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Jennifer J Tate, Rajendra Rai, Terrance G Cooper, More than One Way in: Three Gln3 Sequences Required To Relieve Negative Ure2 Regulation and Support Nuclear Gln3 Import in Saccharomyces cerevisiae, Genetics, Volume 208, Issue 1, 1 January 2018, Pages 207–227, https://doi.org/10.1534/genetics.117.300457
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Abstract
Gln3 is responsible for Nitrogen Catabolite Repression-sensitive transcriptional activation in the yeast Saccharomyces cerevisiae. In nitrogen-replete medium, Gln3 is cytoplasmic and NCR-sensitive transcription is repressed. In nitrogen-limiting medium, in cells treated with TorC1 inhibitor, rapamycin, or the glutamine synthetase inhibitor, methionine sulfoximine (Msx), Gln3 becomes highly nuclear and NCR-sensitive transcription derepressed. Previously, nuclear Gln3 localization was concluded to be mediated by a single nuclear localization sequence, NLS1. Here, we show that nuclear Gln3-Myc13 localization is significantly more complex than previously appreciated. We identify three Gln3 sequences, other than NLS1, that are highly required for nuclear Gln3-Myc13 localization. Two of these sequences exhibit characteristics of monopartite (K/R-Rich NLS) and bipartite (S/R NLS) NLSs, respectively. Mutations altering these sequences are partially epistatic to a ure2Δ. The third sequence, the Ure2 relief sequence, exhibits no predicted NLS homology and is only necessary when Ure2 is present. Substitution of the basic amino acid repeats in the Ure2 relief sequence or phosphomimetic aspartate substitutions for the serine residues between them abolishes nuclear Gln3-Myc13 localization in response to both limiting nitrogen and rapamycin treatment. In contrast, Gln3-Myc13 responses are normal in parallel serine-to-alanine substitution mutants. These observations suggest that Gln3 responses to specific nitrogen environments likely occur in multiple steps that can be genetically separated. At least one general step that is associated with the Ure2 relief sequence may be prerequisite for responses to the specific stimuli of growth in poor nitrogen sources and rapamycin inhibition of TorC1.
GLN3 is one of two transcriptional activators responsible for nitrogen catabolite repression (NCR) sensitive gene expression in Saccharomyces cerevisiae (Hofman-Bang 1999; Cooper 2002, 2004; Magasanik and Kaiser 2002; Broach 2012; Ljungdahl and Daignan-Fornier 2012; Conrad et al. 2014; Swinnen et al. 2014; González and Hall 2017). In adverse, derepressive nitrogen environments (proline, allantoin), downregulation of TorC1, upregulation of Gcn2 kinase, and limited glutamine production act independently, but synergistically, to maintain Gln3 in the nucleus (Cherkasova and Hinnebusch 2003; Hinnebusch 2005; Staschke et al. 2010; Lageix et al. 2015; Rai et al. 2015; Tate et al. 2017; Yuan et al. 2017). Bmh1/2 are also required for this nuclear Gln3 localization (Tate et al. 2017). There, Gln3 activates NCR-sensitive gene expression. In contrast, in nitrogen-replete, repressive environments, TorC1 is upregulated, Gcn2 is downregulated and glutamine levels are high, causing Gln3 to relocate to the cytoplasm where it is sequestered and interacts with Ure2 (for reviews, see Hofman-Bang 1999; Cooper 2002, 2004; Magasanik and Kaiser 2002; Binda et al. 2010; Broach 2012; Ljungdahl and Daignan-Fornier 2012; Conrad et al. 2014; Swinnen et al. 2014; González and Hall 2017). In this way, Gln3 can quickly and finely respond to multiple distinct physiological conditions: rapamycin-mediated TorC1 inhibition, limiting amino acid-dependent Gcn2 activation, methionine sulfoximine (Msx) inhibition of glutamine synthesis, overall short- and long-term nitrogen starvation, and nitrogen limitation (growth in proline medium) (Tate and Cooper 2013; Tate et al. 2017).
In addition to the TorC1 and Gcn2 kinases, two phosphatases play equally important roles in NCR (DiComo and Arndt 1996; Beck and Hall 1999; Jiang and Broach 1999; Bertram et al. 2000; Wang et al. 2003; Cox et al. 2004; Tate et al. 2006, 2009, 2010, 2015; Yan et al. 2006; Georis et al. 2011b; Tate and Cooper 2013). A Gln3 response to rapamycin requires both PP2A and Sit4 phosphatases, whereas short-term nitrogen starvation, or growth under nitrogen-limiting conditions requires only Sit4 (Tate and Cooper 2013). Long-term nitrogen or glutamine starvation requires neither phosphatase (Tate and Cooper 2013). In contrast to the TorC1-regulated Sch9 phosphorylation, Gln3 cytoplasmic sequestration does not respond to leucine starvation (Binda et al. 2009, 2010; Bonfils et al. 2012; Tate and Cooper 2013). Furthermore, cytoplasmic Gln3 sequestration and repressed NCR-sensitive transcription occur readily in mutants lacking multiple components of the Gtr-Ego complex responsible for TorC1 activation (Tate et al. 2015).
Cytoplasmic Gln3 sequestration and NCR-sensitive transcription have long been known to require Ure2 (Lacroute 1971; Drillien and Lacroute 1972; Drillien et al. 1973; Cooper 1982, 2004; Courchesne and Magasanik 1988; Coffman et al. 1994; Cunningham et al. 2000; Magasanik and Kaiser 2002; Feller et al. 2013). This negative regulation correlates with the formation of a Ure2-Gln3101–150 complex (Blinder et al. 1996; Beck and Hall 1999). Our previous experiments have demonstrated that multiple distinct, genetically separable, regions in Gln3 are required for its intracellular distribution in response to the above physiological conditions (Rai et al. 2013, 2014, 2015, 2016). However, we have little idea which, if any, additional Gln3 sequences or regulatory processes beyond the Gln3101–150–Ure2 interaction participate in dissociating this complex in nitrogen-limiting, or any other, conditions that bring about Gln3 relocation to the nucleus.
To effectively investigate this question, detailed identification of all Gln3 sequences required for its nuclear localization is necessary. Such accounting is required because mutational inactivation of a critical nuclear localization sequence (NLS) conceptually yields the same phenotype as a mutation abolishing the ability of Gln3 to disengage normally from negative regulation by Ure2. In both cases, constitutively cytoplasmic Gln3 localization would be expected. Two previous reports provide a starting point for our accounting investigation.
The first sequence shown to be required for nuclear Gln3 localization was EGFP-Gln3344–365 (Kulkarni et al. 2001). This sequence contained a highly K/R-rich region (Gln3351–360). A later report, based on data derived from a series of deletions in full length Gln31–730-Myc9, concluded that Gln3388–394 (NLS1) was the crucial sequence responsible for nuclear Gln3-Myc9 localization (Carvalho and Zheng 2003). Gln3571–577 (NLS2) was also concluded to exhibit homology to a classical NLS consensus sequence. However, its deletion (Gln3571–577Δ) did not exhibit a demonstrable effect on Gln3 localization (Carvalho and Zheng 2003). In that study, all of the constructs were C-terminal tagged.
The present work demonstrates that Gln3 release from negative regulation by Ure2 and subsequent nuclear localization are more complex than previously appreciated. We show that three distinct Gln3 sequences are required for nuclear Gln31–730-Myc13 localization. The first two sequences are K/R-rich: Gln3351–360 and previously uninvestigated Gln3279–307. These sequences are predicted to be homologous to monopartite and bipartite NLS sequences, respectively. We were unable to demonstrate the necessity of Gln3 NLS1 or NLS2 for nuclear Gln3-Myc13 localization in derepressive proline medium. In this respect, the Gln3 NLSs are somewhat similar to the situation observed with the functional Aspergillus ortholog of Gln3 and Gat1, AreA. AreA contains six NLS-homologous sequences, five of which can be collectively abolished without interfering with nuclear AreA import (Hunter et al. 2014). A third, and also uninvestigated Gln3 sequence, is designated the Ure2 relief sequence (Gln3247–282). This sequence is required for nuclear Gln3-Myc13 localization irrespective of whether triggered by rapamycin treatment or growth in derepressive proline medium. Phosphomimetic aspartate substitutions in the Ure2 relief sequence eliminate nuclear Gln3-Myc13 localization. Parallel alanine substitutions yield wild-type Gln3 responses. These observations lead us to conclude that Gln3 responses to specific nitrogen environments potentially occur in multiple discrete steps that can be genetically separated. At least one general step, that performed by the Ure2 relief sequence, appears to be prerequisite for Gln3 responses to specific stimuli, growth in poor nitrogen sources, or rapamycin inhibition of TorC1, in wild-type, but not ure2Δ, cells.
Materials and Methods
Strains and culture conditions
The S. cerevisiae strains used in this work appear in Table 1. Transformants, prepared by the lithium acetate method (Ito et al. 1983), were used as soon as possible after transformation (5 days or fewer).
Saccharomyces cerevisiae strains used in this work
Strain . | Pertinent genotype . | Parent . | Complete genotype . |
---|---|---|---|
JK9-3da | Wild type | MATa, leu2-3,112, ura3-52, trp1, his4, rme1, HMLa | |
RR215 | ure2Δ transformation recipient | JK9-3da | MATa, leu2-3,112, ura3-52, trp1, his4, rme1, HMLa, ure2Δ:[KanMX] |
KHC2 | gln3Δ transformation recipient | TCY1 | MATa, lys2, ura3, gln3Δ::hisGRai et al. (2014) |
TCY1 | Wild type | GC210 | MATa, lys2, ura3 |
Strain . | Pertinent genotype . | Parent . | Complete genotype . |
---|---|---|---|
JK9-3da | Wild type | MATa, leu2-3,112, ura3-52, trp1, his4, rme1, HMLa | |
RR215 | ure2Δ transformation recipient | JK9-3da | MATa, leu2-3,112, ura3-52, trp1, his4, rme1, HMLa, ure2Δ:[KanMX] |
KHC2 | gln3Δ transformation recipient | TCY1 | MATa, lys2, ura3, gln3Δ::hisGRai et al. (2014) |
TCY1 | Wild type | GC210 | MATa, lys2, ura3 |
GC210 is a spontaneously derived, isogenic derivative of Σ1278b (1982-2-1088).
Strain . | Pertinent genotype . | Parent . | Complete genotype . |
---|---|---|---|
JK9-3da | Wild type | MATa, leu2-3,112, ura3-52, trp1, his4, rme1, HMLa | |
RR215 | ure2Δ transformation recipient | JK9-3da | MATa, leu2-3,112, ura3-52, trp1, his4, rme1, HMLa, ure2Δ:[KanMX] |
KHC2 | gln3Δ transformation recipient | TCY1 | MATa, lys2, ura3, gln3Δ::hisGRai et al. (2014) |
TCY1 | Wild type | GC210 | MATa, lys2, ura3 |
Strain . | Pertinent genotype . | Parent . | Complete genotype . |
---|---|---|---|
JK9-3da | Wild type | MATa, leu2-3,112, ura3-52, trp1, his4, rme1, HMLa | |
RR215 | ure2Δ transformation recipient | JK9-3da | MATa, leu2-3,112, ura3-52, trp1, his4, rme1, HMLa, ure2Δ:[KanMX] |
KHC2 | gln3Δ transformation recipient | TCY1 | MATa, lys2, ura3, gln3Δ::hisGRai et al. (2014) |
TCY1 | Wild type | GC210 | MATa, lys2, ura3 |
GC210 is a spontaneously derived, isogenic derivative of Σ1278b (1982-2-1088).
Cultures (50 ml) were grown to midlog phase (A600 nm ∼0.5) in Yeast Nitrogen Base (YNB, without amino acids or ammonia) minimal medium containing the indicated nitrogen source (final concentration, 0.1%). Leucine (120 μg/ml), histidine (20 μg/ml), lysine (40 μg/ml), tryptophan (20 μg/ml), and uracil (20 μg/ml) were added as needed to cover auxotrophic requirements. For experiments assaying EGFP-Gln3 intracellular distribution, 2% raffinose replaced glucose, and 0.2% proline was used in place of the normal 0.1%. Cells were treated with 200 ng/ml rapamycin for 15 or 20 min, and 2 mM methionine sulfoximine (Msx) for 30 min (Georis et al. 2011a).
Plasmid construction
Gln3-Myc13 plasmids were constructed in CEN-based vectors as previously described (Kulkarni et al. 2001, 2006; Rai et al. 2013, 2014, 2016). Pertinent primers are shown in Table 2. Unless indicated otherwise, all plasmids contained a full-length GLN3 gene. Transcription was driven by the native wild-type GLN3 promoter, or, in the case of EGFP-GLN3, a GAL1,10 promoter. The structures of all constructs were verified by restriction mapping and DNA sequence analyses.
Primer sets used to construct plasmids employed in this work
Plasmids . | Sequence changes . | Primer sets . |
---|---|---|
pRR536 | Gln31–730 (F.L. Wild Type) | 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
Rai et al. (2013) | ||
pRR609 | Gln31–496 | 5′-CGCGGATCCGGATGAAGATTTACTGGAACTTGAGGTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR610 | Gln31–476 | 5′-CGCGGATCCAAAATTAGGAGTAACAGTGTTCGAATTGTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR611 | Gln31–384 | 5′-CGCGGATCCTGTAGTGGTTACTGAAGTTGAGGCAGTTGC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR612 | Gln31–400 | 5′-CGCGGATCCAGAGTTTTGTTGTAGTGATTTTTTCCTCGA-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR613 | Gln31–542 | 5′-CGCGGATCCAATTCTTGGTGAGGATGCGACACTATTTCC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
Rai et al. (2013) | ||
pRR620 | Gln31–350 | 5′-CGCGGATCCGATAACGTCCGATTTTAAGGATAATGGCC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR622 | Gln31–584 | 5′-CGCGGATCCCGATGAGGAGTACGATGCATTGCGCGAC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR624 | Gln31–486 | 5′-CGCGGATCCTGAAGTACTACTTCGTCTTGAAGACCTTCT-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR656 | Gln3244–296Δ | 5′-CAATCGGCCGGATAACGTCCGATTTTAAGGATAATGGCCTCATGG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCAAACGGACCCAAACATTGCACAAAAT-3′ | ||
5′-CGCGGATCCAGAGTTTTGTTGTAGTGATTTTTTCCTCGA-3′ | ||
pRR723 | Gln3388–395Δ | 5′-CAATCGGCCGTTTAGCATTTGTAGTGGTTACTGAAG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCTACAACAAAACTCTTTATCTAGAGTG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATT GCT-3′ | ||
pRR725 | Gln3262–271Δ | 5′-CAATCGGCCGATTTGTTGTGTTGGATGAAGATAATCC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGATGTCGTCCACGTCCCTGGCCAATTTC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR754 | Gln3R264D,K265D,S267D | 5′-CAATCGGCCGATGTCGTCCACGTCCCTGGCCAATTTC-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGGATAAGgtcGTTgtcgtcTACAGAATTTG-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR772 | Gln3K281D,K282D,S285D | 5′-CAATCGGCCGgatGTATCTTCCAGTATATCCAAT-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGatcatcGAAATTGGCCAGGGACGT-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR787 | Rai et al. (2015) | |
pRR789 | Gln3K352D,R353D,K356D,K357D | 5′-CAATCGGCCGTTTGTTTGGCgtcatcCTTTGAAATgtcatcTTTGATAACGTCCGATTTTAAGG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCAAACATTGCACAAAATACTCCAAGTGCAC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR792 | Gln3P388D,R392D,K393D | 5′-TCACTCTAGATAAAGAGTTTTGTTGTAGTGATTTgtcatcCGATCGTATgtcTTTAGCATTTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-TCTTTATCTAGAGTGATACCTGAAGAAATCATTAGAG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR823 | Gln3S255D,S256D,S257D,S273D,S274D,S275D,S276D,S287D,S288D,S289D,S291D | 5′-CAATCGGCCGTTTATTTTGTCCTGATGGTTCCATATTggcTATagcggcagcTACTGAGGCAGCTCTTTTGAAATTGGCCAGggcagctgcagcCATTGGCTTGATAAGTGAGTTTTTTCTTACAGAATTTGTTGTGTTggctgcagcTAATCCTATCGAATCGC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCTGATACAATGTTTCAATTGTAAAAC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATT GCT-3′ | ||
pRR826 | Gln3S255A,S256A,S257A,S273A,S274A,S275A,S276A,S287A,S288A,S289A,S291A | 5′-CAATCGGCCGTTTATTTTGTCCTGATGGTTCCATATTgtcTATatcgtcatcTA CTGAGGCAGCTCTTTTGAAATTGGCCAGgtcatcgtcatcCATTGGCTTGATAAGTGAGTTTTTTCTTACAGAATTTGTTGTGTTgtcatcatcTAATCCTATCGAATCGC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCTGATACAATGTTTCAATTGTAAAAC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR834 | Gln3S249A,S251A,S262A,S267A | 5′-CAATCGGCCGTTTCTGAAGAAGgctGATgcgATAGGATTATCTTCATCCAACACAACAAAtgcTGTAAGAAAAAACgcaCTTATCAAG-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGCTGGATATTACTATTGTTGCTATT-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR835 | Gln3S249D,S251D,S262D,S267D | 5′-CAATCGGCCGTTTCTGAAGAAGgacGATgatATAGGATTATCTTCATCCAACACAACAAAtgaTGTAAGAAAAAACgatCTTATCAAG-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGCTGGATATTACTATTGTTGCTATT-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR1034 | Gln3S576D | 5′-GTACGATGCATTGCGgtcCATCTTTCTTCTTGGTATATTC-3′ |
5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACC-3′ | ||
Rai et al. (2014) | ||
pRR1037 | Gln3S576A | 5′-GTACGATGCATTGCGcgcCATCTTTCTTCTTGGTATATTC-3′ |
5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACC-3′ | ||
Rai et al. (2014) | ||
pRR1059 | Gln3S355D | 5′-CGGAACAACAGATCTGGATGAAGATTTACTGGAACTTG-3′ |
5′-CATGGTACCATGAGGCCATTATCCTTAAAATCGGACGTTATCAAAAAGAGGATTgacAAGAAGAGAGCC-3′ | ||
pRR1060 | Gln3S355A | 5′-CGGAACAACAGATCTGGATGAAGATTTACTGGAACTTG-3′ |
5′-CATGGTACCATGAGGCCATTATCCTTAAAATCGGACGTTATCAAAAAGAGGATTgcaAAGAAGAGAGCC-3′ | ||
pRR1293 | Gln3R264A,K265A,S267A | 5′-CAATCGGCCGGATAAGtgcGTTtgctgcTACAGAATTTGTTGTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR1295 | Gln3K281A,K282A,S285A | 5′-CAATCGGCCGATGTCGTCCACGTCCCTGGCCAATTTCgcagcaGCTGCCgcaGTATCTTCCAGTATATC-3′ |
5′-CGCGGATCC TATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGGATAAGTGAGTTTTTTCTTACAGAATTTGTTG-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR1345 | Gln3R572D,R573D,K574D | 5′-CATCCAGATCTGTTGTTCCGAGATATTACCAAAACC-3′ |
5′-GTACGATGCATTGCGCGACATgtcgtcatcTGGTATATTCATTCCTTGAC-3′ | ||
pRR1366 | Gln3S249D,S251D,S256D,S257D | 5′-CAGGACTAGTGGACGACATTGGCTTGATAAGTGAGTTTTTTCTTACAGAATTTGTTGTGTTgtcgtcAGATAATCCTATgtcATCgtcCTTCTTCAGAAAAG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAGGACTAGTCTGGCCAATTTCAAAAGAGCTGCCTCAG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR1368 | Gln3S267D,S273D,S274D,T275D,S276D | 5′-CAGGACTAGTgtcgtcCATTGGCTTGATAAGgtcGTTTTTTCTTACAG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAGGACTAGTCTGGCCAATTTCAAAAGAGCTGCCTCAG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR1370 | Gln3S285D,S287D,S288D,S289D | 5′-CAGGACTAGTCTGGCCAATTTCAAAAGAGCTGCCgacGTAgacgacgacATATCCAATATGGAACC-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAGGACTAGTGGACGACATTGGCTTGATAAGTGAG-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRS316 | Vector | |
pRR482 | Kulkarni et al. (2001) | |
pKA10 | Kulkarni et al. (2001) | |
pKA14 | Kulkarni et al. (2001) | |
pKA36 | Kulkarni et al. (2001) | |
pKA38 | Kulkarni et al. (2001) | |
pKA53 | Kulkarni et al. (2001) |
Plasmids . | Sequence changes . | Primer sets . |
---|---|---|
pRR536 | Gln31–730 (F.L. Wild Type) | 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
Rai et al. (2013) | ||
pRR609 | Gln31–496 | 5′-CGCGGATCCGGATGAAGATTTACTGGAACTTGAGGTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR610 | Gln31–476 | 5′-CGCGGATCCAAAATTAGGAGTAACAGTGTTCGAATTGTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR611 | Gln31–384 | 5′-CGCGGATCCTGTAGTGGTTACTGAAGTTGAGGCAGTTGC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR612 | Gln31–400 | 5′-CGCGGATCCAGAGTTTTGTTGTAGTGATTTTTTCCTCGA-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR613 | Gln31–542 | 5′-CGCGGATCCAATTCTTGGTGAGGATGCGACACTATTTCC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
Rai et al. (2013) | ||
pRR620 | Gln31–350 | 5′-CGCGGATCCGATAACGTCCGATTTTAAGGATAATGGCC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR622 | Gln31–584 | 5′-CGCGGATCCCGATGAGGAGTACGATGCATTGCGCGAC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR624 | Gln31–486 | 5′-CGCGGATCCTGAAGTACTACTTCGTCTTGAAGACCTTCT-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR656 | Gln3244–296Δ | 5′-CAATCGGCCGGATAACGTCCGATTTTAAGGATAATGGCCTCATGG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCAAACGGACCCAAACATTGCACAAAAT-3′ | ||
5′-CGCGGATCCAGAGTTTTGTTGTAGTGATTTTTTCCTCGA-3′ | ||
pRR723 | Gln3388–395Δ | 5′-CAATCGGCCGTTTAGCATTTGTAGTGGTTACTGAAG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCTACAACAAAACTCTTTATCTAGAGTG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATT GCT-3′ | ||
pRR725 | Gln3262–271Δ | 5′-CAATCGGCCGATTTGTTGTGTTGGATGAAGATAATCC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGATGTCGTCCACGTCCCTGGCCAATTTC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR754 | Gln3R264D,K265D,S267D | 5′-CAATCGGCCGATGTCGTCCACGTCCCTGGCCAATTTC-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGGATAAGgtcGTTgtcgtcTACAGAATTTG-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR772 | Gln3K281D,K282D,S285D | 5′-CAATCGGCCGgatGTATCTTCCAGTATATCCAAT-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGatcatcGAAATTGGCCAGGGACGT-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR787 | Rai et al. (2015) | |
pRR789 | Gln3K352D,R353D,K356D,K357D | 5′-CAATCGGCCGTTTGTTTGGCgtcatcCTTTGAAATgtcatcTTTGATAACGTCCGATTTTAAGG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCAAACATTGCACAAAATACTCCAAGTGCAC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR792 | Gln3P388D,R392D,K393D | 5′-TCACTCTAGATAAAGAGTTTTGTTGTAGTGATTTgtcatcCGATCGTATgtcTTTAGCATTTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-TCTTTATCTAGAGTGATACCTGAAGAAATCATTAGAG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR823 | Gln3S255D,S256D,S257D,S273D,S274D,S275D,S276D,S287D,S288D,S289D,S291D | 5′-CAATCGGCCGTTTATTTTGTCCTGATGGTTCCATATTggcTATagcggcagcTACTGAGGCAGCTCTTTTGAAATTGGCCAGggcagctgcagcCATTGGCTTGATAAGTGAGTTTTTTCTTACAGAATTTGTTGTGTTggctgcagcTAATCCTATCGAATCGC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCTGATACAATGTTTCAATTGTAAAAC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATT GCT-3′ | ||
pRR826 | Gln3S255A,S256A,S257A,S273A,S274A,S275A,S276A,S287A,S288A,S289A,S291A | 5′-CAATCGGCCGTTTATTTTGTCCTGATGGTTCCATATTgtcTATatcgtcatcTA CTGAGGCAGCTCTTTTGAAATTGGCCAGgtcatcgtcatcCATTGGCTTGATAAGTGAGTTTTTTCTTACAGAATTTGTTGTGTTgtcatcatcTAATCCTATCGAATCGC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCTGATACAATGTTTCAATTGTAAAAC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR834 | Gln3S249A,S251A,S262A,S267A | 5′-CAATCGGCCGTTTCTGAAGAAGgctGATgcgATAGGATTATCTTCATCCAACACAACAAAtgcTGTAAGAAAAAACgcaCTTATCAAG-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGCTGGATATTACTATTGTTGCTATT-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR835 | Gln3S249D,S251D,S262D,S267D | 5′-CAATCGGCCGTTTCTGAAGAAGgacGATgatATAGGATTATCTTCATCCAACACAACAAAtgaTGTAAGAAAAAACgatCTTATCAAG-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGCTGGATATTACTATTGTTGCTATT-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR1034 | Gln3S576D | 5′-GTACGATGCATTGCGgtcCATCTTTCTTCTTGGTATATTC-3′ |
5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACC-3′ | ||
Rai et al. (2014) | ||
pRR1037 | Gln3S576A | 5′-GTACGATGCATTGCGcgcCATCTTTCTTCTTGGTATATTC-3′ |
5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACC-3′ | ||
Rai et al. (2014) | ||
pRR1059 | Gln3S355D | 5′-CGGAACAACAGATCTGGATGAAGATTTACTGGAACTTG-3′ |
5′-CATGGTACCATGAGGCCATTATCCTTAAAATCGGACGTTATCAAAAAGAGGATTgacAAGAAGAGAGCC-3′ | ||
pRR1060 | Gln3S355A | 5′-CGGAACAACAGATCTGGATGAAGATTTACTGGAACTTG-3′ |
5′-CATGGTACCATGAGGCCATTATCCTTAAAATCGGACGTTATCAAAAAGAGGATTgcaAAGAAGAGAGCC-3′ | ||
pRR1293 | Gln3R264A,K265A,S267A | 5′-CAATCGGCCGGATAAGtgcGTTtgctgcTACAGAATTTGTTGTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR1295 | Gln3K281A,K282A,S285A | 5′-CAATCGGCCGATGTCGTCCACGTCCCTGGCCAATTTCgcagcaGCTGCCgcaGTATCTTCCAGTATATC-3′ |
5′-CGCGGATCC TATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGGATAAGTGAGTTTTTTCTTACAGAATTTGTTG-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR1345 | Gln3R572D,R573D,K574D | 5′-CATCCAGATCTGTTGTTCCGAGATATTACCAAAACC-3′ |
5′-GTACGATGCATTGCGCGACATgtcgtcatcTGGTATATTCATTCCTTGAC-3′ | ||
pRR1366 | Gln3S249D,S251D,S256D,S257D | 5′-CAGGACTAGTGGACGACATTGGCTTGATAAGTGAGTTTTTTCTTACAGAATTTGTTGTGTTgtcgtcAGATAATCCTATgtcATCgtcCTTCTTCAGAAAAG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAGGACTAGTCTGGCCAATTTCAAAAGAGCTGCCTCAG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR1368 | Gln3S267D,S273D,S274D,T275D,S276D | 5′-CAGGACTAGTgtcgtcCATTGGCTTGATAAGgtcGTTTTTTCTTACAG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAGGACTAGTCTGGCCAATTTCAAAAGAGCTGCCTCAG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR1370 | Gln3S285D,S287D,S288D,S289D | 5′-CAGGACTAGTCTGGCCAATTTCAAAAGAGCTGCCgacGTAgacgacgacATATCCAATATGGAACC-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAGGACTAGTGGACGACATTGGCTTGATAAGTGAG-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRS316 | Vector | |
pRR482 | Kulkarni et al. (2001) | |
pKA10 | Kulkarni et al. (2001) | |
pKA14 | Kulkarni et al. (2001) | |
pKA36 | Kulkarni et al. (2001) | |
pKA38 | Kulkarni et al. (2001) | |
pKA53 | Kulkarni et al. (2001) |
Plasmids . | Sequence changes . | Primer sets . |
---|---|---|
pRR536 | Gln31–730 (F.L. Wild Type) | 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
Rai et al. (2013) | ||
pRR609 | Gln31–496 | 5′-CGCGGATCCGGATGAAGATTTACTGGAACTTGAGGTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR610 | Gln31–476 | 5′-CGCGGATCCAAAATTAGGAGTAACAGTGTTCGAATTGTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR611 | Gln31–384 | 5′-CGCGGATCCTGTAGTGGTTACTGAAGTTGAGGCAGTTGC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR612 | Gln31–400 | 5′-CGCGGATCCAGAGTTTTGTTGTAGTGATTTTTTCCTCGA-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR613 | Gln31–542 | 5′-CGCGGATCCAATTCTTGGTGAGGATGCGACACTATTTCC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
Rai et al. (2013) | ||
pRR620 | Gln31–350 | 5′-CGCGGATCCGATAACGTCCGATTTTAAGGATAATGGCC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR622 | Gln31–584 | 5′-CGCGGATCCCGATGAGGAGTACGATGCATTGCGCGAC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR624 | Gln31–486 | 5′-CGCGGATCCTGAAGTACTACTTCGTCTTGAAGACCTTCT-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR656 | Gln3244–296Δ | 5′-CAATCGGCCGGATAACGTCCGATTTTAAGGATAATGGCCTCATGG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCAAACGGACCCAAACATTGCACAAAAT-3′ | ||
5′-CGCGGATCCAGAGTTTTGTTGTAGTGATTTTTTCCTCGA-3′ | ||
pRR723 | Gln3388–395Δ | 5′-CAATCGGCCGTTTAGCATTTGTAGTGGTTACTGAAG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCTACAACAAAACTCTTTATCTAGAGTG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATT GCT-3′ | ||
pRR725 | Gln3262–271Δ | 5′-CAATCGGCCGATTTGTTGTGTTGGATGAAGATAATCC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGATGTCGTCCACGTCCCTGGCCAATTTC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR754 | Gln3R264D,K265D,S267D | 5′-CAATCGGCCGATGTCGTCCACGTCCCTGGCCAATTTC-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGGATAAGgtcGTTgtcgtcTACAGAATTTG-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR772 | Gln3K281D,K282D,S285D | 5′-CAATCGGCCGgatGTATCTTCCAGTATATCCAAT-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGatcatcGAAATTGGCCAGGGACGT-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR787 | Rai et al. (2015) | |
pRR789 | Gln3K352D,R353D,K356D,K357D | 5′-CAATCGGCCGTTTGTTTGGCgtcatcCTTTGAAATgtcatcTTTGATAACGTCCGATTTTAAGG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCAAACATTGCACAAAATACTCCAAGTGCAC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR792 | Gln3P388D,R392D,K393D | 5′-TCACTCTAGATAAAGAGTTTTGTTGTAGTGATTTgtcatcCGATCGTATgtcTTTAGCATTTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-TCTTTATCTAGAGTGATACCTGAAGAAATCATTAGAG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR823 | Gln3S255D,S256D,S257D,S273D,S274D,S275D,S276D,S287D,S288D,S289D,S291D | 5′-CAATCGGCCGTTTATTTTGTCCTGATGGTTCCATATTggcTATagcggcagcTACTGAGGCAGCTCTTTTGAAATTGGCCAGggcagctgcagcCATTGGCTTGATAAGTGAGTTTTTTCTTACAGAATTTGTTGTGTTggctgcagcTAATCCTATCGAATCGC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCTGATACAATGTTTCAATTGTAAAAC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATT GCT-3′ | ||
pRR826 | Gln3S255A,S256A,S257A,S273A,S274A,S275A,S276A,S287A,S288A,S289A,S291A | 5′-CAATCGGCCGTTTATTTTGTCCTGATGGTTCCATATTgtcTATatcgtcatcTA CTGAGGCAGCTCTTTTGAAATTGGCCAGgtcatcgtcatcCATTGGCTTGATAAGTGAGTTTTTTCTTACAGAATTTGTTGTGTTgtcatcatcTAATCCTATCGAATCGC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCTGATACAATGTTTCAATTGTAAAAC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR834 | Gln3S249A,S251A,S262A,S267A | 5′-CAATCGGCCGTTTCTGAAGAAGgctGATgcgATAGGATTATCTTCATCCAACACAACAAAtgcTGTAAGAAAAAACgcaCTTATCAAG-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGCTGGATATTACTATTGTTGCTATT-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR835 | Gln3S249D,S251D,S262D,S267D | 5′-CAATCGGCCGTTTCTGAAGAAGgacGATgatATAGGATTATCTTCATCCAACACAACAAAtgaTGTAAGAAAAAACgatCTTATCAAG-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGCTGGATATTACTATTGTTGCTATT-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR1034 | Gln3S576D | 5′-GTACGATGCATTGCGgtcCATCTTTCTTCTTGGTATATTC-3′ |
5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACC-3′ | ||
Rai et al. (2014) | ||
pRR1037 | Gln3S576A | 5′-GTACGATGCATTGCGcgcCATCTTTCTTCTTGGTATATTC-3′ |
5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACC-3′ | ||
Rai et al. (2014) | ||
pRR1059 | Gln3S355D | 5′-CGGAACAACAGATCTGGATGAAGATTTACTGGAACTTG-3′ |
5′-CATGGTACCATGAGGCCATTATCCTTAAAATCGGACGTTATCAAAAAGAGGATTgacAAGAAGAGAGCC-3′ | ||
pRR1060 | Gln3S355A | 5′-CGGAACAACAGATCTGGATGAAGATTTACTGGAACTTG-3′ |
5′-CATGGTACCATGAGGCCATTATCCTTAAAATCGGACGTTATCAAAAAGAGGATTgcaAAGAAGAGAGCC-3′ | ||
pRR1293 | Gln3R264A,K265A,S267A | 5′-CAATCGGCCGGATAAGtgcGTTtgctgcTACAGAATTTGTTGTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR1295 | Gln3K281A,K282A,S285A | 5′-CAATCGGCCGATGTCGTCCACGTCCCTGGCCAATTTCgcagcaGCTGCCgcaGTATCTTCCAGTATATC-3′ |
5′-CGCGGATCC TATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGGATAAGTGAGTTTTTTCTTACAGAATTTGTTG-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR1345 | Gln3R572D,R573D,K574D | 5′-CATCCAGATCTGTTGTTCCGAGATATTACCAAAACC-3′ |
5′-GTACGATGCATTGCGCGACATgtcgtcatcTGGTATATTCATTCCTTGAC-3′ | ||
pRR1366 | Gln3S249D,S251D,S256D,S257D | 5′-CAGGACTAGTGGACGACATTGGCTTGATAAGTGAGTTTTTTCTTACAGAATTTGTTGTGTTgtcgtcAGATAATCCTATgtcATCgtcCTTCTTCAGAAAAG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAGGACTAGTCTGGCCAATTTCAAAAGAGCTGCCTCAG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR1368 | Gln3S267D,S273D,S274D,T275D,S276D | 5′-CAGGACTAGTgtcgtcCATTGGCTTGATAAGgtcGTTTTTTCTTACAG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAGGACTAGTCTGGCCAATTTCAAAAGAGCTGCCTCAG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR1370 | Gln3S285D,S287D,S288D,S289D | 5′-CAGGACTAGTCTGGCCAATTTCAAAAGAGCTGCCgacGTAgacgacgacATATCCAATATGGAACC-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAGGACTAGTGGACGACATTGGCTTGATAAGTGAG-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRS316 | Vector | |
pRR482 | Kulkarni et al. (2001) | |
pKA10 | Kulkarni et al. (2001) | |
pKA14 | Kulkarni et al. (2001) | |
pKA36 | Kulkarni et al. (2001) | |
pKA38 | Kulkarni et al. (2001) | |
pKA53 | Kulkarni et al. (2001) |
Plasmids . | Sequence changes . | Primer sets . |
---|---|---|
pRR536 | Gln31–730 (F.L. Wild Type) | 5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
Rai et al. (2013) | ||
pRR609 | Gln31–496 | 5′-CGCGGATCCGGATGAAGATTTACTGGAACTTGAGGTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR610 | Gln31–476 | 5′-CGCGGATCCAAAATTAGGAGTAACAGTGTTCGAATTGTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR611 | Gln31–384 | 5′-CGCGGATCCTGTAGTGGTTACTGAAGTTGAGGCAGTTGC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR612 | Gln31–400 | 5′-CGCGGATCCAGAGTTTTGTTGTAGTGATTTTTTCCTCGA-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR613 | Gln31–542 | 5′-CGCGGATCCAATTCTTGGTGAGGATGCGACACTATTTCC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
Rai et al. (2013) | ||
pRR620 | Gln31–350 | 5′-CGCGGATCCGATAACGTCCGATTTTAAGGATAATGGCC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR622 | Gln31–584 | 5′-CGCGGATCCCGATGAGGAGTACGATGCATTGCGCGAC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR624 | Gln31–486 | 5′-CGCGGATCCTGAAGTACTACTTCGTCTTGAAGACCTTCT-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR656 | Gln3244–296Δ | 5′-CAATCGGCCGGATAACGTCCGATTTTAAGGATAATGGCCTCATGG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCAAACGGACCCAAACATTGCACAAAAT-3′ | ||
5′-CGCGGATCCAGAGTTTTGTTGTAGTGATTTTTTCCTCGA-3′ | ||
pRR723 | Gln3388–395Δ | 5′-CAATCGGCCGTTTAGCATTTGTAGTGGTTACTGAAG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCTACAACAAAACTCTTTATCTAGAGTG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATT GCT-3′ | ||
pRR725 | Gln3262–271Δ | 5′-CAATCGGCCGATTTGTTGTGTTGGATGAAGATAATCC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGATGTCGTCCACGTCCCTGGCCAATTTC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR754 | Gln3R264D,K265D,S267D | 5′-CAATCGGCCGATGTCGTCCACGTCCCTGGCCAATTTC-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGGATAAGgtcGTTgtcgtcTACAGAATTTG-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR772 | Gln3K281D,K282D,S285D | 5′-CAATCGGCCGgatGTATCTTCCAGTATATCCAAT-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGatcatcGAAATTGGCCAGGGACGT-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR787 | Rai et al. (2015) | |
pRR789 | Gln3K352D,R353D,K356D,K357D | 5′-CAATCGGCCGTTTGTTTGGCgtcatcCTTTGAAATgtcatcTTTGATAACGTCCGATTTTAAGG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCAAACATTGCACAAAATACTCCAAGTGCAC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR792 | Gln3P388D,R392D,K393D | 5′-TCACTCTAGATAAAGAGTTTTGTTGTAGTGATTTgtcatcCGATCGTATgtcTTTAGCATTTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-TCTTTATCTAGAGTGATACCTGAAGAAATCATTAGAG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR823 | Gln3S255D,S256D,S257D,S273D,S274D,S275D,S276D,S287D,S288D,S289D,S291D | 5′-CAATCGGCCGTTTATTTTGTCCTGATGGTTCCATATTggcTATagcggcagcTACTGAGGCAGCTCTTTTGAAATTGGCCAGggcagctgcagcCATTGGCTTGATAAGTGAGTTTTTTCTTACAGAATTTGTTGTGTTggctgcagcTAATCCTATCGAATCGC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCTGATACAATGTTTCAATTGTAAAAC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATT GCT-3′ | ||
pRR826 | Gln3S255A,S256A,S257A,S273A,S274A,S275A,S276A,S287A,S288A,S289A,S291A | 5′-CAATCGGCCGTTTATTTTGTCCTGATGGTTCCATATTgtcTATatcgtcatcTA CTGAGGCAGCTCTTTTGAAATTGGCCAGgtcatcgtcatcCATTGGCTTGATAAGTGAGTTTTTTCTTACAGAATTTGTTGTGTTgtcatcatcTAATCCTATCGAATCGC-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAATCGGCCGCTGATACAATGTTTCAATTGTAAAAC-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR834 | Gln3S249A,S251A,S262A,S267A | 5′-CAATCGGCCGTTTCTGAAGAAGgctGATgcgATAGGATTATCTTCATCCAACACAACAAAtgcTGTAAGAAAAAACgcaCTTATCAAG-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGCTGGATATTACTATTGTTGCTATT-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR835 | Gln3S249D,S251D,S262D,S267D | 5′-CAATCGGCCGTTTCTGAAGAAGgacGATgatATAGGATTATCTTCATCCAACACAACAAAtgaTGTAAGAAAAAACgatCTTATCAAG-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGCTGGATATTACTATTGTTGCTATT-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR1034 | Gln3S576D | 5′-GTACGATGCATTGCGgtcCATCTTTCTTCTTGGTATATTC-3′ |
5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACC-3′ | ||
Rai et al. (2014) | ||
pRR1037 | Gln3S576A | 5′-GTACGATGCATTGCGcgcCATCTTTCTTCTTGGTATATTC-3′ |
5′-CATCCAGATCTGTTGTTCCGATATTACCAAAACC-3′ | ||
Rai et al. (2014) | ||
pRR1059 | Gln3S355D | 5′-CGGAACAACAGATCTGGATGAAGATTTACTGGAACTTG-3′ |
5′-CATGGTACCATGAGGCCATTATCCTTAAAATCGGACGTTATCAAAAAGAGGATTgacAAGAAGAGAGCC-3′ | ||
pRR1060 | Gln3S355A | 5′-CGGAACAACAGATCTGGATGAAGATTTACTGGAACTTG-3′ |
5′-CATGGTACCATGAGGCCATTATCCTTAAAATCGGACGTTATCAAAAAGAGGATTgcaAAGAAGAGAGCC-3′ | ||
pRR1293 | Gln3R264A,K265A,S267A | 5′-CAATCGGCCGGATAAGtgcGTTtgctgcTACAGAATTTGTTGTG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR1295 | Gln3K281A,K282A,S285A | 5′-CAATCGGCCGATGTCGTCCACGTCCCTGGCCAATTTCgcagcaGCTGCCgcaGTATCTTCCAGTATATC-3′ |
5′-CGCGGATCC TATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAATCGGCCGGATAAGTGAGTTTTTTCTTACAGAATTTGTTG-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRR1345 | Gln3R572D,R573D,K574D | 5′-CATCCAGATCTGTTGTTCCGAGATATTACCAAAACC-3′ |
5′-GTACGATGCATTGCGCGACATgtcgtcatcTGGTATATTCATTCCTTGAC-3′ | ||
pRR1366 | Gln3S249D,S251D,S256D,S257D | 5′-CAGGACTAGTGGACGACATTGGCTTGATAAGTGAGTTTTTTCTTACAGAATTTGTTGTGTTgtcgtcAGATAATCCTATgtcATCgtcCTTCTTCAGAAAAG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAGGACTAGTCTGGCCAATTTCAAAAGAGCTGCCTCAG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR1368 | Gln3S267D,S273D,S274D,T275D,S276D | 5′-CAGGACTAGTgtcgtcCATTGGCTTGATAAGgtcGTTTTTTCTTACAG-3′ |
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
5′-CAGGACTAGTCTGGCCAATTTCAAAAGAGCTGCCTCAG-3′ | ||
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
pRR1370 | Gln3S285D,S287D,S288D,S289D | 5′-CAGGACTAGTCTGGCCAATTTCAAAAGAGCTGCCgacGTAgacgacgacATATCCAATATGGAACC-3′ |
5′-CGCGGATCCTATACCAAATTTTAACCAATCCAATTCGTCAGCAATTGCT-3′ | ||
5′-CAGGACTAGTGGACGACATTGGCTTGATAAGTGAG-3′ | ||
5′-ATCCCCGCGGGACGTCAACTCCATAGAAGTGACTTTTCCG-3′ | ||
pRS316 | Vector | |
pRR482 | Kulkarni et al. (2001) | |
pKA10 | Kulkarni et al. (2001) | |
pKA14 | Kulkarni et al. (2001) | |
pKA36 | Kulkarni et al. (2001) | |
pKA38 | Kulkarni et al. (2001) | |
pKA53 | Kulkarni et al. (2001) |
Bioinformatic analyses
S. cerevisiaeGln3 sequence YER040W was from the Saccharomyces Genome Database. Sequences for S. paradoxus (AABY01000062.1), S. mikatae (AABZ01000001.1), S. kudriavzevii (AACI02000852.1), S. bayanus (AACG02000070.1), S. pastorianus (ABPO01000005.1), S. (AACF01000032.1), Vanderwaltozyma polyspora (XM001643411.1), and Kluyveromyces waltii (AADM01000165.1) were from Whole Genome Shotgun (WGS) section of NCBI. The sequence for Kluyveromyces thermotolerans (XM_002555052.1) was from GenBank. These sequences were found by Blast searching these databases, beginning with yeast GLN3. Bioinformatic analyses were performed using available web-based programs employing default parameters. (Kosugi et al. 2009; Nguyen Ba et al. 2009; Sigrist et al. 2010). Predicted disorder of the Gln3 protein was derived from Ishida and Kinoshita (2007).
Gln3-Myc13 and EGFP-Gln3 localization
Cell collection and Gln3-Myc13 visualization by indirect immunofluorescence microscopy were performed as described (Feller et al. 2013). Intracellular EGFP-Gln3 peptide localization was determined and scored as described in Tate et al. (2010, 2015). As described earlier, only two- category, rather than three-category, scoring is possible when EGFP is used as the fluorescent tag.
Image processing
Microscopic images for presentation were prepared using Adobe Photoshop and Illustrator programs. Level settings (shadow and highlight only) were altered where necessary to avoid any change or loss in cellular detail relative to that observed in the microscope; changes were applied uniformly to the image presented, and were similar from one image to another. Midtone, gamma settings were never altered. These processed images were used for illustrative presentation only, NOT for scoring Gln3-Myc13 intracellular distributions.
Determination of intracellular Gln3-Myc13 localization
Gln3-Myc13 intracellular localization was manually scored in 200 or more cells for each data point. Unaltered, primary .zvi image files viewed with Zeiss AxioVision 3.0 and 4.8.1 software were exclusively used for scoring purposes. Cells were classified into one of three categories: cytoplasmic (cytoplasmic fluorescent material only, red histogram bars), nuclear-cytoplasmic (fluorescent material appears in both the cytoplasm and colocalizing with DAPI-positive material, DNA, yellow bars), or nuclear (fluorescent material colocalizing only with DAPI-positive material, green bars). Representative “standard” images and detailed descriptions of these categories appear in Figure 2 of Tate et al. (2009). The precision of our scoring has been repeatedly documented (Tate et al. 2006, 2010; Rai et al. 2013, 2014). In the present work, the SD of the data, with one exception (ammonia, 13%), was ∼8% for N = 9–11 independent experiments performed over 10 years (Figure 1). In most cases, similar levels of precision were obtained with the mutants. In all cases, the greatest variation was observed when Gln3-Myc13 was significantly localized to more than one cellular compartment. Experiment-to-experiment variation can also be assessed by comparing data obtained with wild type pRR536 in transformants cultured in glutamine, glutamine plus rapamycin, proline, ammonia, and ammonia plus Msx since a separate wild-type culture accompanied each of the mutants.
Images accompanying the histograms were chosen on the basis that they exhibited intracellular Gln3-Myc13 distributions as close as possible to those observed by quantitative scoring. However, identifying a field that precisely reflected the more quantitative scoring data were sometimes difficult unless the tagged protein was situated in a single cellular compartment.
Data availability
Following publication, strains and plasmids will be provided upon request, but only for noncommercial purposes. Commercial and commercial-development uses are prohibited. Materials provided may not be transferred to a third party without written consent. This will be done in accordance with NIH guidelines. The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.
Results
Bioinformatic identification of Gln3 sequences homologous to monopartite and bipartite NLSs
Current and previous bioinformatic NLS analyses identified four potential “NLS” candidates in Gln3: Gln3388–394 (previously designated NLS1), Gln3571–577 (previously designated as NLS2), Gln3353–361 (previously designated the K/R-rich region, Gln3351–361), and Gln3279–307 contained within a previously uninvestigated region, Gln3247–307 (designated the S/R region in this work) (Figure 2D) (Kosugi et al. 2009; Nguyen Ba et al. 2009; Sigrist et al. 2010, default parameters; Carvalho and Zheng 2003). The K/R-rich sequence, Gln3351–360, was predicted to be homologous to monopartite NLSs by two programs Gln3353–359 (Kosugi et al. 2009; Nguyen Ba et al. 2009), or to a bipartite NLS in one (Gln3333–359) (Kosugi et al. 2009). Additionally, a sequence homologous to Gln3318–359 was reported to function as a bipartite NLS in the Aspergillus AreA protein (Hunter et al. 2014). All of these regions are highly conserved among the Saccharomyces species (Figure 2, A–D).
Interestingly, the previously reported NLS1 did not appear in the current search results, whereas the K/R-rich and Gln3279–307 sequences were not identified in previously reported work (Carvalho and Zheng 2003; Sigrist et al. 2010). In short, sequences predicted to be homologous to NLS elements depend on the programs employed in the search. Therefore, we sought to determine whether any or all of these sequences were required for nuclear Gln3-Myc13 localization, and, if so, whether they were minor or major contributors to the process.
NLS1 can be abolished without significant loss of nuclear Gln3-Myc13 localization
We initially determined the extent to which nuclear Gln3-Myc13 was abolished when NLS1 was deleted as described earlier (Carvalho and Zheng 2003). When we constructed the deletion mutant used in that work, (gln3388–395Δ, pRR723), its localization profile was largely wild type (pRR536) (Figure 3, A–C. pRR536 and pRR723). The only detectable defect was a modest, overall decrease in nuclear Gln3-Myc13 localization in minimal-proline medium. Further, when we substituted aspartates for the canonical Gln3 residues critical to NLS1 function (pRR792, Gln3P388D,R392D,K393D), a similar phenotype was observed (Figure 3, A–C, pRR792). These results were unexpected, and indicated Gln3 likely possessed one or more additional sequences able to support its nuclear localization.
NLS2 resides in a Gln3 domain required for a response to rapamycin treatment
We next investigated the participation of previously reported NLS2 in nuclear Gln3-Myc13 localization (Carvalho and Zheng 2003). Since Gln3S576D and Gln3S576A substitutions in NLS2 exhibited wild type phenotypes (Figure 8, pRR1034 and pRR1037 in Rai et al. 2014), we queried whether more drastic substitutions of aspartates for the basic residues in NLS2 (Gln3R572D,R573D,K574D, pRR1345), would have a greater effect (Figure 2B). Indeed they did, abolishing the rapamycin response (Figure 3A, pRR1345). Additionally, in proline medium, the intracellular distribution of Gln3-Myc13 markedly shifted toward a more cytoplasmic localization, now exhibiting a tripartite distribution (Figure 3B). This tripartite Gln3 distribution is repeatedly observed when the domain required for a Gln3 response to rapamycin or the Gln3-Tor1 interaction domain is altered (Rai et al. 2013, 2014, 2015, 2016). Although the loss of rapamycin-elicited nuclear Gln3-Myc13 localization was convincing in the substitution mutant (pRR1345, Gln3R572D,R573D,K574D), one cannot rigorously conclude that the phenotype derived directly from the loss of NLS function because these substitutions are in the middle of a large domain whose integrity was previously demonstrated to be required for a response to rapamycin (Rai et al. 2014). In fact, the overall phenotypic profile of pRR1345 was identical to multiple rapamycin-response domain mutants situated some distance away from the putative NLS2 (Rai et al. 2014). Based on this reasoning alone, one cannot exclude that NLS2 performs a significant function in the nuclear import process, but subsequent data will show that its loss does not diminish the ability of Gln3 to be transported into the nucleus.
K/R-rich Gln3351–360 is required for nuclear Gln3-Myc13 localization
Literature investigating the third candidate sequence was controversial. The initial report concluding that the K/R-rich region (Gln3344–365) was required for nuclear Gln3 localization employed an EGFP-Gln3344–493 peptide (Kulkarni et al. 2001). This construct lacked the Gln3-Ure2 interaction site (Gln3102–150). A later report, based on data employing a full length Gln3-Myc9 construct containing a Gln3351–361 deletion concluded the K/R-rich region was not essential for nuclear Gln3-Myc9 localization (Carvalho and Zheng 2003). That deletion mutant, however, was resistant to rapamycin treatment—a characteristic that occurs when Gln3 activity or nuclear localization are lost. Both reports presented isolated images to arrive at their conclusions.
Therefore, we constructed three substitution mutants in the K/R-rich region of full-length Gln3-Myc13. We first substituted acidic aspartate residues for the basic ones, Gln3K352D,R353D,K356D,K357D (pRR789) and Gln3S355D (pRR1059) (Figure 3, A–C). We reasoned that such drastic changes would most likely destroy function. We then made a more conservative alanine substitution Gln3S355A (pRR1060).
With the aspartate substitutions, nuclear Gln3-Myc13 localization was greatly reduced in rapamycin-treated, and in proline- or ammonia-grown cells (Figure 3, pRR789 and pRR1059). The more drastic substitutions in pRR789 yielded a stronger phenotype in proline-grown transformants, but the lost responses in rapamycin-treated cells were similar, irrespective of the substitution assayed. With the alanine substitution (pRR1060), the response to rapamycin was similar to that of the aspartate substitutions, whereas the proline-grown transformants exhibited a phenotype that was about half way between that of the analogous aspartate substitution (pRR1059) and wild type (pRR536). The fact that both aspartate and alanine substitutions in pRR1059 and pRR1060 adversely affected nuclear Gln3-Myc13 localization in much the same way suggested that the phenotypes likely derived from structural rather than post-translational modifications. NLS1 and NLS2 sequences were wild type in all three of these mutants, and, hence, would a priori have been expected to support nuclear Gln3-Myc13 entry. The fact that they did not supported the idea that nuclear Gln3 entry was more complex than previously appreciated.
The effects of these, and subsequent, substitutions in ammonia-grown cells treated with Msx completed our standard analysis of mutant phenotypes (Figure 3C). In nearly all cases, Msx-treatment elicited high nuclear Gln3-Myc13 localization. However, this high level nuclear localization must be viewed cautiously. Msx starves the cell for glutamine, necessitating Gln3 to bind to its target promoter sequences in order to exit from the nucleus rather than exiting directly without the need for DNA interaction (Rai et al. 2014). Gln3 no longer rapidly cycles in and out of the nucleus as occurs normally. In the presence of Msx, export is greatly slowed to the point that import becomes almost kinetically irreversible. Therefore, any Gln3 imported into the nucleus piles up there, even in cells cultured under highly repressive conditions (with asparagine as sole nitrogen source) where nuclear Gln3-Myc13 import is minimal (see Figure 11C in Rai et al. 2015). In other words, Msx treatment elicits results very similar to those observed when nuclear Gln3 export is abolished by alteration of its NES (Rai et al. 2015). As a result, any decrease in the level of nuclear Gln3-Myc13 localization in Msx-treated cells is indicative of exceedingly low import. Stated in another way, even when Gln3 NLS activity is low, Gln3 will still accumulate in the nucleus due to the low level of export.
Nuclear Gln3 import in truncations eliminating NLS1 and NLS2
If multiple sequences participate in nuclear Gln3-Myc13 entry, it can be difficult to confidently conclude which are, and/or are not, actually participating in the process. Such was the case with NLS1, NLS2, and the K/R-rich region. Framed in another way, to what extent could the K/R-rich region alone support nuclear Gln3-Myc13 localization? To address this question, we hypothesized that it might be possible to construct functional C-terminal Gln3 truncations that retained Gln3 activity but eliminated potential NLS1 and NLS2 participation from consideration. Supporting the feasibility of this approach, we previously demonstrated that a Gln3600–730 C-terminal truncation complemented a gln3Δ (Rai et al. 2013). Therefore, we constructed truncations spanning Gln3 from its C-terminal residue, 730, to residue 350 and followed Gln3-Myc13 localization in each of them.
Gln3 truncations from Gln3730 to residues 542, 496, 486, and 476, which retain NLS1 and the K/R-rich region but lack NLS2, the Gln3-Tor1 interaction, rapamycin response, and cytoplasmic sequestration domains, resulted in moderately decreased cytoplasmic Gln3-Myc13 sequestration, and complete loss of a Gln3-Myc13 response to rapamycin addition (Figure 4, pRR613, pRR609, pRR624, and pRR610). Nuclear Gln3-Myc13 localization in derepressive proline medium, however, remained wild type, as occurs when any of these domains are individually abolished by either amino acid substitutions or deletion (Rai et al. 2013, 2014, 2016). Further, these results suggested that NLS2 (Gln3568–581) did not strongly contribute to nuclear Gln3-Myc13 localization in derepressive medium.
Gln31–400 (pRR612), retains NLS1 and the K/R-rich region, whereas Gln31–384 (pRR611) additionally lacks NLS1. Both truncations yielded phenotypes largely similar to those of the earlier truncations, except that the percentage of cells with nuclear Gln3-Myc13 increased in glutamine-grown cells both in the absence or presence of rapamycin (Figure 4). With derepressive proline as nitrogen source, Gln3-Myc13 was highly nuclear irrespective of whether or not NLS1 was present. Importantly, Gln31–384 (pRR611) lacked both NLS1 and NLS2, indicating that, even when they were absent, Gln3-Myc13 was still capable of entering the nucleus, thus confirming the existence of previously unrecognized sequences supporting nuclear Gln3 entry. In sharp contrast with above results, truncation of 34 additional residues containing the K/R-rich sequence (Gln31–350), almost completely abolished nuclear Gln3-Myc13 localization in both rapamycin-treated and proline-grown cells (Figure 4, pRR620).
The localization profile observed with Gln31–384 (pRR612) could be interpreted in two ways because it contained not only Gln3350–361, but also Gln3333–359 predicted to be homologous to mono- and bipartite NLS elements. To assess whether or not to consider the possibility of Gln3350–361 functioning as a mono-partite NLS, we assayed two truncations (EGFP-Gln31–365, EGFP-Gln31–301) and plasmids encoding EGFP-Gln3 peptides (Gln3344–493, Gln3384–493) transformed into wild-type cells. Transformants were then grown in YNB-raffinose-proline medium, because expression of the mutant gln3 constructs was under the control of a galactose-inducible promoter (Figure 5). EGFP-Gln3 was nuclear in EGFP-Gln31–730 and EGFP-Gln31–365, both of which contained the K/R-rich sequence (Figure 5B, quantitation of images presented in Figure 5A). However, truncation to residue 301, thereby eliminating the K/R-rich sequence, abolished nuclear EGFP-Gln3 localization. A EGFP-Gln3344–493 (pKA36) peptide also supported nuclear EGFP-Gln3 localization. In contrast, a EGFP-Gln3384–493 (pKA38) peptide, which eliminated the K/R-rich region, was no longer able to support nuclear localization. This was true even though pKA38 contained NLS1. To further test this result, we substituted glutamate for the basic residues in the K/R-rich region of Gln3344–493 (pKA53). Again, nuclear Gln3 localization was nearly abolished, indicating that the K/R-rich sequence was required for the peptide’s nuclear localization.
Gln31-384 but not Gln1–350 is sufficient to support growth in minimal medium
While most of the regulatory domains are eliminated in the Gln31–384 construct (pRR611), it still retained those conceptually necessary for Gln3 function, i.e., Gln3 activation, DNA-binding domains, and K/R-rich sequence shown to be required for nuclear Gln3-Myc13 localization. This in turn suggested that these constructs might be able to complement a gln3Δ, thus affording another way of testing whether Gln3 could enter the nucleus in the absence of NLS1 and NLS2.
We tested this hypothesis by transforming gln3Δ strain KHC2 with several of the above plasmids (Rai et al. 2014). Each transformant was streaked on the same plate as a transformant containing wild-type Gln3 in pRR536 for purposes of direct comparison. All of the truncations, up to and including Gln31–384 (pRR611) and pRR723 (NLS1 deletion), were able to grow in YNB-ammonia medium as well as their wild-type partners (Figure 6). However, there was no detectable complementation when the gln3Δ was transformed with the empty vector (pRS316)—a zinc finger mutant that no longer binds to DNA (pRR787) (Rai et al. 2015)—or the Gln31–350 truncation (pRR620). Together, these data argued that Gln31–384 retained demonstrable Gln3 function, and, hence, an ability to enter the nucleus even though both NLS1 and NLS2 were absent. Further, the inability of pRR620 (Gln31–350) to complement a gln3Δ correlated with the loss of the K/R-rich sequence (Gln3351–360), positively correlating with the Gln3-Myc13 localization results shown in Figure 4.
NLS homology in a region of Gln3 containing multiple basic residue repeats
Although the above data demonstrated that K/R-rich Gln3351–360 exhibited NLS activity, an important caveat remained. If two or more regions within Gln31–384 [at least one of them N-terminal to Gln3350] participated in nuclear Gln3-Myc13 localization, they would have escaped identification. Supporting this possibility was the fact that bioinformatic analyses of Gln3 had identified a fourth sequence (Gln3279–307) with predicted homology to bipartite NLSs (Kosugi et al. 2009). That sequence or variations of it appeared multiple times in the bioinformatic analysis indicating an increased probability that significant NLS homology might exist (Kosugi et al. 2009).
Three additional characteristics, however, beyond the possible bioinformatic relationship of Gln3279–307 to a bipartite NLS, piqued our interest in the Gln3247–307 sequence. We designated this sequence the S/R region (Figure 2E). (i) It was more highly conserved among Saccharomyces species than most other Gln3 sequences, save the Gln3 zinc-finger and DNA-binding domains. (ii) It contained four pairs of basic residues (KK/KR repeats) equally spaced 15, 15, and 17 residues apart. (iii) It was highly enriched in serine and threonine residues that could potentially be phosphorylated (19 of 55 in Gln3247–307), and hydrophobic residues that could potentially participate in intramolecular or intermolecular interactions (22 of 55). Therefore, the S/R region (Gln3247–307) merited closer investigation.
S/R region Gln3247–307 is required for nuclear Gln3-Myc13 localization
Our first look at the S/R region (Gln3247–307) was a large deletion eliminating all but the last pair of lysine residues (Figure 7, Gln3244–296Δ, pRR656). The last pair of lysines were spared because of their proximity to the DNA-binding zinc-finger, just four residues away. This was followed by a much more conservative elimination of residues Gln3262–271Δ (pRR725).
Nuclear Gln3-Myc13 localization in response to rapamycin-treatment or in ammonia-grown cells was completely abolished irrespective of the deletion length. Both constructs also exhibited a substantial loss of Gln3-Myc13 nuclear localization in more derepressive, proline or ammonia medium (Figure 7 A–C). In contrast, the two deletions yielded significantly different phenotypes in Msx-treated cells. In the more extensive deletion (pRR656) the response to Msx-treatment was substantially reduced, indicating that nuclear Gln3-Myc13 import was decreased beyond the point where inhibition of nuclear export that occurs in Msx-treated cells could mask it (see earlier discussion of Msx effect). In contrast, nuclear Gln3-Myc13 localization was recovered to wild-type levels in the shorter deletion (pRR725). It is important to note that the larger deletion (pRR656, Gln3244–296Δ) extended into residues predicted to be homologous to a bipartite NLS, Gln3279–307, whereas the smaller one, Gln3262–271Δ, was outside of sequences homologous to the predicted bipartite NLS (Gln3279–307).
Parsing the S/R region to further localize the sequence potentially acting as an NLS
Recognizing that results with deletions—especially large ones—can at times be misleading, we decided to more closely define the sequences required for nuclear Gln3-Myc13 localization. To this end, we substituted aspartate for the N-terminal most pair of basic residues covered by the large deletion in pRR656, (Gln3K247D,K248D,S251D, pRR770). This construct failed to yield a detectable protein, perhaps suggesting that the mutant Gln3 protein might not have survived modification. However, analogous alanine substitutions, Gln3K247A,K248A,D251A (pRR1292), yielded a wild-type phenotype (data not shown).
We experienced greater success with the second pair of basic residues, and an adjacent serine (Gln3R264D,K265D,S267D, pRR754; Gln3R264A,K265A,S267A, pRR1293). The lysine/serine to aspartate substitutions abolished nuclear Gln3-Myc13 localization in response to rapamycin addition, as well as that observed in ammonia- or proline-grown cells, even though these substitutions were well upstream of the sequence with homology to a bipartite NLS (Gln3279–307) (Figure 8, A–C, pRR754). The Msx response, however, was largely unaffected. Parallel alanine substitutions (pRR1293) yielded responses similar to those with pRR754 in all but proline medium where the tripartite distribution of Gln3-Myc13 was observed (Figure 8, pRR1293). These observations supported the contention that the N-terminal portion of the S/R region was important for nuclear Gln3-Myc13 localization, but was unlikely to be participating as an NLS.
Substitutions in the third pair of basic residues, (Figure 8, A–C, Gln3K281D,R282D,S285D, pRR772; Gln3K281A,R282A,S285A, pRR1295) yielded responses nearly identical to those with pRR754 and pRR1293, respectively. However, just as observed with the large deletion (pRR656), the Msx response with pRR772 was substantially decreased, indicating that nuclear Gln3-Myc13 import was slowed to the point that it could not be masked by the effects of Msx on nuclear export. It is pertinent that substitutions in pRR772 abolished the first pair of basic residues of the predicted bipartite NLS (Gln3279–307). We again did not substitute the last pair of basic residues in the S/R region because of their proximity to the zinc-finger motif. Alterations in the zinc-finger DNA-binding domain alters nuclear Gln3-Myc13 localization by an intranuclear mechanism (Rai et al. 2015).
Epistasis relationships between a ure2Δ and gln3 alterations in the K/R-rich and S/R regions
To further test the above conclusion, we asked whether the gln3 mutations in pRR620 and pRR789 (alterations in the K/R-rich region) or pRR754 and pRR772 (alterations in the S/R region) were epistatic to a ure2Δ? Such epistasis would be the expected outcome if one or more of the residues substituted in those mutants directly participated in the nuclear localization process itself, downstream of Ure2 function. Alternatively, if the ure2Δ was epistatic to the gln3 mutations contained in the plasmids, it would suggest that Gln3-Myc13 release from Ure2-mediated cytoplasmic sequestration was somehow damaged in the mutants rather than loss of nuclear import itself. Recall that gln3 loss of function mutations are epistatic to a ure2Δ (Courchesne and Magasanik 1988).
We first transformed the ure2Δ with pRR789, which is a full length Gln3-Myc13 construct with substitutions abolishing the K/R-rich NLS and pRR620 (Gln31–350), lacking the K/R-rich NLS and all residues C-terminal of it. With glutamine as nitrogen source, mutations in pRR789 were partially epistatic to those in the ure2Δ (Figure 9, A and B, pRR789), an expected result if the K/R-rich region acts downstream of Ure2. In contrast, the ure2Δ could be considered substantially epistatic to mutations in the pRR620 construct (Figure 9, A and B, pRR620), leading one to legitimately ask why deleting vs. substituting residues in the K/R-rich NLS did not yield the same results? We speculate that it is because the pRR620 truncation additionally removed the Gln3 regulatory domains, i.e., the cytoplasmic sequestration, rapamycin response and Gln3-Tor1 interaction. The cytoplasmic sequestration and Gln3-Tor1 interaction domains are required to downregulate nuclear Gln3 localization in rich nitrogen sources. All of these domains are wild type in the pRR789 construct.
Most important, Gln31–350 (pRR620) was able to enter the nucleus of the ure2Δ in the absence of the NLS1, NLS2, and K/R-rich NLS candidates when there was a nutrient-associated signal to do so, i.e., with ammonia- or proline-grown cells, or those treated with rapamycin (Figure 9, A and B). Thus Gln31–350 must contain an NLS element. Here, it is pertinent to point out a negative correlation. There are only seven isolated basic residues N-terminal of the S/R region, residues 9, 23, 35, 91, 93, 179, and 217, whereas NLS motifs require multiple basic residues in close proximity to one another. Therefore, by exclusion, one may reasonably conclude that a sequence required for nuclear Gln3-Myc13 import was situated between Gln3247 and Gln3350. Also potentially pertinent, the bioinformatics programs failed to identify any NLS homologous sequences in Gln3118–278, leading us to conclude that the Gln3 sequence predicted to be a bipartite NLS was situated between Gln3279–350. We emphasize, however, that nuclear Gln3-Myc13 localization was only obtained (pRR789) in the absence of Ure2 and the presence of a specific signal for nuclear localization, i.e., rapamycin treatment or nitrogen sources requiring NCR-dependent transcription. In wild-type cells, Gln31–350 (pRR620) was largely cytoplasmic.
We next transformed wild-type and ure2Δ recipients with pRR754 or pRR772, containing alterations in the S/R region. The ure2Δ was epistatic to alterations carried in the N-terminal portion of the S/R region, i.e., pRR754 (Gln3R264D,K265D,S267D), in glutamine-grown cells. Gln3-Myc13 was highly nuclear (Figure 9, C and D, pRR754). This argued that the N-terminal portion of the S/R region likely functioned upstream rather than downstream of Ure2 and was not a good NLS candidate.
In contrast, alterations in the C-terminal portion of the S/R region (pRR772, Gln3K281D,R282D,S285D) were partially epistatic to a ure2Δ, indicating that the gln3 alterations in that plasmid adversely affected processes downstream of Ure2. Thus substitutions exhibiting the greatest epistasis to the ure2Δ were in those sequences bioinformatically identified as NLS candidates, Gln3279–307 and Gln3351–360. Further, the loss of nuclear EGFP-Gln31–301 (pKA10, Figure 5, A and B) localization indicated that sequences beyond Gln3301 were required for it, which correlated with the bioinformatics prediction that Gln3279–307 were homologous to a bipartite NLS. Finally, the ability of pRR620, in which the K/R-rich region of Gln3 was eliminated (Gln3351–360) also eliminated the possibility that the K/R-rich region was participating in Gln3 localization as a bipartite NLS.
The N-terminal portion of the S/R region may serve a regulatory role in nuclear Gln3 localization
The above data show that Gln3247–282, was necessary for nuclear Gln3-Myc13 localization, but was doubtfully participating in the process as an NLS. This reasoning raised the possibility that the N-terminal portion of the S/R region might be functioning in a regulatory role. In addition, the high concentration of serine and threonine residues, over one-third of the total in the S/R region, raised the possibility that their phosphorylation might influence intracellular Gln3 distribution.
To test this possibility, we substituted phosphomimetic aspartate for Gln3 serine/threonine residues, 255–257, 273–276, 287–289, and 291 (pRR826) (Figure 10, Top). Responses to rapamycin-treatment or growth in ammonia or proline media were abolished, whereas the response following Msx-treatment was not demonstrably affected (Figure 10, A–C, pRR826). In contrast, substituting alanine, which cannot be phosphorylated, for these residues, had little, if any, demonstrable effect irrespective of whether nuclear Gln3-Myc13 localization was triggered by treating the cells with rapamycin or growing them in derepressive, proline medium. The mutant responses were nearly indistinguishable from wild type (Figure 10, A–C, pRR823).
To refine the analysis, we substituted aspartate for serine residues in the N-terminal half of this region, Gln3S249D,S251D,S262D,S267D (pRR835) (Figure 10, A–C). Responses to rapamycin and growth in ammonia medium were again abolished. The response to growth in proline medium, however, was only decreased to a tripartite intracellular Gln3-Myc13 distribution characteristic of mutants where one of the major Gln3 regulatory systems is abolished. In contrast, analogous alanine substitutions (Gln3S249A,S251A,S262A,S267A, pRR834) again yielded largely wild type intracellular Gln3-Myc13 distribution profiles (Figure 10, pRR834).
Finally, we constructed aspartate substitutions for Ser/Thr residues contained between individual KK/RK/KR/KK elements of the S/R region: Gln3S249D,S251D,S256D,S257D (pRR1366); Gln3S267D,S273D,S274D,T275D,S276D (pRR1368), and Gln3S285D,S287D,S288D,S289D (pRR1370) (Figure 11, Top). At this resolution, substitutions between the successive repeats responded differently. Phosphomimetic aspartate substitutions between the most N-terminal two KK/RK repeats abolished nuclear Gln3-Myc13 in response to rapamycin-treatment or growth in ammonia medium, and nearly abolished it in proline-grown cells (Figure 11, A–C, pRR1366). Substitutions between the second and third RK/KR repeats (pRR1368) generated a Gln3-Myc13 intracellular distribution profile nearly indistinguishable from transformants containing pRR1366 (Figure 11, pRR1366 and pRR1368).
In contrast, serine to aspartate substitutions between the third and fourth KR/KK repeats, the region with homology to a bipartite NLS, yielded a largely wild type Gln3-Myc13 distribution profile (Figure 11, A–C, pRR1370). These data were consistent with the idea that if serine residues situated in the S/R region (Gln3247–282) were phosphorylated, Gln3-Myc13 would be unable to overcome cytoplasmic sequestration by Ure2, in spite of the fact that all of the sequences C-terminal of Gln3276, including the Gln3279–307, K/R-rich (Gln3351–360), NLS1, and NLS2, and regulatory regions, were intact.
To assess whether these serine to aspartate substitutions were interfering with nuclear Gln3-Myc13 import itself, we transformed the ure2Δ with the multiple phosphomimetic ser/thr to asp mutant gln3 plasmid pRR826 (Figure 10, Top). The ure2Δ was as epistatic to the gln3 mutations in pRR826 as it was to wild-type GLN3, indicating that nuclear Gln3-Myc13 import remained intact as long as Ure2 failed to function (Figure 12, A and B).
Discussion
The experiments in this work demonstrate that nuclear Gln3-Myc13 localization is significantly more complicated than previously appreciated. We identified three Gln3 sequences required for nuclear import of full-length Gln3-Myc13, whether triggered by rapamycin addition or growth under derepressive conditions. This trigger-independence suggests that the overall relief of negative Ure2 regulation and import processes themselves are affected by the substitutions we analyzed rather than regulation associated with responses to distinct physiological situations, as occurs with substitutions in the C-terminal regions of Gln3 (Rai et al. 2013, 2014, 2016).
The first two sequences [Gln3351–360 (previously designated the Gln3351–361 K/R-rich region), and Gln3279–307] are predicted to be homologous to monopartite and bipartite NLSs, respectively (Figure 13). They are both strongly required for nuclear Gln3-Myc13 localization in full-length Gln3 even though NLS1 and NLS2 sequences remain intact. Especially important, Gln31–350, containing Gln3279–307, supports nuclear Gln3 localization in the absence of all other reported and predicted NLS sequences. As expected for processes downstream of Ure2, mutations resulting in alterations of the monopartite and bipartite NLS-homologous sequences were each partially epistatic to a ure2Δ. Thus, both sequences have the predicted structures and demonstrated functions of NLSs. While both sequences are required to be intact in otherwise wild-type cells, either of them will function alone when Ure2 is abolished.
The third sequence, the Ure2 relief sequence (Gln3247–282), is necessary for nuclear Gln3-Myc13 localization, but only in the presence of functional Ure2 (Figure 13, top). Both aspartate and alanine substitutions of basic residues in this sequence abolish or diminish the ability of Gln3 to relocate to the nucleus, leading us to conclude that they represent general loss of function alterations. On the other hand, wild-type Gln3 responses to rapamycin treatment and derepressive growth conditions are abolished by ser > asp but not ser > ala substitutions, leading us to speculate that phosphorylation of the serines between the paired basic residues of the Ure2 relief sequence may be important for Ure2’s negative regulation of Gln3.
We speculate that the putatively phosphorylated form of Gln3247–282 participates with Ure2 to inhibit Gln3’s ability to relocate to the nucleus by preventing the overall operation of the Gln3279–307 and Gln3351–360 NLS sequences. By this reasoning, the putatively unphosphorylated form of the Ure2 relief sequence is prerequisite for Gln3-Myc13 to overcome negative Ure2 regulation, but without influencing Gln3’s specific responses to rapamycin or limiting nitrogen. A corollary of this reasoning is that Gln3 responses to distinct nitrogen environments consist of multiple independent, genetically separable steps, rather than a single concerted event such as rapamycin-elicited Sit4 dephosphorylation of Gln3.
K/R-rich region
Conclusions derived from data with Gln3 K/R-rich, Gln3351–360 substitutions require additional comment. First, there is a clear difference between results reported here and in an earlier work (Kulkarni et al. 2001) vs. those of a report indicating that NLS1 was the predominant Gln3 NLS (Carvalho and Zheng 2003). Since that earlier report was based on quality data, we can only conclude that observed disparities derive from differences in the constructs employed, position of the protein tags and/or the methods used to evaluate Gln3 localization in the three reports. A significant difference in evaluation methods was the use of microscopic images containing only a few cells in both of the early reports compared with the present use of semiquantitative scoring methods involving over 200 cells per data point. Second, present data do not eliminate the possibility that K/R-rich Gln3351–360 functions as both a mono-partite NLS as well as the C-terminal half of a bipartite NLS (Gln3333–359), each supporting nuclear Gln3 localization. Effectively addressing this possibility will require an ability to unambiguously parse the effects of a possible bipartite NLS from simultaneous destruction of the Gln3 GATA DNA binding domain.
S/R region structure/function
Layout of the Gln3 S/R region is intriguing, and may offer some insight into how it potentially functions. The overall S/R sequence (Gln3247–307) structure is KK-X15-RK-X15-KR-X17-KK, with the sequences between the first three basic residue repeats rich in serine and hydrophobic residues. Additionally, the C-terminal portion of the S/R region, the Gln3247–282Ure2 Relief Sequence, partially overlaps the predicted bipartite NLS, Gln3279–307.
That the N-terminal portion of the S/R region performs a second function is concluded from the fact that structurally modifying Gln3247–267, well outside of the region predicted to be the bipartite NLS, resulted in a Ure2-dependent loss of Gln3’s ability to be imported into the nucleus. Elimination of Ure2 concomitantly abolished a need of the Ure2 relief sequence for nuclear Gln3-Myc13 localization, leading us to suggest that the relief sequence functions in facilitating or overcoming the negative regulation of Gln3 by Ure2 rather than directly participating downstream of it in the actual nuclear import process.
Gln3 regulation is mediated by its disordered regions
A pertinent question is justifiably posed with respect to the data presented in present and past experiments investigating Gln3 regulation. Why was it possible to so cleanly identify nitrogen-responsive targets on the Gln3 molecule? Stated another way, why did substitutions in one region of Gln3 fail to telegraph their effects to the rest of the molecule, as would a priori be expected of a globular protein?
We suggest the answer to those questions derives from the unique structure of this transcription activator. Gln3 is predicted to be very highly disordered (Figure 13). The major functional domains of the protein, i.e., zinc-finger DNA-binding, transcriptional activation and Ure2 interaction sites are situated as small structured islands of the protein. These islands are dispersed in the remaining portions of the protein predicted to be highly disordered. There are, however, short sequences in these disordered regions that are predicted to fold into α-helices (Rai et al. 2013, 2014, 2015). It is reasonable to suggest that these predicted helices form the nucleation points for interaction with the various nitrogen-influenced regulatory proteins responsible for the Gln3’s responses to signals associated with each of the five physiological nitrogen-related conditions. We speculate that once initial interaction occurs, the disordered Gln3 regions become more ordered. Consistent with this view, Gln3 is extraordinarily sensitive to proteolysis in vitro (Rai et al. 2015). In sum, the data presented expand our understanding of the structure-function relationships and complexity of Gln3 control and set the stage for biochemical studies focused on the Gln3-Ure2 interaction and its regulation.
Acknowledgments
The authors thank the University of Tennessee Molecular Resource Center Sequencing Core and Dr. Thomas Cunningham who performed all of the DNA sequencing associated with this work. We also appreciate plasmids and assistance from Drs. Ajit Kulkarni and David Nelson. Supported by National Institutes of Health grant GM-35642-27.
Footnotes
Communicating editor: A. Mitchell
Literature Cited
Author notes
These authors contributed equally to this work.