Phosphorylation Regulates the Interaction between Gln3p and the Nuclear Import Factor Srp1p*

John CarvalhoDagger , Paula G. BertramDagger , Susan R. Wente§, and X. F. Steven ZhengDagger

From the Departments of Dagger  Pathology and Immunology and § Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, April 5, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha /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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta  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 alpha  and karyopherin beta  (also termed importins) (extensively reviewed in Refs. 26, 31, and 34). In the cytoplasm, karyopherin alpha  serves as an adaptor to directly bind the NLS, whereas karyopherin beta  binds to karyopherin alpha . 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 beta  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 beta  Pse1p and inhibits its nuclear import (46, 47).

In this study, we found that at least two nuclear transport factors, karyopherin alpha /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 alpha /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 alpha /Srp1p.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

                              
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Table I
Yeast strains used in this study

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-beta -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 beta -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta s and one karyopherin alpha  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-alpha /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.

To confirm the above findings, we directly examined the effects of srp1cs and crm1-1ts mutations on the localization of Gln3p by indirect immunofluorescence. In agreement, we found that Gln3p remained in the cytoplasm in the absence and the presence of rapamycin in the srp1cs mutant at the nonpermissive temperature (20 °C) in high quality nitrogen (Fig. 2a). In contrast, Gln3p localization was normal at the permissive temperature (30 °C) (Fig. 2a). We also showed that Gln3p was exclusively localized to the nucleus both in the absence and the presence of rapamycin in the crm1-1ts mutant at 35 °C (Fig. 2b). Taken together, our data indicate that Srp1p and Crm1p are the karyopherins that mediate Gln3p nuclear entry and exit. Under physiological conditions, nitrogen sources and levels control Gln3p localization. To further establish the role of Srp1p in nitrogen signal transduction, we examined Gln3p localization in the srp1cs mutant strain under different nitrogen conditions (Fig. 3). We found that Gln3p was primarily located in the cytoplasm in rich nitrogen but rapidly accumulated in the nucleus during nitrogen starvation in the wild type strain at both 30 °C and 20 °C and in the srp1cs mutant strain at 30 °C (permissive temperature). In contrast, Gln3p remained in the cytoplasm under nitrogen starvation in the srp1cs mutant strain at the nonpermissive temperature (20 °C). Therefore, Srp1p is required for the regulated Gln3p localization by nitrogen conditions.


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Fig. 2.   Srp1p and Crm1p are required for the nuclear import and export of Gln3p, respectively. a, Gln3p is constitutively cytoplasmic in the absence or the presence of rapamycin in the srp1cs mutant. Exponentially growing wild type (WT) and srp1cs mutant cells expressing Gln3p-MYC9 were shifted to nonpermissive temperature (20 °C) for 2 h and then treated with 200 nM rapamycin for 30 min. The localization of Gln3p-MYC9 was examined by indirect immunofluorescence staining with mAb 9E10. Yeast nuclei were stained with DAPI. b, Gln3p accumulates in the nucleus of crm1-1ts arrested cells. Exponentially growing wild type and crm1-1ts mutant yeast cells expressing Gln3p-MYC9 were shifted to nonpermissive temperature (35 °C) for 2 h and then treated with 200 nM rapamycin for 30 min. The localization of Gln3p-MYC9 was examined by indirect IF staining with mAb 9E10. The nucleus was stained with DAPI.


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Fig. 3.   Nuclear accumulation of Gln3p during nitrogen starvation requires a functional karyopherin alpha /Srp1p. Exponentially growing wild type (WT) and srp1cs mutant cells expressing Gln3p-MYC9 were shifted to nonpermissive temperature (20 °C) for 2 h and then switched to synthetic complete (+N) medium or synthetic complete medium without nitrogen sources (-N) for 30 min. The localization of Gln3p-MYC9 was examined by indirect immunofluorescence staining with mAb 9E10. Yeast nuclei were stained with DAPI.

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-beta (Kap95).


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Fig. 4.   Gln3p nuclear translocation is dependent on the RanGTP/GDP cycle. a, the rna1-1ts mutation in yeast RanGAP abolishes GAP1 gene expression. Exponential wild type (WT) and mutant yeast cultures were shifted to nonpermissive temperature (37 °C) for 2 h and then treated with 200 nM rapamycin for 30 min. Total RNA were prepared and analyzed by Northern blotting with 32P-labeled GAP1 or ACT1 probes. b, the rna1-1ts mutation abrogates normal Gln3p translocation into the nucleus at the restrictive temperature. Exponentially growing wild type and mutant yeast cells expressing Gln3p-MYC9 were shifted to nonpermissive temperature (37 °C) for 2 h and then treated with 200 nM rapamycin for 30 min. The localization of Gln3p-MYC9 was examined by IF staining with mAb 9E10. The nucleus was stained with DAPI.

Karyopherin beta s are known to directly bind either to their cargo proteins or to an adaptor. To establish that Srp1p is an adaptor for Gln3p import and to understand the mechanism of regulated nuclear import of Gln3p, we investigated possible protein-protein interactions between Srp1p and Gln3p. We prepared extracts from log phase yeast cells in high quality nitrogen-containing medium. Under such yeast growth condition, Gln3p was present predominantly as hyperphosphorylated forms (Fig. 5a, lane 1, arrow, and Ref. 17). However, there was still a residual amount of Gln3p in the dephosphorylated form (Fig. 5a, lane 1, double arrow, and Ref. 17). This is consistent with the observation that a small amount of Gln3p is present in the nucleus even when yeast are grown in the nutrient-rich medium, presumably because of the need of limited Gln3p molecules for the basal expression of Gln3p-dependent genes (17). We incubated the yeast extracts with bacterially produced, affinity-purified GST on glutathione-Sepharose beads or GST-Srp1p on glutathione-Sepharose beads. We found that Gln3p specifically bound to GST-Srp1p but not to GST alone (Fig. 5a). More interestingly, GST-Srp1p only bound to the dephosphorylated but not the hyperphosphorylated Gln3p (Fig. 5a). In this experiment, the same amount of GST-Srp1p was used in each sample as indicated by Coomassie Blue staining (Fig. 5b).


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Fig. 5.   Srp1p interacts with Gln3p. a, Gln3p binds Srp1p in vitro. Extracts of yeast expressing Gln3p-MYC9 were incubated with either GST-Srp1p or GST bound to glutathione-agarose beads. GST fusion proteins and bound materials were separated by SDS-PAGE and detected by Western blotting with anti-MYC mAb 9E10. Input, one-fifth of the total input of Gln3p-MYC9 used to bind to GST or GST-Srp1p. b, GST and GST-Srp1p fusion protein used for binding to Gln3p-MYC9. Proteins were separated on SDS-polyacrylamide gel and stained with Coomassie Blue. Mr., molecular weight marker.

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 alpha /Srp1p and that phosphorylation of Gln3p inhibits this interaction.


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Fig. 6.   Srp1p interacts with the hypophosphorylated but not hyperphosphorylated Gln3p. a, Srp1p binds preferentially to dephosphorylated Gln3p. Extracts of yeast expressing Gln3p-MYC9 were treated with a control buffer, CIP alone, and CIP plus Na4P2O7. The samples were then incubated with either GST-Srp1p or GST bound to glutathione-agarose beads. GST fusion proteins and bound materials were separated by SDS-PAGE and detected with mAb 9E10 via Western blotting. Total lysates containing Gln3p-MYC9 were used as controls. b, GST and GST-Srp1p fusion protein used for binding to Gln3p-MYC9. Proteins were separated on SDS-polyacrylamide gel and stained with Coomassie Blue.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

Crm1p is required for the nuclear export of many proteins, including Hog1p (51), the HIV1 Rev protein (38), Dbp5p (66), and Yap1p (67, 68). We found that the crm1-1ts mutation caused constitutive nuclear retention of Gln3p at the restrictive temperature in high quality nitrogen in the absence of rapamycin (Fig. 2b). Consequently, there was a high expression level of GAP1 under such conditions (Fig. 1). Many Crm1p substrates contain a leucine-rich nuclear export sequence (L-X2-3-(F/I/L/V/M)-X2-3-L-X-(L/I)) (36-40, 69). Interestingly, Gln3p also contains a motif (335LHGTMRPLSL) characteristic of a leucine-rich nuclear export signal. Deletion of this leucine-rich sequence causes nuclear accumulation of Gln3p in nitrogen-rich medium and constitutive expression of GAP1.2 Taken together, our results indicate that Crm1p is the nuclear export factor for Gln3p. A defect in nuclear export as a result of the crm1-1ts mutation is sufficient to cause nuclear accumulation of Gln3p in nitrogen-rich medium, suggesting that a small amount of Gln3p shuttles between the cytoplasm and the nucleus when yeast are grown in nitrogen-rich medium. This observation is also consistent with the presence of a small amount of dephosphorylated Gln3p in the same conditions (Fig. 5a).

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.


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Fig. 7.   Model for Gln3p nuclear translocation involving Srp1p and Crm1p. When yeast are grown in high quality nitrogen, TOR is active and phosphorylates Gln3p to sequester it in the cytoplasm. When yeast are grown in poor nitrogen sources or in nitrogen starvation conditions, Gln3p is dephosphorylated and preferentially binds to its import factor Srp1p. Srp1p presumably interacts with Kap95p. This complex of Gln3p, Srp1p, and Kap95p then translocates to the nuclear interior where Gln3p eventually up-regulates transcription of NCR-sensitive genes such as GAP1 and GLN1. Gln3p is later exported from the nucleus by the exportin Crm1p.


    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported by grants from the National Institutes of Health (to S. R. W. and X. F. S. Z.) and by a Howard Hughes Medical Institute New Investigator Award (to X. F. S. Z.).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, 1030 CSRBNT, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110. E-mail: zheng@pathology.wustl.edu.

Published, JBC Papers in Press, April 30, 2001, DOI 10.1074/jbc.M103050200

2 J. Carvalho and X. F. S. Zheng, unpublished data.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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