Imported:YPM/Tec1 synthesis/degradation

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Tec1 synthesis/degradation

• Fus3 activity (but not Kss1 ativity) in response to pheromone interferes with Ste12's ability to associate with filamentation-specific promoters. Zeitlinger et al. 2003 PMID 12732146
  • Deletion of Fus3, or expression of kinase dead Fus3(K42R), results in significant increase in Ste12 binding to filamentation promoters in response to pheromone.
  • Deletion of Kss1 results in no significant change in Ste12 binding to filamentation promoters in response to pheromone.

• Fus3 binds directly to the N-terminal 280 residues of Tec1, whereas Kss1 cannot associate directly with Tec1. Bruckner et al. 2004 PMID 15558284
  • Tec1 co-purifies with GST-Fus3 in WT and ste12Δ cells.
  • Tec1 co-purifies with GST-Kss1 in WT cells, but not in ste12Δ cells. This suggests that Kss1 can only associate with Tec1 via Ste12.
  • Tec1(1-280) co-purifies with GST-Fus3 in WT cells.

• Fus3 (but not Kss1) phosphorylates Tec1. Bruckner et al. 2004 PMID 15558284
  • Neither GST-Fus3 nor GST-Kss1 purified from vegitatively growing cells are able to phosphorylate purified MBP-Tec1 to any significant level in vitro.
  • GST-Fus3 (but not GST-Kss1) purified from pheromone-treated cells is able to phosphorylate purified MBP-Tec1 in vitro.

• Fus3 mediated phosphorylation of Tec1 mediates Tec1 degradation. Bruckner et al. 2004 PMID 15558284
  • In kss1Δ and WT cells, Myc3-Tec1 is undetectable in cells after 1h of pheromone treatment.
  • In fus3Δ cells, Myc3-Tec1 levels are elevated (with respect to pre-pheromone levels) after 1h of pheromone treatment.
  • When Myc3-Tec1 is expressed off of a constitutive promoter (to avoid pheromone-mediated transcription of Tec1), deletion of Ste7 or Fus3 results in no change in Myc3-Tec1 levels. Tec1 mRNA level are unchanged by pheromone treatment (as expected).
  • When Myc3-Tec1 is expressed off of a constitutive promoter (to avoid pheromone-mediated transcription of Tec1), deletion of Kss1 results in undetectable levels of Myc3-Tec1. Tec1 mRNA level are unchanged by pheromone treatment (as expected).
  • Since Tec1 mRNA levels are constant, presumably Tec1 is being synthesized at the same rate in all these experiments, so degradation must be accelerated in a manner that is dependent on activation of Fus3 but not on activation of Kss1.

• Fus3 mediates Tec1 degradation by phosphorylating Tec1 on T273. Bruckner et al. 2004 PMID 15558284
  • Mutation of T273 on Tec1 to methionine or alanine is sufficient to eliminate downregulation of Tec1 levels in response to pheromone.

• Tec1 levels drop off rapidly after pheromone exposure. This effect is eliminated by deletion of Fus3, but not Kss1. Chou et al. 2004 PMID 15620356
  • When Tec1 is expressed from its native promoter, it is almost undetectable 15 minutes after treatment with saturating amounts of pheromone.

• Tec1 is ubiquitinated (and labeled for degradation) by the SCF-Cdc4 ubiquitin ligase. Chou et al. 2004 PMID 15620356
  • Deletion of Cdc4 (F-box protein) almost completely eliminates the pheromone-dependent degradation of Tec1.
  • Deletion of Grr1 or Dia2 (both F-box proteins whose deletions are known to activation filamentation) did not stabilize Tec1 upon pheromone treatment.
  • A Cdc4 phospho-degron was identified centered around T273 (LLTP). The T273V mutation stabilizes Tec1 against pheromone-dependent degradation.

• Fus3 phosphorylates Tec1 at T2763. Chou et al. 2004 PMID 15620356
  • Purified MBP-Tec1-FLAG (expressed in E. coli) was phosphorylated in vitro at T273 by Fus3-myc (purified from pheromone-treated cells), but not by kinase dead Fus3(K42R)-myc (detected my mass spectrometry).
  • The mass spec analysis also identified S86, T297 and S325 as sites that are phosphorylated.

• Tec1 is degraded to background levels within 5 minutes of pheromone treatment. Bao et al. 2004 PMID 15620357

• Tec1 is ubiquitinated in response to pheromone treatment. Bao et al. 2004 PMID 15620357
  • A proteolysis-resistant His6-tagged ubiquitin is coexpressed with FLAG-tagged Tec1. Ubiquitin labeled Tec1 is purified via anickel-NTA column from pheromone treated cells, run out on a gel, and probed with anti-FLAG antibody to detect Tec1. Tec1 is detected in a series of bands with lower mobility than Tec1. These bands are absent when the cells aren't pre-treated with pheromone.

• Deletion of Fus3, or replacement of WT Fus3 with kinase dead Fus3(K42R), completely eliminates the pheromone dependent degradation of Tec1. Bao et al. 2004 PMID 15620357

• Fus3 phosphorylates Tec1 at T2763. Bao et al. 2004 PMID 15620357
  • Purified MBP-Tec1-FLAG (expressed in E. coli) was phosphorylated in vitro at T273 by Fus3-myc (purified from pheromone-treated cells) (detected my mass spectrometry).
  • The mass spec analysis also identified S269, T276, T289, T297, S399, T401, and S421 as sites that are phosphorylated.

• Tec1 is ubiquitinated (and labeled for degradation) by the SCF-Dia2 ubiquitin ligase. Bao et al. 2004 PMID 15620357
  • Deletion of Dia2 (F-box protein) almost completely eliminates the pheromone-dependent degradation of Tec1. Tec1 is still phosphorylated in these cells.
  • Use of temperature sensitive Cdc53 (Cullin subunit of SCF) and Cdc34 (principle E2 of SCF) alleles results in elimination of Tec1 degradation upon pheromone-treatment at the restrictive temperatures.

• The authors state that sumoylation of Tec1 stabilizes it, but the measured half-life (after cyclohehimide treatment) from their plot is ~12min for Myc-Tec1, and ~14min for Myc-SUMO-Tec1. This difference is not convincing to me. Wang and Dohlman. 2005 PMID 16306045

• Tec1 appears to have a half-life of 5-10 minutes in the absence of pheromone, and <5 minutes in the presence of pheromone. Wang and Dohlman. 2005 PMID 16306045
  • In fus3Δ cells, Tec1 appears to have a half-life of 15-20 minutes (relatively unchanged by pheromone treatment.

Reaction Definition

We will not model Tec1, and filamentation gene expression.