The Four Faces of Gln3 and Its Response to Nitrogen Catabolite Repression
Authors: Cooper Terrance G. , Tate Jennifer J. , Rai Rajendra 
Published: November 2023

When S. cerevisiae cells are cultured in adverse nitrogen environments, transcription of the genes whose products are responsible for the uptake and catabolism of poorly utilized nitrogen sources are derepressed, thereby facilitating the use of these poor nitrogen sources during hard times. Conversely, in good times when nitrogen is in excess, this transcription is repressed. Over three decades ago, this regulatory phenomenon was termed nitrogen catabolite repression or NCR. We know today that NCR is achieved by regulating the intracellular localization and function of the transcription activators responsible for NCR‐sensitive transcription, Gln3 and Gat1. In nitrogen excess they are sequestered in the cytoplasm, whereas in nitrogen limitation they relocate to the nucleus and activate NCR‐sensitive gene expression. At one time NCR was thought to be achieved exclusively through nitrogen‐responsive control of Target of Rapamycin (Tor) activity. However, we now know that nitrogen responsiveness derives from 5 distinct environments all but the last one of which elicit nuclear Gln3 localization: (a) nitrogen limitation by growth in a poor nitrogen source [proline] or short‐term starvation, (b) long‐term nitrogen starvation, (c) glutamine starvation, i.e. methionine sulfoximine (Msx) inhibition of glutamine synthetase, (d) rapamycin inhibition of TorC1 and (e) amino acid starvation‐mediated inactivation of TorC1. Distinct phosphatase requirements are associated with the responses to each of these conditions. Our goal has been to distinguish whether the common nuclear Gln3 localization elicited by the 4 distinct environments derived from a single regulatory signal or from multiple signals that cumulatively control Gln3 localization. To this end, we predicted that if Gln3 individually responds to each of the 4 above nitrogen environments, then each of them should possess an identifiable target on the Gln3 molecule itself through which it acts. Supporting this prediction, we show that a serine/threonine‐rich Gln3 tridecapeptide is required for its effective cytoplasmic sequestration in excess nitrogen. Alanine substitutions for these serines largely abolish cytoplasmic Gln3 sequestration, whereas aspartate substitutions have no effect, raising the possibility that serine/threonine residues in this tridecapeptide may be phosphorylated in nitrogen‐rich medium. Most importantly, the above substitutions abrogating NCR‐sensitive cytoplasmic Gln3 sequestration have no effect on Gln3's responses to rapamycin or methionine sulfoximine. These data join previous demonstrations of other equally unique Gln3 sites: one required exclusively for Gln3's rapamycin‐responsiveness, another specifically for the Gln3's interaction with Tor1 and a third which in response to glutamine itself determines whether or not Gln3 must bind to its promoter targets before exiting from the nucleus. Together these findings support the contention that NCR‐sensitive GATA factor localization and transcription are controlled by multiple distinct regulatory pathways. Each one responds to a specific nitrogen environment and then they cumulatively coalesce at the level of the Gln3 molecule itself to control nitrogen catabolism.

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Supported by NIH grant GM‐35642.