General Amino Acid Control and 14‐3‐3 Proteins Bmh1/2 Are Required for Nitrogen Catabolite Repression‐Sensitive Regulation of Intracellular Gln3 and Gat1 Localization

Abstract

Nitrogen Catabolite Repression (NCR), the ability of Saccharomyces cerevisiae to utilize good nitrogen sources in preference to poor ones, derives from nitrogen‐responsive regulation of the GATA family transcription activators Gln3 and Gat1. In nitrogen replete conditions the GATA factors are cytoplasmic and NCR‐sensitive transcription minimal. When only poor nitrogen sources are available, Gln3 is nuclear dramatically increasing GATA factor‐mediated transcription. In addition, to these two growth conditions, Gln3 localization responds to short‐ and long‐term nitrogen starvation as well as rapamycin and methionine sulfoximine (glutamine synthetase inhibitor) treatments. Originally these responses were all attributed to mTorC1‐mediated control of Gln3. We subsequently showed that this was not the case, but that there were at least three distinct regulatory pathways controlling Gln3 localization. Two regulatory systems act cumulatively to maintain cytoplasmic Gln3 sequestration; one is TorC1. The second regulatory system has remained elusive. A third regulatory pathway takes over once Gln3 enters the nucleus. This pathway is glutamine specific. When glutamine levels are low, Gln3 must bind to its DNA target promoter sequences (GATAA) before it can exit from the nucleus. On the other hand, when glutamine (or glutamine analogue) levels are high, Gln3 can exit from the nucleus in the absence of DNA binding. Present experiments identify Gcn2‐dependent general amino acid control as the regulatory system that has thus far escaped detection. We show that the uncharged tRNA‐activated protein kinase Gcn2 is required for NCR‐sensitive nuclear Gln3‐Myc13 localization but not for short‐ and long‐term nitrogen starvation or responses to Msx and rapamycin treatments. From epistasis experiments, it appears that Gcn2 functions upstream of Ure2. Ure2 sequesters Gln3 in the cytoplasm in nitrogen replete conditions. 14‐3‐3 proteins Bmh1/2 are also required for nuclear Gln3‐Myc13 localization and GATA factor‐mediated transcription. In contrast with Gcn2, however, Bmh1/2 appear to function downstream of Ure2. Overall Gln3 phosphorylation levels decrease upon loss of either Gcn2 or Bmh1/2. Our results add a new dimension to the cumulative participation of these three major pathways in the nitrogen‐responsive control of GATA transcription factor localization and NCR‐sensitive gene expression in yeast.Support or Funding InformationNIH GM‐35642‐27