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. 2018 Jan;208(1):207-227.
doi: 10.1534/genetics.117.300457. Epub 2017 Nov 7.

More Than One Way In: Three Gln3 Sequences Required To Relieve Negative Ure2 Regulation and Support Nuclear Gln3 Import in Saccharomyces cerevisiae

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Free PMC article

More Than One Way In: Three Gln3 Sequences Required To Relieve Negative Ure2 Regulation and Support Nuclear Gln3 Import in Saccharomyces cerevisiae

Jennifer J Tate et al. Genetics. .
Free PMC article

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.

Keywords: Gln3; NLS; TorC1; Ure2; methionine sulfoximine; nitrogen catabolite repression; nuclear localization sequence; rapamycin.

Figures

Figure 1
Figure 1
Precision of the assay data presented in this work. The histograms depict the means of the 9–11 independent experiments in this work conducted over a 10 year period, measuring Gln31–730-Myc13 localization in wild type pRR536 transformants of JK9-3da. Error bars represent SD around the means. Cells were grown in YNB-glutamine (Gln), -ammonia (Am.), or -proline (Pro) medium. Rapamycin or Methionine sulfoximine was added where indicated (+Rap, or +Msx). Gln3-Myc13 localization was scored as cytoplasmic (red bars), nuclear-cytoplasmic (fluorescent material appearing 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).
Figure 2
Figure 2
Conservation of amino acid sequences functionally associated with nuclear Gln3 import among various yeast strains (A-D) and their relation to amino acid substitutions in the mutant plasmids used in this work (A-C, E). Truncation plasmids are listed in the text where appropriate. Predicted secondary structures and functional domains are indicated by labeled black rectangles or brackets. Residues situated at the C-terminus of the Gln3 DNA binding domain are indicated as unlabeled black boxes just above the K/R-rich sequence.
Figure 3
Figure 3
Effects of amino acid substitutions on Gln3-Myc13 intracellular localization in sequences previously reported to be associated with nuclear Gln3-Myc13 import. Gln3 subscripts indicate the functional domain being altered, with the pertinent plasmid numbers depicted below them in parentheses. Wild-type and mutant sequences appear at the top of the figure. Transformants (JK9-3da recipient) were cultured in YNB-glutamine ± Rap (A), YNB-proline (B), or YNB-ammonia ± Msx (C) medium. Red bars indicate the percentage of total cells in which Gln3-Myc13 was exclusively cytoplasmic. Yellow bars indicate Gln3-Myc13 was present in both cytoplasm and nucleus, whereas green bars indicate Gln3-Myc13 localized exclusively in the nucleus colocalizing with DAPI-positive DNA. See Materials and Methods for scoring criteria.
Figure 4
Figure 4
Effects of C-terminal truncations on the intracellular distribution of Gln3-Myc13. The peptide coordinates of each truncation mutant appear as subscripts of Gln3, and the plasmid numbers are in parentheses next to or below them. Vertical, red dashed lines denote the positions of the C-termini of the truncations with respect to Gln3 functional domains. The transformation recipient, experimental format, and data presentation are as described in Figure 3.
Figure 5
Figure 5
(A) Intracellular distribution of EGFP-Gln3 peptides. Wild type JK9-3da cells were transformed with the plasmids indicated in the figure. Transformants were grown in YNB-raffinose-proline medium to A600 nm = 0.4. Galactose (4% final concentration) was then added to the medium and cellular distribution assessed 3 hr later. Crude quantitation (B) was obtained by scoring the distribution of >100 cells per peptide. This was lower than our standard 200 scored cells per point because these were live cultures with cells present at much lower densities. The cellular distributions of all of the EGFP-peptides, prior to galactose addition were the same as observed with pRR482 (data not shown).
Figure 6
Figure 6
Growth of gln3Δ KHC2 transformed with wild type GLN3 (pRR536, left side of each plate) or gln3 mutant (right side of each plate) plasmids. The residues deleted (pRR723) or coordinates of the peptides remaining after truncation are shown as subscripts of Gln3. Gln3 Zn Finger mutant (pRR787) is a substitution mutant, Gln3L332D,L336D,M340D,L343D-Myc13 (Rai et al. 2015). Cells were cultured on YNB-ammonia medium for 92 hr.
Figure 7
Figure 7
Deletions in the S/R region of Gln3 abolish the response to rapamycin addition (A) and significantly reduce the Gln3 response to growth in nitrogen sources that depend on NCR-sensitive transcription (B and C). The transformation recipient, experimental format and data presentation were as described in Figure 3.
Figure 8
Figure 8
Substitution of the KK or RK repeats in the S/R region of Gln3 abolish nuclear Gln3-Myc13 localization in response to rapamycin treatment (A) or in nitrogen sources that depend on NCR-sensitive transcription (B,C), but not Msx treatment (C). The transformation recipient, experimental format, and data presentation were as described in Figure 3.
Figure 9
Figure 9
Epistasis relationships between mutations causing alterations in the S/R (A,B) and K/R (C,D) regions of Gln3 and a ure2Δ. The ure2Δ used as the transformation recipient in this experiment was RR215. The wild-type transformation recipient, experimental format, and data presentation were as described in Figure 3.
Figure 10
Figure 10
The effects of substituting aspartate or alanine for groups of serine residues throughout the S/R region of Gln3 (A–C). The transformation recipient, experimental format, and data presentation were as described in Figure 3.
Figure 11
Figure 11
The effects of substituting aspartate for serine residues between the paired basic residues in the S/R region of Gln3. The transformation recipient, experimental format, and data presentation were as described in Figure 3.
Figure 12
Figure 12
Epistasis relationships observed between a ure2Δ and mutations resulting in the substitution of phosphomimetic aspartate for the serine residues between the first, second, and third pairs of basic residues of the S/R region in Gln3, respectively (A,B). The transformation recipients, experimental format, and data presentation were as described in Figure 9.
Figure 13
Figure 13
Function domains and degree of disorder in the Gln3 protein. Gln3 is predicted to be a highly disordered protein (Ishida and Kinoshita 2007). Functional domains are indicated (top) with yellow boxes. They are not drawn to scale. Data upon which the conclusion that Gln3 is likely to be a highly disordered protein appears in the plot of disorder probability as a function of Gln3 residue number. Colored boxes within the plot indicate the boundaries of sequences shown to be required for the function indicated. Precise locations of the most N- and C-terminal substitutions are indicated with corresponding colored arrows.

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