Effects of Abolishing Whi2 on Nitrogen Catabolite Repression‐Sensitive GATA‐Factor Localization and Protein Production

Abstract

Gln3 and Gat1 are the transcriptional activators of nitrogen catabolite repression‐ (NCR‐) sensitive gene expression in Saccharomyces cerevisiae. In nitrogen replete medium, these activators are sequestered in the cytoplasm. As environmental nitrogen supplies dwindle, Gln3 and Gat1 enter the nucleus. There, they activate expression of the autophagy PI3‐kinase subunit gene ATG14 and the genes required to transport and catabolize whatever environmental nitrogen sources are still present. Two global protein kinase regulators, TorC1 and Gcn2, are major contributors to NCR‐sensitive regulation. Active TorC1, via Tor Associated Protein Tap42, binds to and inhibits two protein phosphatases, Sit4 and PP2A, required for nuclear Gln3 and Gat1 entry. When TorC1 is down‐regulated by limiting nitrogen or rapamycin treatment, the phosphatase‐Tap42 complexes are released and dephosphorylate Gln3 and Gat1, which can then enter the nucleus and mediate NCR‐sensitive transcription. Recently, a new regulator of TorC1 activity has been reported, Whi2 (Chen et al., 2018). The Whi2‐Psr1/2 complex down‐regulates TorC1 activity when primarily leucine concentrations are low. One of the reporters used to characterize Whi2 actions was an NCR‐sensitive DAL80‐GFP promoter fusion. We were puzzled by the results obtained with this reporter because Dal80‐GFP protein was detectable when cultures were grown in synthetic complete (SC) medium. We, a priori, expected GFP production to be undetectable in such nitrogen‐rich medium, because DAL80 expression is highly NCR‐sensitive. Therefore, we investigated the source of the GFP production and the NCR‐sensitive proteomes of wild type and whi2Δ cells. In the SC medium, Gln3 was efficiently sequestered in the cytoplasm and NCR‐sensitive DAL80 expression was only minimally present in either wild type or whi2Δ cells. The small amount of DAL80 expression observed could be attributed to Gat1‐mediated activation since this activator is more resistant to NCR‐sensitivity and hence is partially nuclear in nitrogen‐rich medium. Overall, however, proteins encoded by 109 of the combined 125 NCR‐sensitive genes, putatively NCR‐sensitive genes or genes containing GATA elements (the binding sites for Gln3 and Gat1) in their promoters were unaffected or completely absent when cells were cultured in the rich SC medium. The 16 proteins that did respond in the Whi2 experiments were largely those associated with amino acid transport and metabolism as might be expected from the high amino acid content of SC medium. We conclude that while Whi2 can be convincingly shown to down‐regulate TorC1 activity, it is not a significant regulator of NCR‐sensitive gene expression. These observations raise the important question of how a major downstream target of TorC1 remains immune to its down‐regulation by Whi2.