Domains of Gln3p Interacting with Karyopherins, Ure2p, and the
Target of Rapamycin Protein*
John
Carvalho and
X. F. Steven
Zheng
From the Department of Pathology and Immunology, Washington
University School of Medicine, St. Louis, Missouri 63110
Received for publication, January 15, 2003, and in revised form, February 17, 2003
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ABSTRACT |
Gln3p is a GATA-type transcription factor
responsive to the quality of nitrogen and carbon. In preferred nitrogen
such as glutamine, Gln3p is phosphorylated and sequestered in the
cytoplasm in a manner that is dependent on the target of rapamycin
(TOR) protein and Ure2p. In nonpreferred nitrogen or nitrogen
starvation, Gln3p is dephosphorylated and imported into the nucleus via
karyopherin
/Srp1p. Upon reintroduction of preferred nitrogen, Gln3p
is exported from the nucleus by Crm1p/Xpo1p. Although recent work has
provided insights into Gln3p, a more detailed understanding is needed
to elucidate the mechanism of its localization and function. In this study, we show that Gln3p contains canonical nuclear localization signal and nuclear export signal sequences necessary for its
localization and interaction with its relevant karyopherins. In
addition, we identify an N-terminal domain of Gln3p interacting with
Ure2p and a C-terminal region for binding to TOR. Finally, we find a lysine/arginine-rich domain essential for the rapamycin-sensitive function, but dispensable for its localization. Our results reveal key
domains of Gln3p important for its function and regulation.
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INTRODUCTION |
Carbon and nitrogen are two basic nutrient sources used by
eukaryotic cells to produce energy and synthesize a wide range of
biomolecules important for cell growth and proliferation. In response
to the quality of carbon and nitrogen, cells can regulate the
expression of genes involved in different metabolic pathways. A classic
example is nitrogen catabolite repression
(NCR)1 in the yeast
Saccharomyces cerevisiae, where cells regulate expression of
genes involved in utilization and transport of available nitrogen nutrients (1, 2). A number of NCR transcription factors have been
identified, including the GATA-type transcription activators Gln3p and
Gat1p/Nil1p, and the repressors Dal80p and Nil2p/Gzf3p (3-5). Gln3p
has been the primary focus for previous studies, partly due to interest
in the prion precursor Ure2p, which interacts with Gln3p and sequesters
it in the cytoplasm when cells are grown in preferred nitrogen (6, 7).
In addition, it was recently shown that the target of rapamycin (TOR)
protein-nitrogen (8-11) and Snf1-glucose signaling pathways regulate
Gln3p (12).
Rapamycin is an antibiotic currently undergoing advanced clinical
trials for cancer treatment (13). The target of rapamycin protein is a
serine/threonine protein kinase highly conserved in eukaryotes. Tor1p
and Tor2p are the target of rapamycin proteins in yeast. Like their
paralogues in animals, they are key players of nutrient-mediated signal
transduction to control cell growth and proliferation (14-17).
Although Tor1p and Tor2p redundantly regulate growth in a
rapamycin-sensitive manner, Tor2p additionally regulates polarization
of the actin cytoskeleton in a rapamycin-insensitive manner (18-20).
The major NCR transcription factors, including Gln3p, interact with
Tor1p and Tor2p (8, 9). Nitrogen starvation or inhibition of TOR by
rapamycin causes rapid dephosphorylation and nuclear accumulation of
Gln3p in vivo as well as expression of a wide range of NCR
genes (8, 10, 21, 22). In addition, TOR appears to be responsible for
Gln3p phosphorylation and may also regulate Gln3p dephosphorylation (8,
9). Deletion of GLN3 confers rapamycin resistance,
indicating that Gln3p plays an important role in rapamycin-sensitive
TOR signaling (8, 23). Apart from the GATA factors, TOR was shown to
mediate nitrogen signaling to Rtg1/3p, transcription factors involved
in regulation of several genes of the tricarboxylic acid cycle (24), as
well as Msn2/4p, two transcription factors involved in carbon signaling (9). Recent evidence suggests that a conserved TOR-interacting protein
named Kog1p is also involved in TOR signaling to Gln3p (25, 26).
Recent studies have provided insights into the mechanism of
TOR-regulated Gln3p localization. Two nuclear transport factors in
yeast, karyopherin
/Srp1p and Crm1p/Xpo1p, are involved in the
nuclear import and export of Gln3p, respectively (11). Mutation in
Srp1p blocks the nuclear import of Gln3p and the expression of
Gln3p-dependent genes by rapamycin treatment or nitrogen
starvation (11). In contrast, mutation in Crm1p results in nuclear
accumulation of Gln3p and up-regulation of Gln3p-dependent
genes (11). The transport of Gln3p also involves the Ran GTPase pathway
as a mutation in the yeast Ran GTPase activating protein, Rna1p,
causes Gln3p to remain in the cytoplasm in the presence of rapamycin
(11). Srp1p preferentially interacts with the hypophosphorylated Gln3p (11), suggesting that phosphorylation controls Gln3p nuclear import by
regulating the interaction between Gln3p and Srp1p.
Although previous research has revealed key insights into Gln3p, a
detailed understanding of the mechanism by which TOR and Ure2p regulate
Gln3p nuclear translocation is lacking. In this study, we performed
structure-function analysis on Gln3p. Here we report that Gln3p
contains classic nuclear localization signal (NLS) and nuclear export
signal (NES) sequences that are necessary for its interaction with
Srp1p and Crm1p, respectively, and TOR-regulated nuclear transport. We
also identified an N-terminal region of amino acids critical for Gln3p
interaction with its inhibitor Ure2p and a C-terminal Tor1p-binding
domain. Finally, we localized a lysine/arginine-rich motif crucial for
a rapamycin-sensitive function that is unrelated to Gln3p localization.
These results provide a detailed understanding of Gln3p structure and function.
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MATERIALS AND METHODS |
Plasmids, Strains, and Yeast Two-hybrid Interaction
Assay--
Yeast media and culture conditions are followed according
to standard procedures (27). The
pRS315-GLN3-MYC9 plasmid was published
previously (11). Systematic N-terminal and internal deletions of
GLN3 were generated by PCR using the Vent thermally stable
DNA polymerase (New England BioLabs) with the
pRS315-GLN3-MYC9 plasmid as a template. All
constructs were sequenced and showed proper expression.
Gal4p DNA-binding domain (BD)-Tor1p fusion and Gal4p activation domain
(AD)-Ure2p fusion were used in two-hybrid assays previously (8).
Fragments of GLN3 were cloned into either pAS2-1 or pACT2 (Clontech). To generate the yeast two-hybrid
plasmid constructs for SRP1, CRM1, and
CSE1, the open reading frame for each gene was amplified
from genomic DNA by PCR and cloned into the pACT2 plasmid. The
combination of Gal4p BD and AD fusion proteins were expressed in the
yeast strain PJ69-4a. Their interaction was tested by the ability of
the yeast strain PJ69-4a to grow on synthetic complete (SC)
Leu
Trp
Ade
and SC
Leu
Trp
His
plus 3-AT (3 mM) plates.
Cell Lysis and Western Blotting Analysis--
Mid-log phase
yeast cells (A600 = 0.4) were harvested
and lysed with glass beads by vortexing in disruption buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 50 mM
-glycerophosphate, 10 mM NaF, 100 nM
microcystin LR, 1 mM phenylmethylsulfonyl fluoride, plus a
protease inhibitor mixture from Roche Applied Science). Crude extracts
were cleared by centrifugation twice at 20,800 × g for
20 min. For Western blotting analysis, protein samples (20 µg) were
separated by SDS-polyacrylamide gel electrophoresis and transferred
onto an Immobilon-P membrane. Myc-tagged proteins were recognized by
mAb 9E10 and detected by ECL enhanced chemiluminescence system
(Amersham Biosciences).
GST Fusion Proteins and in Vitro Binding Assay--
Purification
of GST-Ure2p and the GST-pull-down assays were performed as described
previously (8).
Immunofluorescence--
The gln3
strains
expressing the wild type or mutant Gln3p-MYC9 proteins were
grown to early log phase (A600 = 0.2) at
30 °C in synthetic complete (SC) media and then treated with and without rapamycin (200 nM) for 10 min. The cells were
fixed, stained with monoclonal antibody (mAb) 9E10 and Texas
Red-conjugated rabbit anti-mouse secondary antibody, and analyzed by
indirect immunofluorescence (IF) microscopy as described before
(8).
The Rapamycin Sensitivity Assay--
Cultures of the
gln3
strain expressing wild type or mutant
Gln3p-MYC9 proteins in SC media were normalized to the same
density, serially diluted (10-fold), and spotted onto SC plates
containing 10 nM rapamycin or no rapamycin, and incubated
for 2 days (no rapamycin) or 4 days (10 nM rapamycin).
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RESULTS |
To define sequences that are important for Gln3p localization and
function, we performed systematic deletion analysis of
Gln3p-MYC9 (Fig.
1A).
GLN3-MYC9 under the GLN3 promoter on
a single copy plasmid is expressed at the physiological level and can
complement the gln3
mutation in all possible tests (11).
Wild type and mutant GLN3-MYC9 were expressed in
the gln3
strain. We have previously found that the
gln3
mutant is significantly resistant to rapamycin (Fig.
1B) (8, 12, 23). Expression of
GLN3-MYC9 restored rapamycin sensitivity to the
wild type strain level (Fig. 1A). Therefore, we decided to
use the rapamycin sensitivity phenotype to evaluate the function of
different Gln3p mutants in this study. We found that cells expressing a
Gln3p-MYC9 mutant without the first 101 amino acids were
still sensitive to rapamycin (Fig. 1, A and B).
However, cells expressing Gln3p mutants with a deletion of 200 amino
acids or more from the N terminus were as highly rapamycin-resistant as
the gln3
strain (Fig. 1, A and B).
A common feature of these rapamycin-resistant mutants is that the Gln3p transcriptional activation domain (aa 126-140) is deleted, suggesting that transcriptional activation is critical for the rapamycin-sensitive function of Gln3p. We also tested several Gln3p-MYC9
mutants with internal deletions. As expected, deletion of amino acids
102-498 that encompass the transcriptional activation domain causes
rapamycin resistance (Fig. 1, A and B). To our
surprise, cells carrying Gln3p-MYC9 mutants with internal
deletions from amino acids 330-600 and 388-600, which did not affect
the transcriptional activation domain, were also highly resistant to
rapamycin (Fig. 1, A and B). This observation
suggests that there may be one or more other domains critical for the
rapamycin-sensitive function within these regions.

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Fig. 1.
Systematic deletional analysis of Gln3p.
A, summary of the systematic deletion analysis of Gln3p.
Systematic N-terminal and internal deletion mutants of
Gln3p-MYC9 used in this experiment are shown on the
left. GLN3-MYC9 is under the control
of GLN3 promoter on the CEN plasmid vector
pRS315. Shown in the middle is the localization of different
Gln3p-MYC9 proteins in cells without rapamycin or treated
with rapamycin (200 nM, 10 min). Shown on the
right is the rapamycin sensitivity of the gln3
strain expressing different Gln3p-MYC9 proteins.
AD, the transcriptional activation domain; GATA,
the C-4 zinc finger DNA-binding domain; C, cytoplasmic;
N, nuclear; S, sensitive; R,
resistant; Rap, rapamycin. B, rapamycin
sensitivity of the N-terminal and internal deletion mutants of
Gln3p-MYC9. Cultures of yeast expressing the wild type
(WT) and mutant Gln3p-MYC9 proteins were
serially diluted (10-fold) and spotted onto SC medium minus leucine in
the absence or presence of 10 nM rapamycin. Plates were
incubated at 30 °C for 2 ( Rapamycin) and 4 (+Rapamycin) days, respectively. C, the effect of
different deletions on Gln3p localization. Yeast cells expressing
different Gln3p-MYC9 proteins were treated without or with
200 nM rapamycin ( /+Rap) (10 min). The
localization of Gln3p-MYC9 was examined by indirect
immunofluorescence (IF) staining with monoclonal antibody (mAb) 9E10.
Yeast nuclei were stained with DAPI.
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To further investigate the nature of the rapamycin-sensitive
phenotypes, we performed localization studies of the
Gln3p-MYC9 mutants by indirect immunofluorescence (IF)
microscopy in the absence or presence of rapamycin. The results of
these studies are summarized in Fig. 1A. In agreement with
previous findings, Gln3p-MYC9 was localized to the
cytoplasm in the absence of rapamycin and accumulated in the nucleus
within 10 min of rapamycin treatment (Fig. 1, A and
C) (8, 11). In contrast, all the mutants with N-terminal
deletion up to amino acid 387 were nuclear in the absence or presence
of rapamycin treatment (Fig. 1, A and C). Further deletion up to amino acid 466 caused Gln3p to remain in the cytoplasm even in the presence of rapamycin (Fig. 1, A and
C). In addition, internal deletions of amino acids 102-498,
330-600, and 388-600 also resulted in constitutive cytoplasmic
localization of Gln3p (Fig. 1, A and C). These IF
results reveal two distinct domains important for Gln3p localization.
The constitutive nuclear localization mutants lacking up to 387 amino acids suggest that there are one or more domains within amino
acids 1-387, such as the NES, which are inhibitory to Gln3p nuclear
localization. On the other hand, the constitutive cytoplasmic mutants
indicate that there is a sequence crucial for nuclear localization
between amino acids 388 and 466, possibly an NLS (Fig. 1, A
and C). The lack of nuclear localization of these mutants in
the presence of rapamycin is consistent with the idea that nuclear
localization and transcriptional activation are necessary for the
rapamycin-sensitive function of Gln3p (Fig. 1, A and
C).
We further sought to identify the Gln3p NLS and NES sequences. Upon
scanning the amino acid sequence of Gln3p by the PSORT sequence
analysis program, we found one motif
(336LHGTMRPLSL345)
characteristic of the canonical leucine-rich nuclear export signal
(Fig. 2A) (28, 29). The fact
that Gln3p nuclear export is dependent on the classic nuclear export
pathway involving Crm1p (11) suggests that this motif is a likely NES.
To confirm this, we deleted this motif and tested for its effect on
Gln3p localization. As would be expected for a mutation in the NES,
this mutant localized to the nucleus even in the absence of rapamycin
(Fig. 2B). Given that Crm1p can interact with the classic
leucine-rich NES sequences (28, 29), we investigated the interaction
between Crm1p and Gln3p in the presence or absence of the putative NES
sequence. By the yeast two-hybrid interaction assay, we found that the
Gal4p activation domain (AD)-Crm1p fusion protein interacted with the Gal4p DNA-binding domain (BD)-Gln3p fusion protein (Fig.
2C). In contrast, Gal4p AD-Crm1p failed to interact with the
Gal4p AD-Gln3p fusion protein missing the putative NES (Fig.
2D). These data suggest that the leucine-rich motif is most
likely the NES sequence for Gln3p.

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Fig. 2.
Gln3p contains a classic nuclear export
signal (NES) sequence at amino acids 336-345 essential for Gln3p
export and interaction with the export factor Crm1p. A,
sequence comparison between the NES consensus and the leucine-rich
motif in Gln3p. Underlined amino acids are consensus
residues defining the classic NES signature. B, the putative
NES is essential for Gln3p nuclear export. Yeast cells expressing
either wild type Gln3p-MYC9 protein or mutant
Gln3p-MYC9 protein with an internal deletion of amino acids
336-345 were treated without or with rapamycin (200 nM, 10 min) ( /+Rap). The localization of Gln3p-MYC9
was examined by IF with mAb 9E10. Yeast nuclei were stained with DAPI.
C, Gln3p interacts with Crm1p in the yeast two-hybrid assay.
Gal4p AD-Crm1p specifically interacts with Gal4p DNA
BD-Gln3p-(141-731) but not with Gal4p BD alone. The yeast two-hybrid
reporter strain (PJ69-4a) expressing different BD and AD fusion
proteins was assayed for growth on an adenine dropout plate or a
histidine dropout plate containing 3-AT (3 mM). The
interaction between SV40 large T antigen (LgT) and p53 was
used as a positive control. D, the putative Gln3p NES is necessary for
Gln3p interaction with Crm1p. Gal4p AD-Crm1p fails to interact with
Gal4p BD-Gln3p-(141-731) without the NES. The yeast two-hybrid
reporter strain (PJ69-4a) expressing different BD and AD fusion
proteins was assayed for growth on an adenine dropout plate or a
histidine dropout plate containing 3-AT (3 mM).
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We have also used the PSORT sequence analysis program to identify the
NLS sequence(s) and found two motifs
(388PIRSRKK394, designated as NLS1) and
(571PRRKMSR577, designated as NLS2) that obeyed
the classic NLS consensus (four consecutive K/R residues, or a P
followed by three consecutive K/R within three amino acids) (Fig.
3A) (30). No bipartite NLS motifs were found. Deletion of NLS1 alone was sufficient to inhibit Gln3p nuclear localization in the presence of rapamycin (Fig. 3,
B and C). Deletion of NLS1 in combination with
other sequences as shown in Figs. 1A and 3B also
resulted in constitutive cytoplasmic localization (Figs. 1A,
3B, and 3C). In contrast, deletion of NLS2 has no
effect on Gln3p localization in the absence or presence of rapamycin
(Fig. 3, B and C). In corroboration with the IF
analysis, the NLS1-deletion mutants were rapamycin-resistant, whereas
the deletions only encompassing NLS2 were still sensitive like wild type (Fig. 3, B and D). There is also a K/R-rich
region at amino acids 351-361 (KKRISKKRAK) that has been suggested as
an NLS for Gln3p (31). Despite its K/R-rich sequence, it does not
follow the NLS consensus sequences (Fig. 3A). Moreover,
deletion of this sequence had no effect on Gln3p localization in the
absence and presence of rapamycin, indicating that it is dispensable
for the TOR-regulated Gln3p localization (Fig. 3, B and
C). Nonetheless, deletion of the K/R-rich region of Gln3p
was rapamycin-resistant (Fig. 3, B and D).
Therefore, it is still important for the rapamycin-sensitive function.

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Fig. 3.
Gln3p contains a classic nuclear localization
signal (NLS) sequence at amino acids 388-394 essential for Gln3p
nuclear localization. A, sequence comparison between
the NLS consensus and the K/R-rich motifs in Gln3p reveal two putative
NLS signals (NLS1 and NLS2). Boxed
amino acids are the consensus residues of classic NLS motifs.
B, Gln3p contains a classic NLS (NLS1) at amino acids 388-394 necessary for its nuclear import and ability to
complement a gln3 strain. Shown on the left
are the deletion mutants and a tabulation of their localization pattern
after cells were treated without or with rapamycin (200 nM,
10 min) and their sensitivity to rapamycin in serial dilution assays.
The boxes indicate different domains of Gln3p:
AD, the transcriptional activation domain; GATA,
the C-4 zinc finger DNA-binding domain; the K/R-rich motif at amino
acids 351-361; NLS1, the first putative NLS motif at amino
acids 388-394; NLS2, the second putative NLS at amino acids
571-577; C, cytoplasmic; N, nuclear;
S, sensitive; R, resistant. C, NLS1,
but not NLS2 or the K/R-rich motif, is essential for Gln3p nuclear
accumulation. Yeast cells expressing different Gln3p-MYC9
proteins were treated without or with rapamycin ( /+Rap)
(200 nM, 10 min). The localization of
Gln3p-MYC9 was examined by IF with mAb 9E10. Yeast nuclei
were stained with DAPI. D, deletion of NLS1 results in a
rapamycin-resistant phenotype. Cultures of yeast expressing different
Gln3p-MYC9 proteins were serially diluted (10-fold) and
spotted onto SC medium minus leucine in the absence or presence of
rapamycin (10 nM). Plates were incubated at 30 °C for 2 ( Rapamycin) and 4 (+Rapamycin) days,
respectively.
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Our earlier study indicates that karyopherin
/Srp1p interacts with
Gln3p and mediates Gln3p nuclear import (11). To further study the
relevance of NLS1, we used the yeast two-hybrid system to determine the
interaction between Srp1p and Gln3p in the presence or absence of NLS1.
We found that Gal4p AD-Srp1p interacted with Gal4p BD-Gln3p but not the
Gal4p BD-Gln3p fusion protein lacking NLS1 (Fig.
4A). In addition to the
srp1 mutation, a temperature-sensitive mutation in
CSE1, which encodes for the export receptor of Srp1p, also
inhibited Gln3p nuclear import in the presence of rapamycin (11).
Although the cse1 mutation is most likely to influence Gln3p
nuclear localization indirectly by blocking Srp1p export, it is
possible that Cse1p acts as a Gln3p importing factor. However, Gal4p
AD-Cse1p did not interact with Gal4p BD-Gln3p (Fig. 4B), providing further support to the model that Cse1p is the Srp1p exporter
as opposed to an import karyopherin for Gln3p.

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Fig. 4.
NLS1 is essential for interaction with import
factor Srp1p. A, NLS1 is essential for Gln3p
interaction with Srp1p. Gal4p AD-Srp1p specifically interacts with
Gal4p BD-Gln3p-(141-731) but not with Gal4p BD-Gln3p-(141-731)
without NLS1. The yeast two-hybrid reporter strain (PJ69-4a) expressing
different BD and AD fusion proteins was assayed for growth on an
adenine dropout plate or a histidine dropout plate containing 3-AT (3 mM). B, Gln3p does not interact with Cse1p, the
nuclear export receptor for Srp1p. Gal4p AD-Cse1p does not interact
with Gal4p BD-Gln3p-(141-731) or with Gal4p BD-Gln3p-(141-731)
without NLS1. The yeast two-hybrid reporter strain (PJ69-4a) expressing
different BD and AD fusion proteins was assayed for growth on an
adenine dropout plate or a histidine dropout plate containing 3-AT (3 mM).
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As shown earlier, deletion of N-terminal amino acids 1-101, 1-200,
and 1-316 resulted in constitutive nuclear localization of Gln3p (Fig.
1, A and C). Because these sequences do not
contain the NES, the results suggest that there is another negative
element for Gln3p nuclear localization within amino acids 1-316. One
possibility is the Ure2p-binding domain (BD). To test this possibility,
we performed protein-protein interaction studies between Gln3p and Ure2p. Yeast cell extracts containing different Gln3p-MYC9
proteins were incubated with bacterially produced GST or GST-Ure2p
bound on glutathione-agarose beads. Gln3p-MYC9 specifically
bound to GST-Ure2p, but not to GST alone, confirming a previous
observation (Fig. 5A) (8). In
addition, GST-Ure2p bound to deletions of Gln3p-MYC9
lacking the NES, the K/R-rich motif, and a protein missing both the NES
and K/R-rich motifs (Fig. 5A), indicating that none of these
regions are necessary for Ure2p binding. In contrast, deletion of amino
acids 1-200 completely abolished the ability of Gln3p to bind to
GST-Ure2p, as did further deletions from the N terminus (Fig.
5A). Deletion of N-terminal amino acids 1-101 resulted in
reduced interaction between GST-Ure2p and Gln3p-MYC9 (Fig.
5A). Therefore, amino acids 102-200 appear to be critical for the Gln3p-Ure2p interaction, possibly directly
involved in binding to Ure2p. The first 101 amino acids may also be
needed for a stable Ure2p-Gln3p interaction, because
Gln3p(
1-101)-MYC9 binds weakly to Ure2p, which could
explain why the 1-101 deletion of Gln3p-MYC9 is localized
to the nucleus even in the absence of rapamycin treatment (Fig. 1,
A and C).

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Fig. 5.
Identification of a Ure2p-binding
domain at the N terminus of Gln3p. A, Gln3p binds Ure2p
in vitro via an N-terminal Ure2p-binding domain. Extracts of
yeast cells expressing different Gln3p-MYC9 proteins were
incubated with either GST-Ure2p or GST bound to glutathione-agarose
beads. GST, GST-Ure2p and bound materials were eluted by
boiling, separated by SDS-PAGE, and detected by Western blotting with
mAb 9E10. Upper panel, GST-Ure2p-bound
Gln3p-MYC9 proteins. Middle panel, total
Gln3p-MYC9 proteins in the cell extracts. *, the
Gln3p-MYC9 mutant proteins at predicted molecular weights.
Certain deletion mutants also show some lower molecular weight forms,
possibly due to proteolysis. Bottom panel, Coomassie Blue
stain of GST and GST-Ure2p used in the binding study. MWM,
molecular weight marker. B, mapping of Gln3p N terminus for
the Ure2p-binding domain. Shown is a summary of regions of Gln3p tested
for interaction with Ure2p in the yeast two-hybrid system.
C, the N-terminal amino acids 1-200 of Gln3p interact with
Ure2p in the yeast two-hybrid assay. Yeast PJ69-4a strain expressing
Gal4p BD-Gln3p-(1-200) and Gal4p AD-Ure2p was assayed for growth on an
adenine dropout plate or a histidine dropout plate containing 3-AT (3 mM). D, the N-terminal amino acids 102-200 of
Gln3p interact with Ure2p in the yeast two-hybrid assay. Yeast PJ69-4a
strain expressing Gal4p BD-Gln3p-(102-200) and Gal4p AD-Ure2p was
assayed for growth on an adenine dropout plate or a histidine dropout
plate containing 3-AT (3 mM). E, the N-terminal
amino acids 102-150 of Gln3p interact with Ure2p. Yeast PJ69-4a strain
expressing Gal4p BD-Gln3p-(102-150) and Gal4p AD-Ure2p was assayed for
growth on an adenine dropout plate or a histidine dropout plate
containing 3-AT (3 mM). F, the N-terminal amino
acids 102-125 and 126-200 of Gln3p do not interact with Ure2p. Yeast
PJ69-4a strain expressing different Gal4p BD-Gln3p fusion proteins and
Gal4p AD-Ure2p was assayed for growth on an adenine dropout plate or a
histidine dropout plate containing 3-AT (3 mM).
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To further characterize the Ure2p-binding domain, we investigated the
interaction between Gln3p and Ure2p using the yeast two-hybrid system.
We constructed a panel of Gal4p BD fusion proteins containing different
regions of Gln3p (Fig. 5B). In this assay, Gal4p AD-Ure2p
specifically interacted with Gal4p BD-Gln3p-(1-200) (Fig. 5,
B and C) but not to Gal4p BD alone (Fig. 5,
C-F). In addition, Gal4p AD-Ure2p interacted with Gal4p
BD-Gln3p-(102-200) (Fig. 5, B and D) and Gal4p
BD-Gln3p-(102-150) (Fig. 5, B and E). By
contrast, it did not interact with Gal4p BD-Gln3p-(102-125) or Gal4p
BD-Gln3p-(126-200) (Fig. 5, B and F). Taken
together, our in vitro and in vivo interaction
data show that the N-terminal region of Gln3p between amino acids
102 and 150 is sufficient for Ure2p binding, although additional
sequence N-terminal to amino acid 102 could be
involved in stabilizing the Ure2p- Gln3p interaction.
Our previous study shows that TOR proteins interact with Gln3p (8). The
yeast two-hybrid library clones that interact with Gal4p BD-Tor1p are
comprised of amino acids 510-731 of Gln3p, suggesting that the C
terminus of Gln3p is responsible for Tor1p binding (8). To further map
the Tor1p-binding domain of Gln3p, we once again used the yeast
two-hybrid system. We constructed a panel of Gal4p AD fusion proteins
containing the C-terminal regions of Gln3p (Fig.
6A). As expected, Gal4p
BD-Tor1p specifically interacted with Gal4p AD-Gln3p and Gal4p
AD-Gln3p-(510-731) (Fig. 6, A and B) but not to
Gal4p AD alone (Fig. 6B). We found that Gal4p BD-Tor1p
interacted with Gal4p AD-Gln3p-(600-731) and Gal4p AD-Gln3p-(600-667)
(Fig. 6, A-C). In contrast, no interaction was observed
between Gal4p BD-Tor1p and Gal4p BD-Gln3p-(667-731) (Fig. 6,
A and C). These data demonstrate that the
C-terminal region of Gln3p between amino acids 600-667 is the Tor1p
BD. Although TOR is clearly a crucial regulator of Gln3p (8, 9, 11), the physiological significance of the TOR-Gln3p interaction is not yet
known. To assess the importance of the TOR-Gln3p interaction in the
regulation of Gln3p, we examined the effect of deletion of the
C-terminal TOR-binding domain on Gln3p localization. We found that
deletion of the TOR-binding domain caused Gln3p to constitutively
localize in the nucleus, which occurred even in rich nutrient
conditions and in the absence of rapamycin (Fig. 6D). This
result suggests that disruption of TOR binding, like rapamycin
inhibition, hinders the ability of TOR to inhibit nuclear accumulation
of Gln3p in rich nutrient conditions.

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Fig. 6.
Gln3p interacts with Tor1p via a C-terminal
TOR-binding domain. A, a summary of the mapping results
of the C terminus of Gln3p for the TOR-binding domain. B,
the C-terminal amino acids 510-731 and 600-731 of Gln3p interact with
Tor1p in the yeast two-hybrid assay. Yeast PJ69-4a strain expressing
Gal4p BD-Tor1p and Gal4p AD-Gln3p fusion proteins was assayed for
growth on an adenine dropout plate or a histidine dropout plate
containing 3-AT (3 mM). C, the C-terminal amino
acids 600-667 of Gln3p are sufficient to interact with Tor1p in the yeast two-hybrid assay. Yeast PJ69-4a strain
expressing Gal4p BD-Tor1p and Gal4p AD-Gln3p fusion proteins was
assayed for growth on an adenine dropout plate or a histidine dropout
plate containing 3-AT (3 mM). D, the TOR-binding
domain (TOR BD) at the C terminus is crucial for maintaining
Gln3p in the cytoplasm under rich nutrient conditions. Yeast cells
expressing the wild type and mutant Gln3p-MYC9 were treated
without or with rapamycin ( /+Rap) (200 nM, 10 min). The localization of Gln3p-MYC9 was examined by IF
with mAb 9E10. Yeast nuclei were stained with DAPI.
|
|
 |
DISCUSSION |
Gln3p is a GATA-type transcription factor originally identified as
a central regulator of nitrogen catabolite-repressible genes (1, 2).
Recent evidence indicates that Gln3p plays a broader role in the
control of gene expression. Gln3p is shown to be important for
autophagy by regulating a number of APG genes such as
APG14 and vacuolar protease genes (21, 32). In addition, Gln3p has been implicated in salt stress response (33). These observations indicate that Gln3p is involved in transcriptional responses to changing nutrient, starvation, and stress conditions. On
the other hand, Gln3p is also an important effector of TOR signaling,
because deletion of GLN3 causes strong
rapamycin resistance (8, 23). Previously, two Gln3p domains involved in
transcriptional regulation of NCR genes were well characterized: a
transcriptional activation domain between amino acids 126 and 140 and a
C-4 zinc finger GATA-DNA-binding domain among residues 306-330
(34-37). In this study, we find that GLN3 mutants with
deletion in the transcriptional activation domain or the C-4 zinc
finger GATA-DNA-binding domain fail to suppress the rapamycin-resistant
phenotype of the gln3
mutation (Fig. 1, A and
B). This observation provides the first evidence that
transcriptional regulation by Gln3p is crucial for its TOR-related
functions. In addition, deletion of the K/R-rich motif does not affect
Gln3p localization (Fig. 3, A and C). However, it
abolishes the ability of Gln3p to suppress the rapamycin-resistant phenotype of the gln3
mutation (Fig. 3, A and
D). Therefore, a likely role of the K/R-rich motif is
involved in gene regulation. Because the K/R-rich motif is in close
proximity to the C-4 zinc finger GATA-DNA-binding domain, it may be
required for Gln3p to recognize and/or bind to the promoter regions of
Gln3p target genes.
The NES domain is a leucine-rich motif at amino acids 336-345 that
obeys the classic NES consensus (Fig. 2A) (28, 29). It is
required for binding to the export factor Crm1p (Fig. 2, C
and D) and is essential for the Crm1p-dependent
nuclear export (Fig. 2B). Hence, this leucine-rich motif is
most likely the Gln3p nuclear export sequence. The NES
mutant is
localized in the nucleus even in the absence of rapamycin or nutrient
starvation (Fig. 2B). Additionally, Gln3p rapidly
accumulates in the nucleus in the crm1-1ts mutant
upon shift to the restrictive temperature (11). In these experiments,
blocking export is sufficient to cause Gln3p to accumulate in the
nucleus, despite the presence of rich nutrients and the absence of
rapamycin. These observations suggest that Gln3p is normally shuttling
between the cytoplasm and nucleus, and the control of Gln3p
localization by nutrients is likely a result of changing the balance
between import and export. This new model is in contrast to the static
models proposed previously (8, 9), which do not satisfactorily explain
the dynamic Gln3p distribution.
In the case of the Gln3p NLS, although there are two structural motifs
that obey the classic NLS consensus, NLS1 (aa 388-394) and NLS2 (aa
571-577), only NLS1 is crucial for Gln3p nuclear localization under
nutrient starvation, or during rapamycin treatment (Fig. 3 and data not
shown). This is also in agreement with the observation that NLS1 is
essential for mediating the interaction between Gln3p and Srp1p (Fig.
4). In addition, a deletion of NLS1, but not NLS2, causes a
rapamycin-resistant phenotype, presumably as a result of blocking Gln3p
from entering into the nucleus (Fig. 3, B and D).
These observations strongly suggest that NLS1 is the Gln3p nuclear
localization sequence. By contrast, the K/R-rich region (aa 351-361)
does not obey the classic NLS consensus and is not required for the
TOR-regulated nuclear localization in the presence of rapamycin (Fig.
3, A and C). It is possible that the K/R-rich
motif and NLS2 are involved in directing Gln3p nuclear localization
under other yet unknown conditions that also regulate Gln3p transport.
Indeed, the K/R-rich motif has been shown to direct a tagged green
fluorescent protein (GFP) into the nucleus (31). However, in the same
study, GFP-tagged full-length Gln3p is found exclusively in the nucleus
even in the apparent rich nutrient conditions. Therefore, GFP-Gln3p
proteins may localize in the nucleus in a K/R-rich
motif-dependent manner unique to the specific experimental condition.
By sequential deletion analysis, we find that the first 200 amino acids
are indispensable for Gln3p to bind to Ure2p (Fig. 5A),
suggesting that the Ure2p-binding domain resides within amino acids
1-200. This hypothesis is confirmed by the interaction between Gal4p
BD-Gln3p-(1-200) and Gal4p AD-Ure2p in the yeast-two hybrid assay
(Fig. 5, B and C). Furthermore, Gal4p
BD-Gln3p-(102-150), but not Gal4p BD-Gln3p-(102-125) or Gal4p
BD-Gln3p-(126-200), interacts with Gal4p AD-Ure2p (Fig. 5,
B, E, and F). Therefore, amino acids
102-150 are necessary and sufficient for Gln3p to interact with Ure2p.
However, Gln3p with a deletion of amino acids 1-101 has a reduced
binding to Ure2p and shows a constitutive nuclear localization
phenotype (Figs. 1C and 5A), suggesting that additional sequence between amino acids 1 and 101 is needed to stabilize the Gln3p-Ure2p interaction, which is important to keep the
majority of Gln3p in the cytoplasm under rich nutrient conditions.
We have previously found that TOR and Gln3p interact with each other
(8). Using the yeast two-hybrid interaction approach, we identify the
TOR-binding domain at the C terminus of Gln3p between amino acids 600 and 667 (Fig. 6, A and C). More importantly, deletion of the TOR-binding domain causes Gln3p to constitutively localize in the nucleus (Fig. 6D). This observation provides
the first evidence that interaction with TOR is important for the control of Gln3p localization. In summary, this study leads to identification of five new key domains of Gln3p: NES (aa 336-345), NLS
(aa 388-394), K/R-rich motif (aa 351-361), Ure2p-binding domain (BD)
(aa 102-150), and TOR-binding domain (BD) (aa 600-667) (Fig. 7). These new domains are shown to play
critical roles in the regulation and function of Gln3p. A detailed
understanding of structure-function should help elucidate the detailed
mechanism that controls Gln3p localization and function by nutritional
cues.

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Fig. 7.
A model of Gln3p structure and regulation by
nutrients. Shown are the structural domains of Gln3p. Based on our
data, we propose the following model for Gln3p regulation and function.
In rich nutrient conditions, Gln3p is rapidly shuttling between the
cytoplasm and nucleus, mediated by Crm1p that binds to the NES (aa
336-345) of Gln3p and by Srp1p that interacts with the NLS (aa
388-394). Ure2p and Tor1p bind to the Ure2p BD (aa 102-150) and TOR
BD (aa 600-667), respectively, resulting in predominant cytoplasmic
distribution of Gln3p. Under nonpreferred nutrient conditions, during
starvation or rapamycin treatment, the ability of TOR and Ure2p to
inhibit Gln3p nuclear import is hindered, leading to predominant
nuclear accumulation of Gln3p. In the nucleus, Gln3p binds to the
promoter sequences and activates Gln3p-dependent genes via
its C4 GATA DNA-binding domain, the activation domain, and possibly the
K/R-rich domain.
|
|
 |
ACKNOWLEDGEMENTS |
We are grateful to D. Dean for use of the
fluorescence microscope and to other members of the Zheng laboratory
for insightful discussions.
 |
FOOTNOTES |
*
This work was supported by Grants R01CA77668 and R01GM62817
from the National Institutes of Health.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.
To whom correspondence should be addressed: Campus Box 8069, Washington University School of Medicine, 660 South Euclid Ave., St.
Louis, MO 63110. Tel.: 314-747-1884; Fax: 314-747-1887; E-mail: zheng@pathology.wustl.edu.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M300429200
 |
ABBREVIATIONS |
The abbreviations used are:
NCR, nitrogen
catabolite repression;
TOR, target of rapamycin;
NLS, nuclear
localization signal;
NES, nuclear export signal;
BD, binding
domain;
SC, synthetic complete medium;
mAb, monoclonal antibody;
GST, glutathione S-transferase;
IF, immunofluorescence;
aa, amino acid(s);
AD, activation domain;
GFP, green fluorescent protein;
DAPI, 4',6-diamidino-2-phenylindole;
3-AT, 3-aminotriazole.
 |
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