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

Jennifer J Tate,Isabelle Georis,Evelyne Dubois,Terrance G Cooper

doi:10.1074/jbc.m109.085712

Abstract

In yeast, rapamycin (Rap)-inhibited TorC1, and the phosphatases it regulates (Sit4 and PP2A) are components of a conserved pathway regulating the response of eukaryotic cells to nutrient availability. TorC1 and intracellular nitrogen levels regulate the localization of Gln3 and Gat1, the activators of nitrogen catabolite repression (NCR)-sensitive genes whose products are required to utilize poor nitrogen sources. In nitrogen excess, Gln3 and Gat1 are cytoplasmic, and NCR-sensitive transcription is repressed. During nitrogen limitation or Rap treatment, Gln3 and Gat1 are nuclear, and transcription is derepressed. We previously demonstrated that the Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 requirements for nuclear Gln3 localization differ. We now show that Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 requirements for NCR-sensitive and Rap-induced nuclear Gat1 localization markedly differ from those of Gln3. Our data suggest that Gln3 and Gat1 localizations are controlled by two different regulatory pathways. Gln3 localization predominantly responds to intracellular nitrogen levels, as reflected by its stronger NCR-sensitivity, weaker response to Rap treatment, and strong response to methionine sulfoximine (Msx, a glutamine synthetase inhibitor). In contrast, Gat1 localization predominantly responds to TorC1 regulation as reflected by its weaker NCR sensitivity, stronger response to Rap, and immunity to the effects of Msx. Nuclear Gln3 localization in proline-grown (nitrogen limited) cells exhibits no requirement for Pph21/22-Tpd3/Cdc55, whereas nuclear Gat1 localization under these conditions is absolutely dependent on Pph21/22-Tpd3/Cdc55. Furthermore, the extent to which Pph21/22-Tpd3-Cdc55 is required for the TorC1 pathway (Rap) to induce nuclear Gat1 localization is regulated in parallel with Pph21/22-Tpd3-Cdc55-dependent Gln3 dephosphorylation and NCR-sensitive transcription, being highest in limiting nitrogen and lowest when nitrogen is in excess.

Highlights

A more careful study is required before they become apparent
A milestone in the study of GATA factor regulation occurred with the discoveries that (i) nitrogen catabolite repression (NCR)-sensitive transcription aberrantly increases after the addition of the TorC1 serine/threonine kinase inhibitor, rapamycin, to cultures growing under the repressive conditions of excess nitrogen [18, 19, 22, 23] and (ii) NCR-sensitive and rapamycin-induced gene expression positively correlates with intracellular Gln3 and Gat1 localization [18, 19, 24, 25]
Time Course of Gat1 Localization in Response to Rapamycin— Confidently establishing protein phosphatase requirements associated with control of intracellular Gat1 localization in response to rapamycin treatment or the environmental nitrogen supply required high quality semiquantitative assays

Summary

Introduction

A more careful study is required before they become apparent This is the case with the Saccharomyces cerevisiae GATA family transcription factors, Gln and Gat1/Nil. This is the case with the Saccharomyces cerevisiae GATA family transcription factors, Gln and Gat1/Nil1 Both Gln and Gat are responsible for nitrogen catabolite repression (NCR)2-sensitive expression of genes encoding the transport and enzyme proteins needed to scavenge poor nitrogen sources (e.g. proline) when more readily usable ones are limiting or unavailable [5,6,7,8]. Work from many laboratories has established Tor1/2 in S. cerevisiae and mTor in mammals as highly conserved global regulators coordinating cell division with nutrient supplies and a wide variety of metabolically related processes ranging from

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