Tate Jennifer J, Georis Isabelle, Feller André, Dubois Evelyne, Cooper Terrance G
Department of Molecular Sciences, University of Tennessee, Memphis, Tennessee 38163, USA.
J Biol Chem. 2009 Jan 23;284(4):2522-34. doi: 10.1074/jbc.M806162200. Epub 2008 Nov 17.
Gln3, the major activator of nitrogen catabolite repression (NCR)-sensitive transcription, is often used as an assay of Tor pathway regulation in Saccharomyces cerevisiae. Gln3 is cytoplasmic in cells cultured with repressive nitrogen sources (Gln) and nuclear with derepressive ones (Pro) or after treating Gln-grown cells with the Tor inhibitor, rapamycin (Rap). In Raptreated or Pro-grown cells, Sit4 is posited to dephosphorylate Gln3, which then dissociates from a Gln3-Ure2 complex and enters the nucleus. However, in contrast with this view, Sit4-dependent Gln3 dephosphorylation is greater in Gln than Pro. Investigating this paradox, we show that PP2A (another Tor pathway phosphatase)-dependent Gln3 dephosphorylation is regulated oppositely to that of Sit4, being greatest in Pro- and least in Gln-grown cells. It thus parallels nuclear Gln3 localization and NCR-sensitive transcription. However, because PP2A is not required for nuclear Gln3 localization in Pro, PP2A-dependent Gln3 dephosphorylation and nuclear localization are likely parallel responses to derepressive nitrogen sources. In contrast, Rap-induced nuclear Gln3 localization absolutely requires all four PP2A components (Pph21/22, Tpd3, Cdc55, and Rts1). In pph21Delta22Delta, tpd3Delta, or cdc55Delta cells, however, Gln3 is dephosphorylated to the same level as in Rap-treated wild-type cells, indicating Rap-induced Gln3 dephosphorylation is insufficient to achieve nuclear localization. Finally, PP2A-dependent Gln3 dephosphorylation parallels conditions where Gln3 is mostly nuclear, while Sit4-dependent and Rap-induced dephosphorylation parallels those where Gln3 is mostly cytoplasmic, suggesting the effects of these phosphatases on Gln3 may occur in different cellular compartments.
谷氨酰胺3（Gln3）是氮分解代谢物阻遏（NCR）敏感转录的主要激活因子，常用于酿酒酵母中Tor信号通路调控的检测。在以抑制性氮源（谷氨酰胺）培养的细胞中，Gln3位于细胞质中；而在以去阻遏性氮源（脯氨酸）培养的细胞中，或在用Tor抑制剂雷帕霉素（Rap）处理谷氨酰胺培养的细胞后，Gln3位于细胞核中。在经雷帕霉素处理或脯氨酸培养的细胞中，推测Sit4会使Gln3去磷酸化，然后Gln3从Gln3 - Ure2复合物中解离并进入细胞核。然而，与这种观点相反的是，Sit4依赖的Gln3去磷酸化在谷氨酰胺培养条件下比脯氨酸培养条件下更显著。为研究这一矛盾现象，我们发现蛋白磷酸酶2A（PP2A，另一种Tor信号通路磷酸酶）依赖的Gln3去磷酸化与Sit4的情况相反，在脯氨酸培养的细胞中最显著，在谷氨酰胺培养的细胞中最不显著。因此，它与细胞核中Gln3的定位以及NCR敏感转录情况相似。然而，由于在脯氨酸培养条件下细胞核中Gln3的定位不需要PP2A，PP2A依赖的Gln3去磷酸化和细胞核定位可能是对去阻遏性氮源的平行反应。相比之下，雷帕霉素诱导的细胞核中Gln3定位绝对需要所有四个PP2A组分（Pph21/22、Tpd3、Cdc55和Rts1）。然而，在pph21Delta22Delta、tpd3Delta或cdc55Delta细胞中，Gln3去磷酸化的水平与雷帕霉素处理的野生型细胞相同，这表明雷帕霉素诱导的Gln3去磷酸化不足以实现细胞核定位。最后，PP2A依赖的Gln3去磷酸化与Gln3主要位于细胞核的条件相似，而Sit4依赖的和雷帕霉素诱导的去磷酸化与Gln3主要位于细胞质的条件相似，这表明这些磷酸酶对Gln3的作用可能发生在不同的细胞区室。
