Domains of Gln3p Interacting with Karyopherins, Ure2p, and the Target of Rapamycin Protein*

John Carvalho and X. F. Steven ZhengDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha /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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha /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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 gln3Delta 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 gln3Delta 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).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 gln3Delta mutation in all possible tests (11). Wild type and mutant GLN3-MYC9 were expressed in the gln3Delta strain. We have previously found that the gln3Delta 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 gln3Delta 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 gln3Delta 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.

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).

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 gln3Delta 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.

Our earlier study indicates that karyopherin alpha /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).

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(Delta 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).

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 gln3Delta 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 gln3Delta 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 NESDelta 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.

Dagger 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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