Rapamycin-induced Gln3 dephosphorylation is insufficient for nuclear localization

Sit4 and PP2A phosphatases are regulated and function differently

Jennifer J. Tate, Isabelle Georis, André Feller, Evelyne Dubois, Terrance Cooper

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

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 Rap-treated 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 pph21Δ22Δ, tpd3Δ, or cdc55Δcells, however, Gln3 is dephosphorylated to the same level as in Rap-treated wildtype 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.

Original languageEnglish (US)
Pages (from-to)2522-2534
Number of pages13
JournalJournal of Biological Chemistry
Volume284
Issue number4
DOIs
StatePublished - Jan 23 2009

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Sirolimus
Phosphoric Monoester Hydrolases
Nitrogen
Catabolite Repression
Transcription
Yeast
Saccharomyces cerevisiae
Cultured Cells
Assays

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Rapamycin-induced Gln3 dephosphorylation is insufficient for nuclear localization : Sit4 and PP2A phosphatases are regulated and function differently. / Tate, Jennifer J.; Georis, Isabelle; Feller, André; Dubois, Evelyne; Cooper, Terrance.

In: Journal of Biological Chemistry, Vol. 284, No. 4, 23.01.2009, p. 2522-2534.

Research output: Contribution to journalArticle

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abstract = "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 Rap-treated 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 pph21Δ22Δ, tpd3Δ, or cdc55Δcells, however, Gln3 is dephosphorylated to the same level as in Rap-treated wildtype 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.",
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