From the Department of Microbiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
Received for publication, October 11, 2000, and in revised form, February 22, 2001
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ABSTRACT |
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The yeast AP-1-like transcription factor, Yap1p,
is essential for the oxidative stress response in budding yeast.
Yap1p is located predominantly in the cytoplasm; however, upon
imposition of oxidative stress, Yap1p concentrates in the nucleus and
activates target genes. Yap1p is constitutively transported in and out
of the nucleus. Oxidative stress inhibits the
Crm1p/Xpo1p-dependent nuclear export step, resulting in
nuclear accumulation of Yap1p. In this study, we examined the mechanism
for Yap1p nuclear import, and determined whether the import step is
affected by oxidative stress. The nuclear accumulation of Yap1p
required the activity of the small GTPase, Ran/Gsp1p. Under conditions
in pse1-1 cells carrying a temperature-sensitive mutation
of the importin Regulation of the nucleocytoplasmic transport of transcription
factors is, in most cases, a crucial step for the transmission of
extracellular signals such as stress and growth stimuli to the nucleus.
When such information is received, transcription factors in the
cytoplasm translocate into the nucleus and become ready to activate
specific gene expressions.
Macromolecules are transported in and out of the nucleus through the
nuclear pore complex (NPC)1
embedded in the nuclear envelope. Transport through the NPC is mediated
by a family of transport receptors (importin The direction of the nucleocytoplasmic transport is thought to be
mediated by the function of small GTPase Ran (4, 5). Ran
GTPase-activating protein and its coactivator, Ran-binding protein 1, are localized in the cytoplasm (6, 7). On the other hand, the guanine
nucleotide exchange factor for Ran is exclusively in the nucleus (8).
This distribution predicts that Ran is in the GTP-loaded form (Ran-GTP)
in the nucleus and in the GDP-loaded form in the cytoplasm. According
to the predicted gradient of different nucleotide-loaded forms of Ran,
it has been suggested that these different forms of Ran can regulate
the association and dissociation of the receptor-cargo complex. The
binding of Ran-GTP to the import receptor facilitates the dissociation
of the import receptor-cargo complex and releases cargo into the nucleus (4, 9, 10). In contrast, the binding of Ran-GTP to the export
receptor is required for the formation of the export receptor-cargo-Ran-GTP complex (11-13). When this trimeric complex is
recognized by Ran GTPase-activating protein and Ran-binding protein 1 in the cytoplasm, it dissociates and releases the cargo into the
cytoplasm (12, 14). Thus, the Ran-GTP gradient is a key determinant for
transport event between the nuclear and cytoplasmic compartments.
It is becoming clear that nuclear translocation of transcription
factors depends on whether transport signals for the receptors are
presented rather than on changing activity of the receptor. Some
examples indicate that the NLSs of transcription factors, which are
normally covered with a higher ordered structure, can be exposed by
extracellular signals transmitted through phosphorylation array (see
Ref. 15 for review).
The yeast AP-1-like transcription factor, Yap1p, which has a basic
leucine-zipper domain and forms a homodimer, is crucial for the
oxidative stress response in the budding yeast (16). In response to
oxidative stress, Yap1p is activated to induce multiple target genes,
which are critical in the cellular defense system for oxidative stress
to increase the levels of reduced thioredoxin and reduced glutathione
in the cells (see Ref. 17 for review). We have shown previously that
Yap1p is mainly localized in the cytoplasm and translocates into the
nucleus under oxidative stress (16). Interestingly, the nuclear
translocation of Yap1p is regulated at the step of nuclear export.
Yap1p is likely to be constitutively imported into the nucleus and
exported to the cytoplasm by the export receptor Crm1p/Xpo1p. When
oxidative stress is imposed, the later step, that is, the interaction
of Crm1p with the cysteine-rich domain at the C terminus of Yap1p, is
inhibited, resulting in increased Yap1p levels in the nucleus (18, 19). Consistent with this result, Yap1p derivatives lacking the
cysteine-rich domain are constitutively localized in the nucleus, and
the resulting Yap1p-dependent transcription of the reporter
gene is increased. In addition to this elevated level of transcription,
however, oxidative stress can further induce reporter gene expression, suggesting that an additional mode of the regulatory system is responsible (16). We have so far found no evidence that the import step
is not responsible for the oxidative stress-induced nuclear
localization of Yap1p.
Here we address the molecular mechanism of the nuclear import of Yap1p
to determine whether oxidative stress affects the Yap1p import step.
Our results indicate that importin Yeast Strains and Cultures--
The yeast strains used in
this study are listed in Table I. Yeast
cells were grown in yeast extract, pepton, adenine, dextrose or
synthetic medium supplemented with amino acids (20). Yeast cells
were exposed to oxidative stress by the addition of diamide to a final
concentration of 1.5 mM or of H2O2
to a final concentration of 0.5 mM, when required, as
described previously (16).
Construction of Plasmids--
Expression plasmids for the
green fluorescent protein (GFP) fused to Yap1p were constructed as
follows: GFPS65T-fused Yap1p expression plasmids, pRS
cpGFP-YAP1, and pRS cpGFP-yap1(1-571) (TRP1 CEN) (16) were
digested with SacI and XhoI and inserted into the
corresponding sites of pRS315 (LEU2 CEN) (21) to generate pRS315-GFP-YAP1 and pRS315-GFP-yap1(1-571), respectively.
To make the plasmid to express Pse1p as a fusion protein to glutathione
S-transferase (GST), a BamHI-MscI
fragment corresponding to an N-terminal region of Pse1p from pPS1567
(22) and an MscI-XhoI fragment corresponding to a
C-terminal region of Pse1p from pPS1066 (22) were ligated into
BamHI and SalI sites of pGEX-6P-2 (Amersham Pharmacia Biotech). The resulting GST-Pse1p expression plasmid was
designated pGEX-PSE1.
The pGEX-KAP123 was constructed as follows: A 5'-coding region of
KAP123 was amplified from pPS1067 containing
KAP123 (22) by polymerase chain reaction (PCR) using
5'-GTCAGTCGACTATGGATCAACAATTTCTAAGTCA-3' and
5'-TTCACCAGAAGCAGTTTGGAT-3' as primers (SalI and
XcmI sites are underlined) and digested SalI and
XcmI; a 3'-coding region of KAP123 was isolated
from pSP1067 with XcmI and AvaII, where the
AvaII site was blunt-ended; and these fragments were
inserted between SalI and blunt-ended NotI of
pGEX-6P-2.
To generate hemagglutinin (HA)-tagged GFP-Yap1p(1-244), a
BamHI-BstEII fragment corresponding to a
C-terminal region of Yap1p from pRS cpGFP-HA-YAP1 (16) was replaced by
that containing yap1(1-244) from pRS cpGFP-yap1(1-244) (16). The
resulting plasmid pRS cpGFP-HA-yap1(1-244) was digested with
PvuII and SalI and inserted into the
blunt-ended EcoRI and SalI sites of pET28b(+) (Novagen) to construct the His-tagged Yap1p(1-244) expression plasmid, pET-yap1(1-244).
By utilizing pPS965 (23) as a template for GSP1, a G21V
mutation was introduced by PCR using the following primers: primer 1, 5'-GTCACATATGTCTGCCCCAGCTGCTAAC-3'; primer 2, 5'-TCAAACTAGTTCTTGTCGGTGATGTCGGTACTGGT-3'; and primer 3, 5'-GACTGTCGACAGCTTGTTCTCGTTTGTCCCTT-3'. A 5'-coding region
of GSP1 was isolated by PCR using primers 1 and 2 (NdeI and SpeI sites are underlined) and digested
with NdeI and SpeI. A 3'-coding region of
GSP1, in which the 21st codon of GGT (Gly) was changed to
GTC (Val) (G21V mutation), was isolated by PCR using primers 2 and 3 (SpeI and SalI sites are underlined) and digested
with SpeI and SalI. Both of these fragments were
ligated between the NdeI and SalI sites of pET15b
(Novagen) to generate the His-tagged Gsp1pG21V expression
plasmid. The resulting plasmid was designated
pET-GSP1G21V.
In the construction of plasmids to identify Yap1p NLS, the following
GFP fusion vector was generated. Plasmids having genes encoding the
duplicate inflame fusion protein of GFP536 (24) as well as the HA tag
were constructed under the regulated constitutive cup1
promoter (16) or inducible met3 promoter (18) in plasmid pRS315 (21) and were designated pRS315 cup1-2xGFP-HA and pRS315 met3-2xGFP-HA, respectively. To generate a series of Yap1p derivatives on the N-terminal region of Yap1p (see Fig. 5A), PCRs using
corresponding synthetic oligonucleotides are carried out or restriction
enzymes are used. The resulting fragments corresponding to various
N-terminal regions of Yap1p were inserted in the 3'-coding region of
duplicated GFP. Expected fusion proteins expressed by these plasmids
are more than 60 kilodaltons.
In Vivo Import Assay--
To determine the minor transport
activity of Yap1p and the effect of oxidative stress on the nuclear
import of Yap1p, we performed an in vivo nuclear import
assay using the inducible met3 promoter to express
2xGFP-fused Yap1p(1-571) or Yap1p(2-59). Yeast cells were cultured
under a condition in which the met3 promoter was suppressed
in the presence of methionine until the mid-log phase. The cells were
then collected by centrifugation, washed once with methionine-depleted
medium, and incubated in the methionine-depleted medium to induce the
expression of 2xGFP-Yap1p derivatives by a met3 promoter.
Oxidative stress and/or temperature shift from the permissive
temperature (25 °C) to the restrictive temperature (37 °C) were
performed at the same time as induction by the met3 promoter. GFP fluorescence, which could be detected from 30 min after
induction, was observed at the indicated time.
Fluorescence Microscopy--
Confocal laser scanning microscopic
analysis was carried out as described previously (16). Briefly, yeast
cells were grown to mid-log phase, and the localization of GFP in live
yeast cells was observed using a confocal laser scanning microscope
(MRC1024, Bio-Rad).
Recombinant Protein Expression and
Purification--
Purification of GST fusion proteins was performed as
described by Isoyama et al. (25). Escherichia
coli BL21 cells were transformed with the pGEX-PSE1 or pGEX-KAP123
plasmids to express fusion proteins to GST and grown at 37 °C in the
presence of ampicillin. At an A600 nm of
0.5, isopropyl- Binding Assay--
For the assay of Yap1p binding to Pse1p or
Kap123p, fusion proteins were expressed and purified from bacterial
lysate as described above. 5 µg of GST-Pse1p, GST-Kap123p, or GST
were bound to 20 µl of the 50% slurry of glutathione-Sepharose beads
by incubating in binding buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol, 10% glycerol, and 0.5 mg/ml bovine
serum albumin) for 2 h at 4 °C. The beads bound to GST fusion
proteins were washed with washing buffer (50 mM Tris-HCl,
pH 7.5, 100 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol, and 10% glycerol) and incubated with
5 µg of His-Yap1p(1-244) in 250 µl of binding buffer for 3 h
at 4 °C. After washing extensively, bound proteins were eluted with
10 mM reduced glutathione and analyzed by
SDS-polyacrylamide gel electrophoresis (PAGE) followed by Coomassie
staining or immunoblotting. His-Yap1p(1-244) was detected with
anti-His-Tag (MBL) and peroxidase-conjugated goat anti-rabbit
immunoglobulins (DAKO) as primary and secondary antibodies,
respectively. ECL Western blotting detection reagents were used
according to the manufacturer's instructions (Amersham Pharmacia
Biotech).
Dissociation Experiment--
His-Gsp1pG21V was
incubated in the presence of 2 mM GTP, 20 mM
EDTA, and 2 mM dithiothreitol for 1 h at room
temperature. After the addition of MgCl2 to a final
concentration of 50 mM, the reaction mixture was incubated
further on ice for 20 min. His-Gsp1pG21V loaded with GTP
(His-Gsp1pG21V-GTP) was purified by chromatography on Fast
Desalting (Amersham Pharmacia Biotech) in 50 mM Tris-HCl,
pH 7.5, 300 mM NaCl, 2 mM MgCl2,
and 2 mM 2-mercaptoethanol. 10 µg of His-Yap1p(1-244)
was incubated with 10 µg of GST-Pse1p or GST-Kap123p prebound to 20 µl of glutathione-Sepharose beads in a reaction volume of 500 µl
for 3 h at 4 °C. The beads were washed with washing buffer, divided into two equal parts, and incubated with 5 µg of
His-Gsp1pG21V-GTP or buffer for 30 min at room temperature.
After centrifugation, the supernatant was collected as unbound protein.
Then, the beads were washed several times with washing buffer, and the
bound protein was dissolved in SDS sample buffer. The samples were
separated by SDS-PAGE and immunoblotted with anti-His-tag antibodies as described above.
Nuclear Accumulation of Yap1p Requires Ran/Gsp1p--
In most
cases, nuclear transport events require the activity of the small
GTPase Ran (4, 9, 10). Therefore, we first examined if the nuclear
import of Yap1p also requires the activity of Ran using a previously
characterized temperature-sensitive mutant (gsp1-1) of
GSP1 encoding the budding yeast homologue of Ran (23). The
constitutive nuclear-localized mutant of Yap1p, GFP-Yap1p(1-571), was
expressed in gsp1-1 cells, and the cells were cultured at
the permissive temperature (25 °C). As we previously observed in the
wild-type cells (16), GFP-Yap1p(1-571) was localized in the nucleus
(Fig. 2A, panel 1). In contrast, this nuclear
accumulation was inhibited at the restrictive temperature (37 °C)
(Fig. 1), indicating clearly that the
nuclear import of Yap1p is mediated in a Ran-dependent
manner.
Nuclear Accumulation of Yap1p Requires Pse1p--
The above data
suggested that, like many other NLS-containing cargo proteins, Yap1p
was imported into the nucleus by a transport receptor(s) that requires
Ran for its transport activity. To identify such a receptor(s), we
examined how the nuclear accumulation of Yap1p was affected using a
panel of yeast strains carrying each mutation of transport receptors
including the importin
Yeast cells carrying a mutation of importin
Intriguingly however, most of the GFP-Yap1p(1-571) was spread in the
cytoplasm in pse1-1 cells at the restrictive temperature and residual fluorescence could be observed in the nucleus (Fig. 2A, panel 3). One possible explanation for
this was that preexisting GFP-Yap1p(1-571) in the nucleus was not
degraded within 1 h after shifting to the restrictive temperature.
To examine this possibility, nuclear accumulation of
Yap1pWT was induced by the imposition of oxidative
stress in pse1-1 cells at the same time as the temperature
shift to the restrictive temperature. Again, the nuclear accumulation
of Yap1pWT induced by diamide was dramatically inhibited
only in pse1-1 cells at the restrictive temperature (Fig.
2B, compare panels 4 and 6 with
panels 3 and 5) but not other mutant cells (Fig. 2B, compare panels 7 and 8; data not
shown). Nevertheless, as shown in Fig. 2B, panel
6, the residual nuclear fluorescence of GFP-Yap1pWT
still could be observed in this condition.
We then speculated that other transport receptors could confer the weak
activity for nuclear import of Yap1p in addition to Pse1p. It has been
suggested that Pse1p and its closely related transport receptor Kap123p
(Yrb4p) can confer overlapping functions for nuclear import,
e.g. ribosomal protein L25 is transported mainly by Kap123p;
however, Pse1p can substitute for Kap123p (10, 26). Therefore, we
predicted that Kap123p might be responsible for the residual nuclear
localization of Yap1p in pse1-1 cells at the restrictive
temperature. To test this possibility, we observed the nuclear
accumulation of Yap1p in yeast cells carrying a KAP123 disruption mutant in the background of pse1-1
(pse1-1 kap123 Direct Binding of Yap1p to Pse1p--
Previous studies have
indicated that transport receptors can directly bind to the import
cargos. To test whether this was the case for the interaction between
Pse1p or Kap123p and Yap1p in an in vitro binding assay,
His-tagged Yap1p(1-244), which has an NLS and can be constitutively
localized in the nucleus (16), and GST-fused Pse1p were expressed in
E. coli and purified. His-Yap1p(1-244) was mixed with
GST-Pse1p or GST-Kap123p prebound to glutathione-Sepharose beads, and
protein bound to the beads was resolved in SDS-PAGE followed by
Coomassie staining to detect Pse1p and Kap123p or immunoblotting with
anti-His-tag antibodies to detect Yap1p. We found that
His-Yap1p(1-244) could bind to GST-Pse1p as well as GST-Kap123p, but
not to GST alone (Fig. 4A),
indicating that Yap1p directly interacts with Pse1p and Kap123p. As
shown in Fig. 4A, lane 3, degradation products of
GST-Kap123p, which were purified by glutathione-Sepharose beads, were
detected. We speculated that these products did not bind to Yap1p
because the degradation might have been limited in the C-terminal
region of Kap123p, which is generally required for cargo binding.
Therefore, it was presumed that His-Yap1p(1-244) bound only to
full-length GST-Kap123p. Despite the minor function of Kap123p on the
nuclear import of Yap1p as described above, the amount of
Yap1p(1-244) binding to Pse1p was similar to that binding to Kap123p
in vitro (Fig. 4A, compare lanes 2 and
3).
Yap1p-Pse1p Complex Is Dissociated by Gsp1p-GTP--
Previous
studies indicate that Ran-GTP can dissociate import receptor-cargo
interaction (4, 9, 10). To determine whether the interaction between
Yap1p and Pse1p or Kap123p could be disrupted by Gsp1p-GTP, we prepared
the G21V mutant of Gsp1p (Gsp1pG21V), where GTP hydrolysis
was disrupted by the mutation (27). As shown in Fig. 4B,
Yap1p(1-244) bound to Pse1p and Kap123p were clearly dissociated by
the addition of Gsp1pG21V-GTP (compare lanes 2 and 4 with lanes 1 and 3). These
results support the idea that the binding of Yap1p to Pse1p or
Kap123p is a specific interaction between the import receptor and cargo and provide further support for the functional relevance of the interaction of Yap1p with Pse1p or Kap123p.
Identification of Yap1p NLS--
We have shown previously that the
Yap1p NLS is located within amino acids 1-244 (16), which contain the
basic DNA binding region as well as the dimerization domain (basic
leucine-zipper). To identify the Yap1p NLS, genes corresponding to the
series of N-terminal regions of Yap1p were fused to duplicate copies of GFP536 genes under the inducible met3 promoter as described
under "Experimental Procedures." As shown in Fig.
5, Yap1p(2-72), Yap1p(2-66), and
Yap1p(2-59) were localized in the nucleus (A and
B, panel 2). However, when this C-terminal
deletion was extended further to amino acid position 50 (Yap1p(2-49)),
the nuclear localization of Yap1p was abolished (Fig. 5B,
panel 3), suggesting that Yap1p amino acids 50-59 are
essential for the NLS function. Actually, Yap1p amino acid region from
50 to 59 (KKKGSKTSKK) is similar to the classical NLS (28). Therefore,
we tested whether this short amino acid sequence could act as an NLS.
Unexpectedly, it failed to confer NLS activity when fused to duplicate
copies of GFP (Fig. 5B, panel 4). Next we
demonstrated the effect of N-terminal deletion for the NLS activity.
Although the deletion of four N-terminal amino acids did not
affect NLS activity, the deletion of nine amino acids strongly
abrogated NLS activity (Fig. 5B, panels 5 and
6). Further deletion did not recover the NLS activity of
Yap1p (Fig. 5A). However, the internal amino acid region
from 17 to 49, but not that from 10 to 49, could be deleted without
having an effect on NLS activity (Fig. 5, A and
B, panels 7 and 8). Taken together, we
conclude that two amino acid regions of Yap1p, 5-16 and 50-59, are
required for the NLS activity. In addition, when the two basic amino
acid residues in this N-terminal region were substituted to alanine
(K7A, R8A), the NLS activity was abrogated (data not shown).
Nuclear Import of Yap1p Is Not Affected by Oxidative
Stress--
We have shown previously that the constitutive
nuclear-localized Yap1p(1-571) can still confer further elevation of
the transcriptional level of the reporter gene in response to oxidative
stress (16). Therefore we performed an in vivo import assay
of Yap1p(1-571) and Yap1p(2-59) under oxidative stress to examine
whether oxidative stress could affect the nuclear import of Yap1p. The
nuclear accumulation of Yap1p(1-571) or Yap1p(2-59) observed at 30, 60, and 90 min after induction of the met3 promoter was not
affected by the oxidative stress imposed by either diamide (Fig.
6) or H2O2 (data
not shown). Therefore, we conclude that oxidative stress does not
affect the nuclear import of Yap1p.
Regulation of the Yap1p transcription factor is mediated mainly at
the level of nuclear localization. Inhibition of the nuclear export
step by oxidative stress results in an increased level of Yap1p in the
nucleus, leading to the activation of target gene transcription. It has
been suggested that an additional activation mechanism confers
enhancement of Yap1p-dependent transcription because the
constitutive nuclear-localized Yap1p mutant (Yap1p(1-571)) can be
further activated by oxidative stress (16). Here we examined the
molecular mechanism of the import step of Yap1p and determined whether
the import step of Yap1p was affected by oxidative stress.
Two lines of evidence indicate that Pse1p mediates the nuclear import
of Yap1p. First, the nuclear transport of Yap1p was strongly prevented
in pse1-1 cells at the restrictive temperature, whereas
none of the other mutant cells carrying each one of the genes encoding
an importin In addition to the Pse1p-mediated nuclear import of Yap1p, we observed
a minor effect of Kap123p on the nuclear import of Yap1p (Fig. 3). More
residual nuclear-localized Yap1p was detected in pse1-1
cells than in pse1-1 kap123 Yap1p now can be included in the list of cargos transported by Pse1p,
which include the transcription factor Pho4p and ribosomal protein L25.
In the case of Pho4p, the import is solely mediated by Pse1p but not by
Kap123p (29). Thus, Yap1p, Pho4p, and ribosomal protein L25 apparently
have different specificities in terms of receptor selectivity. Among
these homologous import receptors (Pse1p and Kap123p), only Pse1p is
essential for yeast cell growth. Although ribosomal proteins are
essential for protein synthesis, cell growth is not affected in the
KAP123 disruption mutant. This was explained by the fact
that the nuclear transport of ribosomal proteins is substituted by
Pse1p as described above. PSE1 is an essential gene for
yeast cell growth, whereas Yap1p and Pho4p transported by Pse1p are not
essential, suggesting that there should be some other essential cargos
that definitely require Pse1p as a nuclear import receptor.
We identified bipartite NLS in Yap1p amino acid regions 5-16 and
50-59, where those respective regions include two basic residues (KR)
and a cluster of basic residues (KKKGSKTSKK) with a 41-amino acid-long
spacer (Fig. 5). There is no sequence homology between the NLS of Yap1p
and that of Pho4p except that the basic amino acids are distributed in
Pho4p NLS (amino acids 140-166) (29). It is becoming clear that many
NLSs, which are recognized directly by the importin Here, we demonstrated that the nuclear import of Yap1p(1-571) as well
as Yap1p(2-59) are neither repressed nor enhanced by oxidative stress
(Fig. 6), suggesting that the enhancement of the transcriptional
activity of the constitutive nuclear-localized Yap1p(1-571) by
oxidative stress should be caused by other step, e.g. the
enhancement of DNA binding activity (39) or transcriptional activity by
modification. It has been shown that Pse1p interacts with a number of
nucleoporins that constitute the NPC, and these interactions can be
dissociated by Ran-GTP (40-42). These events are supposed to be the
mechanism of nuclear transport through the NPC. Therefore, our results
indicate that the course of nuclear import of Yap1p, that is, binding
of Yap1p to Pse1p, interaction of Pse1p with nucleoporins, and the
dissociation of Yap1p from Pse1p, is not affected by oxidative stress
caused by diamide and H2O2.
Regulation of the nuclear import step is generally crucial for most
transcription factors that are activated by extracellular signals. In
contrast, we now provide evidence that Yap1p nuclear localization is
entirely regulated at the step of nuclear export. The interaction
between Yap1p and its export receptor Crm1p is regulated by oxidative
stress (18, 19). This unique strategy for Yap1p regulation might be
significant because this regulatory mechanism seems to be conserved in
Yap1p homologues Pap1 of the fission yeast (43) and Cap1 of
Candida albicans (44). We have shown recently that Yap1p can
directly sense an oxidative stress (redox) signal, and the nuclear
accumulation starts at 1 min.2 This rapid response
might be carried out by inhibition of the nuclear export of preexisting
Yap1p in the nucleus because H2O2 and diamide
might spread rapidly in the cells. Alternatively, it may be required
for yeast cells to sense the oxidative stress (redox) signal in the
nucleus under certain conditions.
In summary, we identified Pse1p as the nuclear transport receptor of
Yap1p and showed that the nuclear import step of Yap1p was not affected
by oxidative stress. Our results and previous studies clearly indicate
that Yap1p subcellular localization in response to oxidative stress is
regulated solely at the step of Crm1p-dependent nuclear export.
family member PSE1/KAP121, nuclear
translocation of Yap1p was inhibited dramatically. In an in
vitro assay, we showed that Yap1p could directly bind to Pse1p
and that this interaction was dissociated by Ran-GTP. These results
indicate that Pse1p is the nuclear import receptor for Yap1p. In
addition to Pse1p, we suggest that Kap123p, which is homologous to
Pse1p, has a minor effect on the nuclear import of Yap1p.
Furthermore, we identified the nuclear localization signal of Yap1p and
demonstrated that the nuclear import of Yap1p was not affected by
oxidative stress.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/karyopherin
family; importins and exportins) that share homology in the Ran binding
domain. The transport receptors recognize cargos through transport
signals such as the nuclear localization signal (NLS) and the nuclear
export signal. It has been shown that multiple pathways of
nucleocytoplasmic transport are carried out by utilizing different
transport receptors and transport signals (see Ref. 1 for review). 14 transport receptors (the importin
family proteins) have been
identified from the genome information of the budding yeast,
Saccharomyces cerevisiae (1-3). To date, those include nine
import receptors, four export receptors, and one other that is
uncharacterized. Intriguingly, only four of the yeast transport
receptors are essential, and the other receptors are not essential
despite the fact that they transport essential cargos. One explanation
for this is that multiple transport receptors may mediate the transport
of a single specific cargo.
family member Pse1p/Kap121p is
the nuclear import receptor for Yap1p. In addition to Pse1p, we suggest
that Kap123p, which is related closely to Pse1p, has a minor effect on
the nuclear import of Yap1p. Moreover, we identified the Yap1p NLS and
showed that oxidative stress does not affect the nuclear import pathway
of Yap1p.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
The yeast strains used in this study
-D-thiogalactoside was added to the
culture at a final concentration of 1 mM and further cultured at 37 °C for 4 h. The cells were harvested by
centrifugation, resuspended in phosphate-buffered saline (140 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, and 1.8 mM
KH2PO4), and disrupted by sonication. The cell
lysates were mixed with Triton X-100 to a final concentration of 1%
and incubated at 4 °C for 30 min. The fusion proteins were purified
by using glutathione-Sepharose 4B according to the manufacturer's instructions (Amersham Pharmacia Biotech). To isolate His-Yap1p(1-244) or His-Gsp1pG21V, E. coli BL21(DE3) cells
carrying pET-yap1(1-244) or pET-GSP1G21V were grown in the
presence of kanamycin or ampicillin, respectively, and the expression
was induced with isopropyl-
-D-thiogalactoside at a final
concentration of 1 mM at 20 °C for 20 h. The cells were harvested and sonicated in lysis buffer (50 mM
Na2HPO4, pH 8.0, 300 mM NaCl, 1 mM 2-mercaptoethanol, and 20 mM imidazole). After the addition of Triton X-100 to a final concentration of 1%, the
lysates were loaded into a column of nickel-nitrilotriacetic acid agarose according to the manufacturer's instructions
(Qiagen). The column was washed, and the fusion proteins were eluted
with elution buffer (lysis buffer containing 250 mM
imidazole). Purified fusion proteins were concentrated by VIVASPIN
(Vivascience) and further purified by chromatography on a Superdex 200 in 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 2 mM 2-mercaptoethanol using the SMART system (Amersham
Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (85K):
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Fig. 1.
Nuclear import of Yap1p requires
Ran/Gsp1p. The gsp1-1 cells carrying a
temperature-sensitive GSP1 allele were transformed with
pRS315-GFP-yap1(1-571). The cells were grown to mid-log phase at the
permissive temperature of 25 °C and further incubated at the
restrictive temperature of 37 °C for 1 h. GFP-Yap1p(1-571) was
detected by confocal microscopy as described under "Experimental
Procedures." The fluorescent images (left panels) and the
transmitted images (right panels) are shown.
family members as well as importin
. Two
different conditions were used to induce the nuclear accumulation, or
in other words, to inhibit the nuclear export step of Yap1p. First, the
constitutive nuclear-localized mutant of Yap1p (GFP-Yap1p(1-571)) was
expressed in mutant cells, and we observed which mutation affected its
localization. Second, wild-type Yap1p (Yap1pWT) fused with
GFP was expressed in each mutant cell, and the specific nuclear export
step of Yap1pWT was inhibited by oxidative stress to induce
nuclear accumulation of Yap1p (18).
(srp1-31)
or importin
(rsl1-4) showed no defect in nuclear
translocation of Yap1p (data not shown), demonstrating that the nuclear
import of Yap1p does not depend on the classical NLS import pathway. Other strains carrying mutations of importin
family member
sxm1
, nmd5
, or mtr10
showed
no defect in the nuclear accumulation of Yap1p (data not shown).
However, we found that nuclear localization of GFP-Yap1p(1-571) was
significantly inhibited in the temperature-sensitive PSE1
mutant (pse1-1) cells under the restrictive temperature of 37 °C (Fig. 2A, compare
panels 2 and 3). In addition, unlike Pse1p, Kap123p did not affect the nuclear localization, although Kap123p is
related closely to Pse1p (Fig. 2A, panel 4).
View larger version (56K):
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Fig. 2.
Nuclear localization of Yap1p requires
Pse1p. The effects of mutation of the import receptor on the
nuclear import of Yap1p were observed using a constitutive
nuclear-localized mutant of Yap1p (A,
Yap1p(1-571)) or by oxidative stress-induced
nuclear localization of wild-type Yap1p (B,
Yap1pWT). A, localization of
Yap1p(1-571) was observed in yeast cells carrying each mutation of
import receptors. Wild-type, pse1-1, or
kap123 cells were transformed with
pRS315-GFP-yap1(1-571) and incubated at 30 °C (panel 1,
wild-type cells; panel 4, kap123
cells), the
permissive temperature of 25 °C (panel 2,
pse1-1 cells), or the restrictive temperature of 37 °C
(panel 3, pse1-1 cells) for 1 h.
B, oxidative stress-induced nuclear localization of
Yap1pWT was observed in wild-type, pse1-1, or
kap123
cells transformed with pRS315-GFP-YAP1. The cells
were incubated at 30 °C (panels 1 and 2,
wild-type cells; panels 7 and 8,
kap123
cells), the permissive temperature of 25 °C
(panels 3 and 4, pse1-1 cells), or
the restrictive temperature of 37 °C (panels 5 and
6, pse1-1 cells) with 1.5 mM diamide
(+, panels 2, 4, 6, and 8)
or without diamide (
, panels 1, 3,
5, and 7) for 1 h. The fluorescent and
transmitted images were detected as described in the legend to Fig.
1.
cells). In this case, we performed an
in vivo import assay to eliminate the effect of possible
artifacts caused by the oxidative stress and protein stability
described above. Expression of 2xGFP-Yap1p(1-571) was repressed with
the addition of methionine and induced by depleting methionine from the
medium at the same time of the shift to the restrictive temperature
(37 °C) as described under "Experimental Procedures." Although
the residual fluorescence of 2xGFP-Yap1p(1-571) still could be
observed in the pse1-1 cells (Fig.
3, panel 2), it was strongly
suppressed in pse1-1 kap123
cells at the restrictive temperature (Fig. 3, compare panels 2 and 4).
Taken together, these results indicate that Pse1p is a nuclear import
receptor responsible for Yap1p; however, Kap123p also has a minor
potential function when the activity of Pse1p is repressed under
restrictive conditions.
View larger version (37K):
[in a new window]
Fig. 3.
Kap123p has a minor potential role in the
nuclear accumulation of Yap1p. Duplicated GFP-fused Yap1p(1-571)
was expressed in pse1-1 or pse1-1 kap123
cells by using an inducible met3 promoter. The cells were
grown to mid-log phase under the repressive condition for the
met3 promoter in the medium containing methionine, washed
with methionine-depleted medium, and incubated further in the
methionine-depleted medium to induce the fusion protein at the
permissive temperature of 25 °C (panels 1 and
3) or the restrictive temperature of 37 °C (panels
2 and 4) for 1 h. The fluorescent and transmitted
images were detected as described in the legend to Fig. 1.
View larger version (12K):
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Fig. 4.
Yap1p interacts directly with Pse1p and
Kap123p, and the interaction is dissociated by Gsp1p-GTP.
A, Yap1p associated with Pse1p and Kap123p. GST, GST-Pse1p,
or GST-Kap123p purified from E. coli were bound to
glutathione-Sepharose beads, incubated with His-Yap1p(1-244) also
purified from E. coli, and washed extensively. Bound
proteins were eluted with 10 mM glutathione, and eluted
proteins were separated on 12% SDS-PAGE and subjected to Coomassie
staining (upper panel) or immunoblotting with anti-His-tag
antibodies (lower panel). His-Yap1p(1-244) bound to GST,
GST-Pse1p, and GST-Kap123p are shown in lanes 1,
2, and 3, respectively. The bands representing
GST, GST-Pse1p, GST-Kap123p, and His-Yap1p(1-244) are indicated by
arrows. The positions of molecular mass markers are
shown on the left of the figure in kilodaltons. The protein of 67 kDa
is bovine serum albumin. It is also noted that some bands (from 55 to
70 kDa) appearing in lane 3 are degradation products of
Kap123p, because these were detected by immunoblotting with anti-GST
antibodies (data not shown). B, Gsp1p-GTP dissociated the
Yap1p-Pse1p and Yap1p-Kap123p complexes. GST fusion proteins of import
receptors bound to glutathione-Sepharose beads and His-Yap1p(1-244)
were incubated and washed extensively. The dissociation reaction was
carried out with His-Gsp1pG21V-GTP (lanes 2,
4, 5, and 6) or buffer alone
(lanes 1 and 3). After centrifugation, the
supernatant was collected as unbound protein (lanes 5 and
6). The beads were washed, and the bound protein was
dissolved in SDS sample buffer (lanes 1-4). The
samples were separated on 12% SDS-PAGE and visualized by
immunoblotting with anti-His-tag antibodies. The bands representing
His-Yap1p(1-244) and His-Gsp1pG21V-GTP are indicated by
the arrows.
View larger version (55K):
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Fig. 5.
Yap1p amino acids 5-59 are necessary for
nuclear localization. A, the localization of a series
of Yap1p deletion mutants as fusion proteins to duplicated GFP was
observed in the wild-type cells. The localization of 2xGFP-Yap1p
derivatives is indicated on the right (N,
nucleus; C, cytoplasm). The sequence of Yap1p amino acids
1-72 is shown above. B, the localization of
2xGFP-Yap1p derivatives is shown as follows: panel 1, images
of yeast cells transformed with pRS315 met3-2xGFP-HA; panel
2, Yap1p(2-59); panel 3, Yap1p(2-49); panel
4, Yap1p(50-59); panel 5, Yap1p(10-59);
panel 6, Yap1p(5-59); panel 7, Yap1p(5-16,
50-59); and panel 8, Yap1p(5-9, 50-59). The fluorescent
and transmitted images were detected as described in the legend to Fig.
1.
View larger version (50K):
[in a new window]
Fig. 6.
Nuclear import of Yap1p is not affected by
oxidative stress. The effect of oxidative stress on the nuclear
import of Yap1p was observed in the wild-type cells. The constitutive
nuclear-localized Yap1p(1-571) (A) and Yap1p(2-59)
(B) were fused with the duplicated copies of GFP and
expressed under the inducible met3 promoter as described
under "Experimental Procedures." To impose oxidative stress, cells
were treated with 1.5 mM diamide (+, panels 3,
5, and 7) or without diamide ( , panels
1, 2, 4, and 6) at the same time
as induction by the met3 promoter. Yap1p derivatives were
observed at 0, 30, 60, and 90 min after induction. The fluorescent and
transmitted images were detected as described in the legend to Fig.
1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
family member as well as an adaptor molecule (importin
) that we tested affected the nuclear import of Yap1p (Fig. 2). In
addition, our genetic studies also showed that the activity of the
small GTPase Ran was required for the nuclear accumulation of Yap1p,
similar to many other nuclear transport cargos (Fig. 1). Second, we
demonstrated that Yap1p can directly interact with Pse1p in
vitro, and Ran-GTP can specifically inhibit this interaction (Fig.
4).
cells at the restrictive temperature. In addition, we showed that Kap123p can also bind directly to Yap1p (Fig. 4A), and the other group showed that
Kap123p can interact with Yap1p by the two- hybrid assay (19). However, our observation indicated the discrepancy between the in
vitro binding ability and the nuclear import ability of Pse1p and
Kap123p. Such a discrepancy may be caused by the inefficiency of the
other nuclear import step after Kap123p-Yap1p binding, e.g.
transport through the NPC and dissociation of the receptor-cargo
complex in the nucleus. Thus, we conclude that Pse1p is a major import receptor for Yap1p, and Kap123p has a minor potential function. A
similar example has been indicated for the case of ribosomal protein
L25 (10). Ribosomal protein L25 can bind to Kap123p and Pse1p in
vitro. Disruption of KAP123 inhibits the nuclear localization of ribosomal protein L25, indicating that Kap123p is a
major import receptor for the ribosomal protein. However, it has
been suggested that Pse1p is a minor import receptor for ribosomal
protein L25 because overexpression of Pse1p induces the nuclear
localization of the ribosomal protein (26).
homologue,
consist of longer amino acid regions rather than a short peptide motif
like the classical NLS recognized by the importin
/
complex (1,
28). This may be explained by the hypothesis that import receptors have
a second function, i.e. receptors bind to cargos and
somewhat cover the cargo molecules to prevent inappropriate interaction
with the cellular components before the cargos reach their final
destination. This hypothesis is consistent with the recent finding that
Ran-GTP-mediated dissociation of the interaction between the
TATA-binding protein and its import receptor, Kap114p, is stimulated by
TATA-containing DNA (30). Interestingly, in the case of many
transcription factors including Yap1p, NLS is located next to the DNA
binding domain (for example, Gal4 DNA binding domain (31), the basic
leucine-zipper domain including the CAAT/enhancer-binding protein (32)
and v-jun (33), basic helix-loop-helix domain (34, 35), homeodomain
(36, 37), and high-mobility group domain (38)). We therefore
speculate that the association of the respective import receptor to the NLS of these transcription factors might protect their DNA binding domains and might inhibit the DNA binding activity until these factors
reach an appropriate DNA binding site, and the binding of the
transcription factor to the DNA binding site might accelerate the
dissociation of the receptor-cargo interaction.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Pamela A. Silver for helpful discussion and for providing yeast strains and plasmids and Ed Hurt for providing yeast strains.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan and the Japan Society for the Promotion of Science.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.
Supported by research fellowships of the Japan Society for the
Promotion of Science for Young Scientists.
§ Present address: Laboratory of Molecular and Biochemical Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aza-Aoba, Aoba-ku, Sendai, Miyagi 980-8578, Japan. To whom correspondence should be addressed: Tel.: 81-22-217-6872; Fax: 81-22-217-6872; E-mail: skuge@mail.pharm.tohoku.ac.jp.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M009258200
2 M. Arita, A. Murayama, K. Maeta, S. Izawa, Y. Inoue, A. Nomoto, and S. Kuge, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NPC, nuclear pore complex; NLS, nuclear localization signal; Ran-GTP, GTP-loaded form of Ran; GFP, green fluorescent protein; GST, glutathione S-transferase; PCR, polymerase chain reaction; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; WT, wild type.
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REFERENCES |
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