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

Abstract

Gln3 is responsible for Nitrogen Catabolite Repression-sensitive transcriptional activation in the yeast 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-Myc localization is significantly more complex than previously appreciated. We identify three Gln3 sequences, other than NLS1, that are highly required for nuclear Gln3-Myc 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 Δ. 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-Myc localization in response to both limiting nitrogen and rapamycin treatment. In contrast, Gln3-Myc 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.

Sit4 and PP2A Dephosphorylate Nitrogen Catabolite Repression-Sensitive Gln3 When TorC1 Is Up- as Well as Downregulated

“…The precision of our scoring has been repeatedly documented with overall SD ,10% for N = 7-10 experiments performed over 9 months in some cases, and up to 3 years in others (Tate et al 2006(Tate et al , 2010(Tate et al , 2018Rai et al 2013Rai et al , 2014. Precision decreases 5% when Gln3 is more or less equally distributed in all three scoring categories.…”

“…To achieve signals of similar intensity, it was necessary to increase the amount of protein by approximately twofold for samples taken beyond 1 hr of starvation irrespective of whether wild-type or mutant cells were employed. This necessity derives from the fact that Gln3 is predicted to be a very highly disordered protein that is exquisitely susceptible to proteolysis (Tate et al 2018). This sensitivity becomes increasingly severe the longer the cells are subjected to adverse growth conditions, and irrespective of whether or not optimal growth conditions are then restored for a short time.…”

The flavonoid 4,4′-dimethoxychalcone promotes autophagy-dependent longevity across species

“…These data suggest, on the one hand, that DMC regulates Gln3 activity TORC1-independently. Indeed, recent evidence indicates that such non-canonical mechanisms of Gln3 regulation may exist 45–47 . On the other hand, it establishes synergistic potential with other anti-ageing interventions that act on the TORC1 inhibitory pathway.…”

Sensing, Uptake and Catabolism of L-Phenylalanine During 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in Saccharomyces cerevisiae

“…However, in the presence of nonpreferred nitrogen sources, limited nitrogen or added rapamycin, Gln3 is dephosphorylated and freed. Then, it is targeted to the nucleus and binds to the GATAA/G motifs of NCR-sensitive gene promoters, activating gene transcription (Beck and Hall, 1999;Cox et al, 2000;Kulkarni et al, 2001;Tate et al, 2018).…”