Differing responses of Gat1 and Gln3 phosphorylation and localization to rapamycin and methionine sulfoximine treatment in Saccharomyces cerevisiae

Abstract

Gln3 and Gat1/Nil1 are GATA-family transcription factors responsible for transcription of nitrogen-catabolic genes in Saccharomyces cerevisiae. Intracellular Gln3 localization and Gln3-dependent transcription respond in parallel to the nutritional environment and inhibitors of Tor1/2 (rapamycin) and glutamine synthetase (L-methionine sulfoximine, MSX). However, detectable Gln3 phosphorylation, though influenced by nutrients and inhibitors, correlates neither with Gln3 localization nor nitrogen catabolite repression-sensitive transcription in a consistent way. To establish relationships between Gln3 and Gat1 regulation, we performed experiments parallel to those we previously reported for Gln3. Gat1 and Gln3 localization are similar during steady-state growth, being cytoplasmic and nuclear with good and poor nitrogen sources, respectively. Localization correlates with Gat1- and Gln3-mediated transcription. In contrast, three characteristics of Gat1 and Gln3 differ significantly: (i) the kinetics of their localization in response to nutritional transitions and rapamycin-treatment; (ii) their opposite responses to MSX-treatment, i.e. that cytoplasmic Gln3 becomes nuclear following MSX addition, whereas nuclear Gat1 becomes cytoplasmic; and (iii) their phosphorylation levels in the above situations. In instances where Gln3 phosphorylation can be straightforwardly demonstrated to change, Gat1 phosphorylation (in the same samples) appears invariant. The only exception was following carbon starvation, where Gat1, like Gln3, is hyperphosphorylated in a Snf1-dependent manner. However, neither carbon starvation nor MSX treatment elicits Snf1-independent Gat1 hyperphosphorylation, as observed for Gln3.

Distinct Phosphatase Requirements and GATA Factor Responses to Nitrogen Catabolite Repression and Rapamycin Treatment in Saccharomyces cerevisiae

“…GATA Factor-specific Responses to Methionine Sulfoximine Inhibition of Glutamine Synthetase-The final set of observations pointing to the distinct regulation of Gln3 and Gat1 localization derive from experiments with Msx (43,44,57). Treating cells with Msx results in rapid and complete nuclear Gln3-Myc 13 and Gln3-GFP localization (Ref.…”

“…Work to this point has provided a detailed view of conditions and phosphatase requirements affecting the intracellular localization of Gln3-Myc 13 . Unfortunately, similar information for Gat1 was lacking except for the observations that rapamycin treatment did not demonstrably affect the electrophoretic mobility of Gat1-Myc 13 (44), and rapamycin-induced nuclear Gat1-Myc 13 localization occurred similarly in wild type and sit4⌬ cells (48).…”

“…Gln3-Myc 13 is hyperphosphorylated in nitrogen-starved, proline-grown, or methionine sulfoximine-treated cells but hypophosphorylated in rapamycin-treated cells. Yet Gln3-Myc 13 is nuclear in all four cases (43)(44)(45). (iii) Sit4-dependent Gln3-Myc 13 dephosphorylation is greater in glutamine-than proline-grown cells (46); i.e.…”

“…18 and data not shown). However, past attempts to detect changes in the electrophoretic mobility of Gat1-Myc 13 either in response to rapamycin treatment or in vitro treatment of cell extracts with calf intestinal alkaline phosphatase have yielded only negative results (44). 3 The above localization requirements identified new conditions under which to search for a change in Gat1-Myc 13 electrophoretic mobility.…”

Nuclear translocation of Gln3 in response to nutrient signals requires Golgi-to-endosome trafficking in Saccharomyces cerevisiae

“…Although Gln3 and Gat1 were thought to be functionally redundant, recent studies have suggested that they respond differentially to various nitrogen conditions (17). For some genes, abolishment of the NCR response is achieved only by mutation of both GLN3 and GAT1 (18).…”