Dataset Information

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 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 ure2?. 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.

SUBMITTER: Tate JJ

PROVIDER: S-EPMC5753859 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Tate Jennifer J JJ Rai Rajendra R Cooper Terrance G TG

Genetics 20171107 1

Gln3 is responsible for Nitrogen Catabolite Repression-sensitive transcriptional activation in the yeast <i>Saccharomyces cerevisiae</i> 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 conclu ...[more]

PMID: 29113979

Similar Datasets

Project description:The peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores. The central role of Pex14p in peroxisomal matrix protein import processes renders it an obvious target for regulatory mechanisms such as protein phosphorylation. To explore this possibility, we examined the state of Pex14p phosphorylation in Saccharomyces cerevisiae. Phos-tag-SDS-PAGE of Pex14p affinity-purified from solubilized membranes revealed Pex14p as multi-phosphorylated protein. Using mass spectrometry, we identified 16 phosphorylation sites, with phosphorylation hot spots located in the N- and C-terminal regions of Pex14p. Analysis of phosphomimicking and non-phosphorylatable variants of Pex14p revealed a decreased import of GFP carrying a peroxisomal targeting signal type 1, indicating a functional relevance of Pex14p phosphorylation in peroxisomal matrix protein import. We show that this effect can be ascribed to the phosphomimicking mutation at serine 266 of Pex14p (Pex14p-S266D). We further screened the subcellular distribution of 23 native GFP-tagged peroxisomal matrix proteins by high-content fluorescence microscopy. Only Cit2p, the peroxisomal isoform of citrate synthase, was affected in the Pex14p-S266D mutant, showing increased cytosolic localization. Cit2p is part of the glyoxylate cycle, which is required for the production of essential carbohydrates when yeast is grown on non-fermentable carbon sources. Pex14p-S266 phosphosite mutants showed reversed growth phenotypes in oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Overexpression of Cit2p rescued the growth phenotype of yeast cells expressing Pex14p-S266D in oleic acid. Our data indicate that phosphorylation of Pex14p at S266 provides a mechanism for controlling the peroxisomal import of Cit2p, which helps S. cerevisiae cells to adjust their carbohydrate metabolism according to the nutritional conditions.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets