region of SPO12 was
PCR-amplified from S. cerevisiae genomic DNA and inserted
into pYX242-GFP (26) using primer-encoded EcoRI and
HindIII sites to generate an in-frame fusion with the green
fluorescent protein (GFP), pSpo12-GFP. pSpo12-GFP derivatives
pSpo121-143-GFP, pSpo121-90-GFP,
pSpo1276-173-GFP, pSpo1291-173-GFP,
pSpo12116-173-GFP, pSpo1276-143-GFP, pSpo1276-130-GFP, pSpo1276-115-GFP, and
pSpo1291-130-GFP were generated similarly. A PCR fragment
containing a deletion of amino acids 76-130 of Spo12p (
Spo12p fragments 1-173, 76-130, and Cell Fractionation and Immunoisolation--
Post-nuclear,
post-ribosomal cytosol was prepared from a strain expressing Spo12p-PrA
grown to an A600 of 1.8 as described (23).
Spo12p-PrA-interacting proteins were immunoisolated by overnight
incubation of cytosol with IgG-Sepharose at 4 °C. After washing with
transport buffer (TB: 20 mM HEPES, pH 7.5, 110 mM KOAc, 2 mM MgCl2, 0.1% Tween
20), proteins were eluted with a step gradient of MgCl2 and
concentrated using methanol-chloroform precipitation prior to SDS-PAGE
analysis. Gels were stained with Coomassie Blue, and proteins of
interest were excised, digested, and analyzed by MALDI-TOF mass
spectrometry (MS) (29). Gels were also transferred to nitrocellulose,
and Western blotting procedures and subsequent ECL detection were as
described (Amersham Pharmacia Biotech).
Recombinant Protein Expression--
Cultures were grown at
37 °C to an A600 of 0.7 in Luria Broth
medium containing 100 µg/ml ampicillin and induced with 1 mM isopropyl-1-thio- Solution Binding Assay--
2 µg of purified recombinant GST
and GST-Spo12p were immobilized on 25 µl of a 50% slurry of
glutathione-Sepharose equilibrated in TB by incubation for 30 min at
room temperature. After washing, Sepharose was incubated with 2 µg of
Kap121p or Kap114p for 30 min in TB containing 3 mM EGTA
and protease inhibitors. The resin was washed three times with 1 ml of
TB, and bound proteins were eluted with sample buffer and analyzed by
SDS-PAGE and Coomassie Blue staining. Binding assays using GST,
GST-Spo12p, GST-Spo12p76-130, and
GST-Spo12p Fluorescence Microscopy--
GFP was visualized after overnight
growth of yeast strains in medium or on agar selective for the
respective plasmid. 5 µl of culture or one colony resuspended in 5 µl of water was placed on a slide. All images were viewed with a 63×
oil objective on a Zeiss Axiophot microscope. Images were collected
with a Hamamatsu video imaging system, using Openlab software, and
transferred to Adobe Photoshop.
Spo12p Binds to Kap121p in Vivo--
Spo12p was previously
localized to the nucleus (13). Kap proteins involved in import form
stable complexes with their substrates in the cytosol, prior to their
import into the nucleus. Once inside the nucleus, specific factors
disrupt the complex, leading to the deposition of the cargo and the
recycling of the empty kap to the cytosol for another round of import.
To determine which karyopherins play a role in Spo12p import, we
isolated proteins interacting with the cytosolic pool of Spo12p. To do
so, we first genomically tagged the Spo12p C terminus in frame with the
IgG binding domain of S. aureus PrA. This resulted in the
endogenous expression of Spo12p-PrA from the SPO12 promoter.
We then prepared a post-nuclear, post-ribosomal cytosolic fraction of
this strain. After incubation with IgG-Sepharose, bound proteins were
eluted with a step gradient of MgCl2. The eluted fractions
were analyzed by SDS-PAGE and Coomassie Blue staining. The results of a
typical experiment are shown in Fig.
1A. Spo12p-PrA, by virtue of
the high affinity of PrA to IgG, is expected to elute only with high salt concentrations. Although a band of the expected size of Spo12-PrA is evident throughout the elution (indicated by an asterisk
in Fig. 1A), immunoblot analysis indicated that Spo12-PrA
eluted only in the expected fractions (Fig. 1B). A
fortuitously comigrating band eluted at lower MgCl2
concentrations. A protein with an apparent molecular mass of ~116 kDa
consistently eluted in the 0.1-1 M fractions, the typical
range of MgCl2 concentrations needed to disrupt the
kap/cargo complex (Fig. 1A). The band was excised, digested
with trypsin, and resulting peptides submitted to MALDI-TOF MS
analysis. 27 peaks of a total of 29 were consistent with the mass of
peptides resulting from the theoretical digest of Kap121p. The amount
of isolated Kap121p appears to be in excess of Spo12p-PrA. However,
since this is a semiquantitative assay, we cannot comment on the
complex stoichiometry. Some proteins have decreased solubility in high
concentrations of MgCl2, which results in a lack of
correlation between the amount of isolated proteins and the amount
visible on the gel.
We also sought to determine what fraction of cytosolic Kap121p was
associated with Spo12p. After isolation of Spo12p-PrA in the manner
described above, fractions were run on an SDS-PAGE gel and transferred
to nitrocellulose. Western blot analysis with a Kap121p antibody show
that the majority of Kap121p present in the cytosol remains in the
unbound fraction and is not, therefore, associated with Spo12p (Fig.
1C).
Spo12p Binds to Kap121p Directly--
Since the Spo12p/Kap121p
complex was isolated from cytosol, it is possible that this interaction
is bridged by another protein. Therefore, we investigated whether the
interaction between Spo12p and Kap121p is direct. IgG-Sepharose-bound
Spo12p-PrA purified from yeast cytosol was incubated with purified
recombinant GST-Kap121p, washed, and bound proteins eluted with sample
buffer. After SDS-PAGE and Coomassie Blue staining, we detected binding
of GST-Kap121p in addition to the endogenous Kap121p (data not shown).
We next immobilized bacterially expressed GST and GST-Spo12p on
glutathione-Sepharose and then incubated with recombinant Kap121p or
Kap114p, which was used as a control. The resin was washed, bound
proteins were eluted with sample buffer and analyzed by SDS-PAGE and
Coomassie Blue staining. As seen in Fig.
2, we found that Kap121p bound to
GST-Spo12p. We did not detect either kap bound to GST alone, while only
a small quantity of Kap114p bound to GST-Spo12p relative to Kap121p. We
conclude that Spo12p and Kap121p interact directly.
Spo12p Is Mislocalized in a Kap121p Mutant Strain--
To
determine the role of Kap121p in Spo12p nuclear import, we examined the
localization of Spo12p fused to GFP in vivo. In an initial
attempt, the fusion was expressed under the control of the SPO12
natural promoter from a low copy number (CEN) plasmid, but no
fluorescence was observed (data not shown). We then constructed a
plasmid to overexpress Spo12p fused to GFP from a high copy number
(2µ) plasmid, which was found to be nuclear in wild-type cells and
kap114 Spo12p Is Mislocalized in pse1-1/pse1-1--
We investigated
whether Kap121p is also responsible for Spo12p import into the nucleus
of diploid cells. We found that Spo12p accumulated in the nucleus of a
wild-type diploid strain and was slightly mislocalized in a
pse1-1/KAP121 heterozygous strain (Fig. 4). However, significant mislocalization
to the cytoplasm was observed in a pse1-1/pse1-1
homozygous strain (Fig. 4), suggesting the import pathway is the same
in both haploid and diploid cells.
We next investigated whether pse1-1/pse1-1 exhibits a
sporulation phenotype similar to that of
spo12 Mapping of Spo12p NLS--
In order to identify regions in Spo12p
that are necessary for import, we constructed several deletion mutants
of Spo12p in frame with GFP and analyzed their localization in
vivo (Fig. 5A). We found
the region containing amino acids 76-130 to be both necessary (Spo12p
The fusion Spo12p76-130-GFP is mislocalized to the
cytosol in pse1-1 (Fig. 5B), indicating that it
is targeted to the nucleus in a Kap121p-dependent manner.
To further address this point, we determined whether this region was
also necessary and sufficient for direct interaction with Kap121p.
Purified recombinant GST, GST-Spo12p, GST-Spo12p76-130,
and GST-Spo12p
In order to more clearly define which residues of
Spo12p76-130 contribute to NLS function, we performed
alanine-scanning mutagenesis by sequentially substituting stretches of
5 residues in this region with alanine. These mutants were expressed in
frame with GFP, and their localization assessed in living cells (Fig. 7A). As shown in Fig.
7B, we found that mutation of residues 91-95 (LVKKT) to
alanine leads to significant mislocalization to the cytoplasm.
Surprisingly, the localization of the other mutants was
indistinguishable from that of the wild-type protein (Fig. 7A).
We present evidence that Kap121p is the kap used for import of
Spo12p. Kap121p was first described as a karyopherin whose overexpression was able to substitute for Kap123p in the import of the
ribosomal protein L25 NLS (14). Later, also suggesting an overlapping
pathway with Kap123p, mRNA export was shown to be defective in a
pse1-1/kap123 We have shown that Spo12p is imported by Kap121p. We have
coimmunoisolated the two proteins from yeast cytosol and reconstituted this complex with recombinant proteins, which indicates this
interaction is direct. Spo12p is mislocalized in pse1-1,
but not in any of the other kap mutant strains tested (including
kap123 Spo12p absence in sporulating diploid cells results in the formation of
dyads (a rather unique characteristic). We found that diminished
activity of Kap121p in sporulating diploid cells leads to an increased
number of asci containing two spores. This observation is consistent
with deficient import of Spo12p in pse1-1/pse1-1; nonetheless, we cannot claim it is directly related. Kap121p associates with a large number of proteins in the cytosol (data not shown). These
proteins might constitute additional substrates. Identification of
these substrates should provide more insight into Kap121p's distinctive cellular roles and why it is essential for viability. Kap121p might be responsible for import of essential substrates or
constitute a backup pathway in the import of a large number of
proteins, and thus be vital in compensating for the loss or malfunction
of other kaps (for example, see Ref. 21).
We have characterized the Spo12p NLS and determined that the region
containing residues 76-130 is both necessary and sufficient for import
and for direct binding to Kap121p. When we attempted to determine which
residues were important for NLS function by alanine scan mutagenesis,
we only identified 1 group (out of 11 generated) of five residues,
which, if mutated, led to mislocalization of Spo12p to the cytoplasm
(91LVKKT95). One explanation for this
observation is that many residues throughout the entire NLS interact
with Kap121p. Since the mapped NLS is large, it could be that mutation
of five residues to alanine was not sufficient in order to observe a
phenotype. On the other hand, the fact that we could not obtain a
shorter deletion mutant with nuclear accumulation could mean that a
large domain of the protein is needed in order to provide the proper
structure necessary for interaction with Kap121p. This study emphasizes
the caution one must use while performing NLS analysis. The need for a
large domain to confer complete nuclear accumulation has been reported previously. For instance, the region containing residues 112-224 of
Lhp1p confers Kap108-dependent import. However, some
cytoplasmic signal is observed, in contrast with the full-length
protein (26). Similarly, residues 283-414 of Npl3p are necessary and
sufficient for import and binding to its import receptor, Kap111p.
Again, more accumulation in the cytoplasm is observed in comparison
with the full-length protein (30). The Spo12p NLS is located between residues 76 and 130, but only a structural analysis will fully elucidate which residues are required for interaction with Kap121p and
thus constitute the actual/minimal NLS. It is, nonetheless, clear that
it is different from the cNLS. It is larger, and even though there are
some stretches of basic residues in the mapped NLS, these are not
sufficient for nuclear accumulation, nor does mutation of these
residues lead to mislocalization. Although a large number of proteins
have predicted cNLSs, it should not be assumed that they will be
imported via the Kap The mapped Spo12p NLS and the Pho4p NLS do not bear any noticeable
resemblance, so a pattern of substrate recognition by Kap121p could not
be derived. The Rpl25p and the Pdr1p NLSs were not shown to bind
Kap121p directly; nonetheless, they also do not reveal any sequence
similarities. NLS mapping of additional substrates might reveal a
pattern of recognition by Kap121p. As is the case for Kap121p, other
kaps likely recognize numerous dissimilar NLSs. It is, however,
probable that only after structures of a large number of kap/substrate
complexes are known might we start to better understand nature's
solution to this daunting molecular recognition problem.
/spo12
. In addition, we
have characterized the Spo12p nuclear localization signal, mapped it to
residues 76-130, and identified residues within this region that are
important for nuclear localization signal function.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 needs an adapter (Kap
) in
order to bind to most substrates. Kap
recognizes proteins containing a classical NLS (cNLS), which consists of one or two stretches of basic
amino acid residues (3, 4). In yeast, there is one Kap
and 14 Kap
s (herein referred to as kaps). The kaps share a low but
significant identity (about 20%), most striking at the amino terminus
(1, 2, 5). To date, all yeast kaps except Kap120p have been shown to
function in import or in export. The involvement of a kap in both
processes has recently been discovered (6), and it remains to be
elucidated whether more share the same ability. There are hundreds of
yeast nuclear proteins or proteins with a nuclear life style, but only
14 kaps. This necessarily means that some kaps will be responsible for
the transport of a large number of cargoes. Sequences of the NLSs that
have been mapped so far do not resemble the cNLS or each other. Hence,
the basis for kap/substrate specificity and the overall mechanism of
substrate recognition by karyopherins remain to be further elucidated.
population (11). Spo12p is synthetically lethal with Dbf2p, a
kinase required for events in anaphase/telophase and has been suggested
by genetic studies to be an activator of Dbf2p and its relative
Dbf20p (12). Its biological function remains unknown, and its
functional differences in mitosis and meiosis await further clarification.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(6), kap123
(14),
pse1-1, PSY580, and PSY581(15), kap122
(20),
kap114
(21), kap119
(22), kap104
(23), kap111
(24), and
kap108
(25) have been described previously. A
pse1-1/KAP121 heterozygous strain was generated by mating
PSY581 with pse1-1. After sporulation and tetrad dissection of pse1-1/KAP121, pse1-1 (mating type
) was
obtained. A pse1-1/pse1-1 homozygous diploid strain was
generated by mating pse1-1 with pse1-1
(
).
76-130) was
created by the overlap extension method and cloned into pYX242-GFP as
described above to generate
pSpo12
76-130-GFP. Alanine-scanning mutants
in the region 76-130 in pSpo12-GFP were generated by the same method.
Oligonucleotides that sequentially introduced five alanine codons in
place of wild-type sequences were used. Detailed information on plasmid
construction is available upon request. Yeast strains were transformed
with plasmids via the lithium acetate method or electroporation
(18).
76-130 were PCR-amplified
from pSpo12-GFP, pSpo1276-130-GFP, and
pSpo12
76-130-GFP, and ligated into the
bacterial expression vector pGEX4TTev (27) using primer-encoded
EcoRI and SalI sites, resulting in N-terminal fusions with GST. All constructs were verified by sequencing analysis. hLa-GFP and Lhp1-GFP (26), GST-Kap114p (21), and pGEX-Kap121p (28) were
described previously. XL1-blue and BL21 gold (Stratagene, La Jolla, CA)
were used for general cloning and expression purposes. Bacterial
strains were transformed by heat shock.
-D-galactopyranoside for
2 h. Cells were harvested and resuspended in NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl,
pH 8.0, 0.5% Nonidet P-40) containing 20 mM
phenylmethylsulfonyl fluoride and protease inhibitors (CompleteTM
EDTA-free protease inhibitor mixture tablets, Roche). After sonication,
cell debris was pelleted at 15,000 rpm for 15 min and the cleared
supernatant incubated with glutathione-Sepharose for 20 min at room
temperature. Beads were washed with NETN and proteins eluted with
glutathione. In the case of Kap121p and Kap114p, beads were washed and
resuspended in TB containing 0.1 M CaCl2 and,
respectively, 5 units of thrombin or 10 units of factor Xa protease.
Cleavage proceeded overnight at 4 °C. Thrombin was deactivated by
addition of 5 units of hirudin. All proteins were quantified by the
Bradford method.
76-130 were performed similarly.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cytosolic Spo12p interacts with Kap121p.
A, cytosol from cells expressing Spo12p-PrA was incubated
with IgG-Sepharose. Bound proteins were washed with TB and eluted with
a step gradient of MgCl2. Proteins of collected fractions,
as well as the final TB wash, were separated by SDS-PAGE and stained
with Coomassie Blue. The band representing Kap121p is indicated. The
predicted molecular size of Spo12p-PrA is marked with an
asterisk. B, the gel from A was
transferred to nitrocellulose and Spo12p detected via the PrA tag. As
expected, it was detected only in the 2.5-4.5 M
MgCl2 fractions. C, Spo12p-PrA was isolated as
in A, and fractions were run on an SDS-PAGE gel, transferred
to nitrocellulose, and probed with a Kap121p antibody.
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Fig. 2.
Spo12p and Kap121p interact directly.
Purified recombinant GST or GST-Spo12p were immobilized on
glutathione-Sepharose and incubated with Kap121p or Kap114p, as a
control. After washing with TB, bound proteins were eluted with sample
buffer, separated by SDS-PAGE, and visualized with Coomassie Blue
staining.
(Fig. 3), as well as
kap120
, kap119
, kap122
, kap142
, kap104
, kap123
,
kap111
, and kap108
(not shown). In contrast, significant mislocalization to the cytoplasm was observed in
pse1-1, a temperature-sensitive strain bearing a mutation
in Kap121p, at the permissive temperature (Fig. 3). Human La and S. cerevisiae Lhp1p (also known as Yla1p), which are
imported via the Kap95p and Kap108p pathways, respectively (26), were correctly targeted to the nucleus in pse1-1, showing that
other import pathways were not affected in this strain (data not
shown).
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Fig. 3.
Spo12p is mislocalized in the
temperature-sensitive Kap121p mutant, pse1-1.
Spo12p localization was assessed in wild-type (DF5a),
kap114 ,and pse1-1 cells grown at RT.
Spo12p-GFP was visualized in living cells by virtue of the fluorescent
GFP (upper panels). Coincident Nomarski (DIC) images are
shown (lower panels).
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Fig. 4.
Spo12p is mislocalized in pse1-1
homozygous diploid cells. Spo12p-GFP localization was
monitored in diploid wild-type (PSY580/PSY581), pse1-1
heterozygous (pse1-1/KAP121), and pse1-1
homozygous (pse1-1/pse1-1) strains, grown at
room temperature. Coincident Nomarski (DIC) images are also shown
(lower panels).
/spo12
. We found that, among the cells
that had sporulated, the number of asci containing two spores was at
least 3 times greater in pse1-1/pse1-1 than in wild-type
cells (Table I).
Percentage of asci with two spores
76-130-GFP) and sufficient (Spo12p76-130-GFP) for import into the nucleus (Fig.
5B). However, we detected some mislocalization of
Spo12p76-130-GFP to the cytoplasm. This was decreased or
abolished when larger constructs were used (Fig. 5A),
suggesting that larger domains might stabilize the kap/substrate
interaction, leading to higher transport efficiency.
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Fig. 5.
Identification of the Spo12p NLS.
A, deletion mutants of Spo12p were cloned in frame with GFP.
Localization of Spo12p proteins was assessed in living cells by virtue
of the fluorescent GFP. Localization varied from exclusively nuclear
(++++) to throughout the cell, with vacuolar exclusion ( ). All
fusions with nuclear accumulation were mislocalized in
pse1-1. N/A, not assayed. B,
localization of Spo1276-130-GFP and
pSpo12
76-130-GFP was assessed in both
wild-type (DF5a) and pse1-1 cells. Nomarski (DIC) images
are also shown. The GFP fusions were expressed at similar levels, as
assayed by Western blot (data not shown).
76-130 were immobilized on
glutathione-Sepharose and incubated with purified recombinant Kap121p.
After washing, bound proteins were eluted with sample buffer and
analyzed by SDS-PAGE and Coomassie Blue staining (Fig.
6). GST-Spo12p76-130 was
purified as a doublet. The doublet was observed even in whole cell
extracts, after detection of the GST tag by Western blot (data not
shown), suggesting that this protein contains a protease
sensitive site. Note that there was binding of Kap121p observed
to this fusion and not to GST-Spo12p
76-130, even though
there was more GST-Spo12p
76-130 than
GST-Spo12p76-130 or even GST-Spo12p.
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Fig. 6.
Kap121p interacts directly with the Spo12p
NLS. Purified recombinant GST, GST-Spo12p,
GST-Spo12p76-130, and GST-Spo12p 76-130
were immobilized on glutathione-Sepharose and incubated with purified
recombinant Kap121p. After washing, bound proteins were eluted with
sample buffer, separated by SDS-PAGE, and visualized by Coomassie Blue
staining.
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Fig. 7.
Mutagenesis analysis of the Spo12p NLS.
A, mutants in the context of full-length Spo12p-GFP were
constructed by alanine scan mutagenesis and their localization assessed
in living cells. Mut8 contained a valine in position 111, as a result
of a random PCR mutation. B, fluorescence localization of
Spo12p-GFP and Mut4-GFP in wild-type cells (PSY580). Coincident
Nomarski (DIC) images are also shown.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
double mutant but not
kap123
or pse1-1 (15). However, it should be
noted that the observed defect may be indirect. It may be that
deficient import of a protein involved in mRNA export in
pse1-1/kap123
would result in the observed phenotype.
More recently, Kap121p was shown to import transcription
factor Pho4p under phosphate starvation conditions. This role was not
dependent on Kap123p, since Pho4p did not interact with Kap123p nor was
it mislocalized in kap123
(16). This was the first time
Kap121p and Kap123p were attributed separate functions. While this
report was under revision, Kap121p was shown to be responsible for
import of transcription factor Pdr1p (17).
) supporting a primary role for Kap121p in Spo12p
import. The fact that some nuclear accumulation of Spo12p was still
observed in pse1-1 remains to be clarified. One hypothesis
is that since almost 50% of Kap121p remains in this strain at the
permissive temperature (15), the Kap121p still present is sufficient
for import of some of the Spo12p-GFP fusion. In fact, when we studied
Spo12p localization in diploid cells, a slight accumulation of the
fusion in the cytosol of pse1-1/PSY581 was observed,
whereas there was none in wild-type cells and comparable levels in
pse1-1/pse1-1 and haploid pse1-1. This seems
to suggest a connection between the amount of Kap121p and the extent of
Spo12p nuclear accumulation, consistent with this hypothesis. There is
also the possibility that another karyopherin imports Spo12p in this
strain. It has been suggested that some kaps can compensate for the
absence of others, suggesting they can function in overlapping pathways
and that family members have redundant functions (14, 21). Since
Kap121p is essential, a complex of Spo12p with another kap in the
absence of Kap121p could not be identified in vivo.
/
heterodimer.
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ACKNOWLEDGEMENTS |
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We thank the Rockefeller University protein/DNA Technology Center for the MS analysis and Dr. Lucy Pemberton, Dr. Jonathan Rosenblum, Dr. Yuh Min Chook, Dr. John Aitchison, Dr. Michael Rout, Dr. Kimihisa Yoshida, Dr. Markus Albertini, Dr. Anton Titov, and Dr. Pamela Silver for plasmids and strains. We also thank Dr. Rick Wozniak for the Kap121p antibody. We appreciate the technical assistance provided by Evette Ellison. We thank Jonathan Rosenblum and other members of the Blobel laboratory, as well as Lucy Pemberton and Mike Rout, for helpful discussions. We are also grateful to Jonathan Rosenblum and Elias Coutavas for review of this manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a fellowship from PRAXIS XXI, Portugal.
§ To whom correspondence should be addressed. Tel.: 212-327-8096; Fax: 212-327-7880; E-mail: blobel@rockvax.rockefeller.edu.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M010760200
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ABBREVIATIONS |
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The abbreviations used are: NPC, nuclear pore complex; kap, karyopherin; NLS, nuclear localization signal; NES, nuclear export sequence; cNLS, classical NLS; PrA, Protein A from S. aureus; MS, matrix-assisted laser desorption ionization/time of flight mass spectrometry; MALDI-TOF , matrix-assisted laser desorption ionization/time of flight; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; GFP, green fluorescent protein; PCR, polymerase chain reaction; TB, transport buffer; DIC, differential interference contrast.
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