Saccharomyces cerevisiae Sit4 phosphatase is active irrespective of the nitrogen source provided, and Gln3 phosphorylation levels become nitrogen source-responsive in a sit4-deleted strain.
Tor1,2 control of type 2A-related phosphatase activities in Saccharomyces cerevisiae has been reported to be responsible for the regulation of Gln3 phosphorylation and intracellular localization in response to the nature of the nitrogen source available. According to the model, excess nitrogen stimulates Tor1,2 to phosphorylate Tip41 and/or Tap42. Tap42 then complexes with and inactivates Sit4 phosphatase, thereby preventing it from dephosphorylating Gln3. Phosphorylated Gln3 complexes with Ure2 and is sequestered in the cytoplasm. When Tor1,2 kinase activities are inhibited by limiting nitrogen, or rapamycin-treatment, Tap42 can no longer complex with Sit4. Active Sit4 dephosphorylates Gln3, which can then localize to the nucleus and activate transcription. The paucity of experimental data directly correlating active Sit4 and Pph3 with Gln3 regulation prompted us to assay Gln3-Myc(13) phosphorylation and intracellular localization in isogenic wild type, sit4, pph3, and sit4pph3 deletion strains. We found that Sit4 actively brought about Gln3-Myc(13) dephosphorylation in both good (glutamine or ammonia) and poor (proline) nitrogen sources. This Sit4 activity masked nitrogen source-dependent changes in Gln3-Myc(13) phosphorylation which were clearly visible when SIT4 was deleted. The extent of Sit4 requirement for Gln3 nuclear localization was both nitrogen source- and strain-dependent. In some strains, Sit4 was not even required for Gln3 nuclear localization in untreated or rapamycin-treated, proline-grown cells or Msx-treated, ammonia-grown cells.
Tate
Jennifer J
JJ
Department of Molecular Sciences, University of Tennessee, Memphis, Tennessee 38163, USA.
Feller
André
A
Dubois
Evelyne
E
Cooper
Terrance G
TG
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics; D017353:Gene Deletion; D005800:Genes, Fungal; D008717:Methionine Sulfoximine / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009584:Nitrogen / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*
D004271:DNA, Fungal; C071664:GLN3 protein, S cerevisiae; D012331:RNA, Fungal; D012333:RNA, Messenger; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D008717:Methionine Sulfoximine; C502440:PPH3 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; C104343:SIT4 protein, S cerevisiae; D009584:Nitrogen; D020123:Sirolimus