Gln3p is a GATA-type transcription
activator of nitrogen catabolite repressible (NCR) genes. Gln3p
was recently found to be hyperphosphorylated in a
TOR-dependent manner and resides in the cytoplasm in high
quality nitrogen. In contrast, during nitrogen starvation or rapamycin
treatment, Gln3p becomes rapidly dephosphorylated and accumulates in
the nucleus, thereby activating nitrogen catabolite repression genes.
However, a detailed mechanistic understanding is lacking for the
regulation of Gln3p nucleocytoplasmic distribution. In this study, we
applied a functional genomics approach to identify the nuclear
transport factors for Gln3p. We found that yeast karyopherin
/Srp1p
and Crm1p are required for the nuclear import and export of Gln3p,
respectively. Similarly, the Ran GTPase pathway is also involved in the
nuclear translocation of Gln3p. Finally, we show that Srp1p
preferentially interacts with the hypophosphorylated versus
the hyperphosphorylated Gln3p. These findings define a possible
mechanism for regulated nucleocytoplasmic transport of Gln3p by
phosphorylation in vivo.
 |
INTRODUCTION |
In response to nitrogen levels and sources, eukaryotic cells
selectively regulate expression of genes involved in utilization and
transport of the available nutrients. In the presence of rich nitrogen
sources, the expression of genes necessary for utilization and
transport of poor nitrogen sources is repressed. These genes become
derepressed in a poor nitrogen source such as glutamate. This
phenomenon is called nitrogen catabolite repression
(NCR)1 (reviewed in Ref. 1).
Genetic analysis in budding yeast led to the identification of
regulatory pathways that detect the quality of nutrients and regulate
gene expression. A number of GATA-type transcription factors have now
been identified, including Gln3p, Nil1p/Gat1p, and Nil2p/Gzf3p (2-4).
In particular, Gln3p has been the primary focus for most subsequent
studies. It is a transcriptional activator of NCR genes when only poor
nitrogen sources are available or during nitrogen starvation. Ure2p is
a yeast pre-prion protein genetically antagonistic to Gln3p (5,
6).
Recent studies have shed light into the regulatory events leading to
activation of Gln3p. TOR is the yeast target of rapamycin (7-12) and a
key player of nutrient-mediated signal transduction (recently reviewed
in Refs. 13-16). Major NCR transcription factors, including Gln3p,
Nil1p/Gat1p, and Nil2p/Gzf3p interact with both Tor1p and Tor2p (17).
Nitrogen starvation or inhibition of TOR by rapamycin causes rapid
dephosphorylation and nuclear accumulation of Gln3p in vivo
(17, 18) and expression of a wide variety of NCR genes (17, 19-21).
TOR is a protein serine/threonine kinase (22-24) and appears to be
responsible for Gln3p phosphorylation (17) and may also regulate Gln3p
dephosphorylation (17, 18). In addition to the classic NCR
transcription factors, TOR also mediates nitrogen signaling to regulate
Rtg1/3p, transcription factors involved in regulation of several genes
of the trichloroacetic acid cycle (25). Although hyperphosphorylation
of Gln3p correlates well with its cytoplasmic retention and
dephosphorylation is consistent with its nuclear accumulation, it
remains to be determined whether and how phosphorylation directly
influences the nuclear accumulation of Gln3p.
Recent progress has defined the basic mechanism of nuclear transport
(26-28). Nuclear import and export factors interact with distinct
targeting signals and share common functional and structural domains.
These are collectively called karyopherins (also importins/exportins, transportins). Analysis of the Saccharomyces data base
revealed a total of 14 proteins belonging to the karyopherin
family, defined partly by a ~150-amino acid region required for
binding to the small GTPase Ran (29, 30). Some of these karyopherin family members have been shown to serve as receptors for the import and/or export of diverse cargo molecules such as proteins and tRNA
(reviewed in Ref. 31). Each karyopherin is likely specific for a
distinct targeting signal. The classical nuclear targeting signal is
typified by the SV40 large T antigen nuclear localization signal (NLS)
that is rich in basic amino acids (32, 33). The import factor
recognizing this NLS is a heterodimer of karyopherin
and
karyopherin
(also termed importins) (extensively reviewed in Refs.
26, 31, and 34). In the cytoplasm, karyopherin
serves as an adaptor
to directly bind the NLS, whereas karyopherin
binds to karyopherin
. The complex then docks at the nuclear pore, translocates across
the pore, and is disassembled in the nucleus. In contrast to the NLS, a
major nuclear export signal is characterized by a small leucine-rich
sequence that is bound by the export factor Crm1/Xpo1 (also called
exportin) and Ran-GTP in the nucleus (35-40). This complex
translocates across the nuclear pore and is disassembled in the
cytoplasm. The small GTPase Ran is thought to impose directionality to
transport processes because its regulators are specifically
compartmentalized within the cell (reviewed in Refs. 26 and 34).
Ran-GTP concentration is presumably high in the nucleus, whereas
Ran-GDP predominates in the cytoplasm. Ran-GTP binds to the karyopherin
family members to facilitate release of import factors from their
cargo in the nucleus (41-43) or to mediate the formation of export
complexes consisting of the export receptor, cargo, and Ran-GTP
(recently reviewed in Ref. 31).
Cells can respond to extracellular signals by modulating gene
expression, which requires the transfer of information from the plasma
membrane to the nucleus. Protein kinase cascades are commonly involved
in transducing these signals, and they typically culminate in the
phosphorylation of transcription factors. Phosphorylation of both
transcription factors and kinases results in regulation of their
nuclear localization, suggesting that control of the subcellular
localization of these proteins is important for the response to
extracellular signals (reviewed in Refs. 44 and 45). Phosphorylation
can directly regulate the recognition of targeting signals by soluble
karyopherins and effectively modulates nucleocytoplasmic translocation
(reviewed in Refs. 44 and 45). For example, phosphorylation of the
phosphate-regulated transcription factor Pho4p at Ser-152 disrupts its
association with the karyopherin
Pse1p and inhibits its nuclear
import (46, 47).
In this study, we found that at least two nuclear transport factors,
karyopherin
/Srp1p and Crm1p/Xpo1p, are required for the import and
export of Gln3p, respectively. Mutation in Srp1p blocked the nuclear
import of Gln3p and the expression of the Gln3p-dependent
gene GAP1 by rapamycin treatment or nitrogen starvation. In
contrast, shifting to the nonpermissive growth temperature in the
crm1-1ts mutant was sufficient to cause nuclear
accumulation of Gln3p and expression of GAP1 in high quality
nitrogen and in the absence of rapamycin. Furthermore, bacterially
produced karyopherin
/Srp1p binds preferentially to the
hypophosphorylated form of Gln3p. Our results indicate that Gln3p is
dynamically shuttling between the cytoplasm and the nucleus.
TOR-dependent phosphorylation appears to inhibit the
ability of Gln3p to enter into the nucleus by reducing its affinity for
the karyopherin
/Srp1p.
 |
MATERIALS AND METHODS |
Yeast Strains and Plasmids--
All of the yeast strains are
shown in Table I. The deletion and
temperature-sensitive mutants for karyopherins have been described previously (40, 48-54). Yeast media and culture conditions are followed according to standard procedures. Rapamycin is dissolved in methanol and stored at
20 °C. The concentration of rapamycin used throughout this study is 200 nM.
For the plasmid expressing GST-Srp1 (pSW347), oligonucleotides were
used in the polymerase chain reaction to generate the SRP1 open reading
frame flanked by BamHI restriction sites. The fragment was
inserted into BamHI-digested pGEX-3x, such that the coding
sequence for GST was in frame with the second codon of SRP1. To
generate pRS315-GLN3-MYC9 and
pRS416-GLN3-MYC9, the chromosomal gene GLN3-MYC9 and its natural promoter were
generated by polymerase chain reaction from genomic DNA prepared from
the GLN3-MYC9 strain (17) and cloned into pRS315
(LEU CEN) or pRS416 (URA CEN) using the
SalI and NotI restriction sites. The resultant
plasmids were transformed into yeast and shown to express
Gln3p-MYC9 at a level comparable with that of the
chromosomal GLN3-MYC9.
pRS315-GLN3-MYC9 and
pRS416-GLN3-MYC9 were transformed
into wild type and mutant strains and used for both phosphorylation and
indirect immunofluorescence (IF) studies.
Northern Blotting Analysis--
Exponential wild type and mutant
yeast cultures were treated with 200 nM rapamycin. Aliquots
of yeast cultures were withdrawn at different times. Total yeast RNAs
were prepared using the phenol freezing extraction method (55). 20-µg
total yeast RNA samples were separated on denaturing agarose gels,
transferred onto nylon filters, hybridized to 32P-labeled
DNA probes, and detected by phosphorus imaging. For the temperature-
and cold-sensitive mutants, yeast were first grown to the early log
phase, shifted to nonpermissive temperatures for 2-3 h, and then
treated with 200 nM rapamycin.
Western Blotting Analysis, Immunoprecipitation, and Phosphatase
Treatment--
Log phase yeast cells were harvested and lysed with
glass beads in disruption buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 plus a
mixture of protease inhibitors; Roche Molecular Biochemicals) by
vortexing. 1 mg of total protein was used for immunoprecipitation with
5 µg of monoclonal antibody (mAb) 9E10 for MYC. For Western blotting
analysis, 20-µg protein samples were used for gel electrophoresis and
detected by ECL (Amersham Pharmacia Biotech) with mAb 9E10. For the
phosphatase treatment, cell extracts containing Gln3p-MYC9
were incubated with CIP buffer alone, 20 units of CIP (Roche Molecular
Biochemicals), or 20 units CIP plus phosphatase inhibitor 10 mM Na4P2O7 for 10 min
at 30 °C.
Immunofluorescence Microscopy--
The wild type and mutant
yeast strains expressing the Gln3p-MYC9 fusions were used
for indirect IF studies. For the cold- and temperature-sensitive
mutants, the yeast cells were grown to mid-log phase at their
permissive temperatures (30 °C for srp1cs;
23 °C for all other mutants) and then shifted to the nonpermissive temperatures for 2-3 h (20 °C for srp1cs,
35 °C for crm1-1ts, and 37 °C for all
other temperature-sensitive mutants). 200 nM rapamycin was
then added for the duration indicated in each experiment. The IF
studies were carried out as described previously (17) using anti-MYC
monoclonal antibody 9E10, and incubation was continued for an
additional time as indicated for each experiment.
GST Fusion Protein and in Vitro Binding Assays--
Bacteria
were grown at 37 °C in 250 ml of liquid broth medium containing 100 µg/ml ampicillin to an optical density of 0.4 and were induced with 1 mM isopropyl-1-thio-
-D-galactopyranoside for
3 h. The culture was chilled on ice for 10 min, and the cells were
pelleted and resuspended in 15 ml of lysis buffer (1×
phosphate-buffered saline, 1 mM phenylmethylsulfonyl
fluoride, 1 mM EDTA, 1× protease inhibitor mixture, and 5 mM
-mercaptoethanol). The suspension was subjected to
two freeze-thaw cycles, and the cells were finally disrupted by
sonication. The cell lysate was clarified by centrifugation for 20 min
at 20,000 × g at 4 °C. The recombinant proteins
were purified by glutathione-Sepharose. The in vitro binding
assays for the recombinant proteins were carried out as described
previously (17).
 |
RESULTS |
Rapamycin treatment or nitrogen starvation leads to nuclear
accumulation of Gln3p and activation of Gln3p-dependent
genes such as GAP1 (general amino
acid permease 1) (17, 18). We reasoned that mutations in the Gln3p import factor would prevent Gln3p
from accumulating in the nucleus in the presence of rapamycin. Under
such conditions, there would be no expression of GAP1. In contrast, dysfunction of the export pathway would cause constitutive nuclear accumulation of Gln3p and a high level of GAP1
expression in the absence of rapamycin. Therefore, GAP1 can
be conveniently used as a reporter to monitor the import/export of
Gln3p by examining the effect of their mutations on GAP1
expression (Fig. 1a). There are a total of 14 karyopherin
s and one karyopherin
in the Saccharomyces cerevisiae genome as revealed by genomic
sequence analysis (30, 56). Therefore, we devised a functional genomics approach to identify the factors that affect Gln3p nuclear transport by
monitoring the expression of GAP1 in individual karyopherin mutants in the presence or the absence of rapamycin.

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|
Fig. 1.
Mutations in SRP1 and
CRM1 yeast karyopherin genes alter GAP1
expression. a, the functional genomics strategy
to identify Gln3p nuclear transport factors. b, exponential
wild type and yeast karyopherin mutant cells were treated with 200 nM rapamycin for 30 min. Total RNA was prepared and
analyzed by Northern blotting with 32P-labeled
GAP1 or ACT1 probes. For the temperature- and
cold-sensitive strains, log phase yeast cultures were shifted to
nonpermissive temperature for 2 h before treatment with 200 nM rapamycin for 30 min. Arrow,
srp1cs and cse1-1cs
mutations completely abolished GAP1 expression when cells
were treated with rapamycin. Double arrow, GAP1
expression remained at a high level in crm1-1ts
even in the absence of rapamycin.
|
|
We have screened all the yeast karyopherin mutants using Northern blot
analysis for GAP1. As expected, GAP1 expression
patterns in a majority of the karyopherin mutants were similar to the
wild type strain; GAP1 expression was essentially
undetectable in the absence of rapamycin but increased significantly in
the presence of rapamycin (Fig. 1b). However,
GAP1 expression remained very low or undetectable both in
the presence and in the absence of rapamycin in two mutant strains,
srp1cs and cse1-1cs (Fig.
1b). Both srp1cs and
cse1-1cs are cold-sensitive mutants. The
induction of GAP1 by rapamycin was normal at the permissive
temperature (30 °C) (data not shown) but not at the nonpermissive
temperature (20 °C) (Fig. 1b). Cse1p is a karyopherin
required for the export of Srp1p from the nucleus to the cytoplasm (57,
58). The cse1-1cs mutation may affect
GAP1 expression indirectly by preventing the return of Srp1p
to the cytoplasm. Kap95p is a co-factor for karyopherin-
/Srp1p.
Therefore, a loss-of-function mutation in KAP95 was also
expected to inhibit GAP1 expression in the presence of
rapamycin. Unfortunately, the two kap95 mutants we analyzed grew extremely poorly, which precluded us from obtaining good quality
RNA from these strains to address this point. Taken together, the
srp1 and cse1 phenotypes suggest that Srp1p
mediates Gln3p import. In contrast, crm1-1ts, a
temperature-sensitive mutation of CRM1, a known exporting factor, led to a high level of GAP1 expression even in the
absence of rapamycin in high quality nitrogen at the restrictive
temperature (35 °C) (Fig. 1b) but not at the permissive
temperature (25 °C) (data not shown). Thus, Crm1p appears to be the
karyopherin responsible for Gln3p nuclear export.
The small GTPase Ran/Gsp1p plays an essential role in nucleocytoplasmic
transport (56, 59). The RanGTP gradient is maintained by two proteins:
RanGAP/Rna1p, which is located in the cytoplasm (60-64), and
RanGEF/Prp20p, which is exclusively found in the nucleus (65).
Disruption of this gradient results in failure of nucleocytoplasmic transport. To determine whether Gln3p transport involves the RanGTP gradient, we investigated the effect of a temperature-sensitive mutation in RanGAP (rna1-1ts) on the expression
of GAP1 and the localization of Gln3p in the absence and the
presence of rapamycin. We found that this mutation completely abolished
the expression of GAP1 (Fig.
4a) and the nuclear import of
Gln3p in the presence of rapamycin at the restrictive temperature
(37 °C) (Fig. 4b). Therefore, the RanGTP gradient is
critical for Gln3p import. This is consistent with the requirement for
Srp1, an adaptor that binds to a Ran-GTP binding karyopherin-
(Kap95).
To directly demonstrate that phosphorylation regulates the interaction
between Gln3p and Srp1p, we treated the cell extracts with CIP. CIP
resulted in dephosphorylation of Gln3p as indicated by the increase of
its gel mobility (Fig. 6a,
lane 2, and Ref. 17). This dephosphorylation was completely
blocked by Na4P2O7, a potent
phosphatase inhibitor. We found that GST-Srp1p indeed preferentially
bound to the dephosphorylated Gln3p (Fig. 6a, lanes 4-6). The small amounts of hypophosphorylated and
dephosphorylated Gln3p in the CIP-untreated or
CIP/Na4P2O7 sample were barely
detectable (because of the short exposure of the Western blot). The
same amount of GST-Srp1p was used for each different sample as
indicated by Coomassie Blue staining (Fig. 6b). Taken
together, these results show that Gln3p binds to karyopherin
/Srp1p
and that phosphorylation of Gln3p inhibits this interaction.
Cellular responses to nitrogen availability and the quality of
nitrogen nutrients require regulated nuclear transport of the GATA-type
transcription factor Gln3p. Gln3p is phosphorylated in a
TOR-dependent manner and localizes predominantly to the
cytoplasm when yeast are grown in high quality nitrogen-containing
medium. In contrast, Gln3 becomes dephosphorylated and accumulates in the nucleus when yeast are grown in poor nitrogen, grown under nitrogen
starvation conditions, or treated with rapamycin (17, 18). In this
study, we sought to understand how phosphorylation of Gln3p regulates
its nucleocytoplasmic transport and the nuclear transport machinery
that moves Gln3p into and out of the nucleus. We found that the
srp1cs mutation specifically blocked the nuclear
import of Gln3p at nonpermissive temperatures in the presence of
rapamycin. In addition, Gln3p binds specifically to bacterially
expressed GST-Srp1p in vitro. Taken together, these results
indicate that Srp1p is the nuclear import factor for Gln3p.
Phosphorylation is broadly involved in the regulated transport of many
important proteins between the cytoplasm and nucleus (reviewed in Refs.
44 and 45). Phosphorylation within or adjacent to the NLS sequence can
dramatically affect recognition of cargo proteins by the karyopherins.
One such example is Pho4p, a phosphate-regulated transcription factor.
Pho4p is phosphorylated and localized in the cytoplasm when yeast is
grown in phosphate-rich medium, whereas in low phosphate medium, Pho4p
is dephosphorylated and localized to the nucleus. Pho4p nuclear
translocation requires the karyopherin Pse1. Phosphorylation decreases
the affinity of Pho4p for Pse1p and results in the cytoplasmic
retention of Pho4p (46, 47). A similar scenario may also be true for
Gln3p and possibly other NCR-sensitive transcription activators and
repressors (a model is shown in Fig. 7).
During nitrogen starvation or rapamycin treatment, TOR-dependent phosphorylation decreases, which is possibly
accompanied by an increase in the phosphatase activity toward Gln3p
(17, 18). As a result, the dephosphorylated form of Gln3p predominates and is imported into the nucleus as a result of its interaction with
Srp1p. Our results provide further support for a common mechanism by
which nutrient-sensing transcription factors are controlled by
phosphorylation-dependent interaction with the nuclear
transport machinery.
We are grateful to A. Hopper, A. Tartakoff,
K. Weis, L. Davis, M. Fitzgerald-Hayes, M. Johnston, and P. Silver for
strains, K. Mishra for construction of pSW347, and D. Dean for use of
the fluorescence microscopes. We also thank other members of the Zheng laboratory for insightful discussions.
The abbreviations used are:
NCR, nitrogen
catabolite repression;
NLS, nuclear localization signal;
GST, glutathione S-transferase;
IF, immunofluorescence;
mAb, monoclonal antibody;
CIP, calf intestine alkaline phosphatase;
HIV1, human immunodeficiency virus, type 1;
DAPI, 4',6-diamidino-2-phenylindole, dialactate.
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