Ste7 synthesis/degradation
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Ste7 degradation
- Ste7 degradation products do not copurify with Ste5. The authors conclude that binding to Ste5 might protect Ste7 from degradation, though other explanations are equally likely (i.e. degradation products quickly dissociate from Ste5, or the only degradation products that do associate with Ste5 do not contain the purification tag). Choi et al. 1994 PMID 8062390
- Ste7 poly-ubiquitinylation and degradation is mediated (at least in part) by the ubiquitin-specific processing protease Ubp3. Wang and Dohlman 2002 PMID 11864977
- A Ste7 mutant that lacks Ste11 phosphorylation sites (S359A T363A) is not ubiquitinated in response to pheromone, whereas a Ste7 mutant that is not phosphorylated by Fus3/Kss1 is uqbiquitinated. Wang et al. 2003 PMID 12668671
- Mutation of the Ste7 sites for Fus3/Kss1-mediate feedback phosphorylation to alanines (S105A T116A S130A T137A T149A S167A S471A) does not prevent the accumulation of ubiquitin-tagged Ste7 in cells lacking Ubp3. This suggests that this feedback phosphorylation is not responsible for marking the Ste7 molecules for ubiquitinylation and degradation. Maleri et al. 2004 PMID 15456892
- Temperature sensitive mutants cdc34-2 and cdc53-1 (components of the SCF complex) block pheromone dependent ubiquitination of Ste7 at the restrictive temperature, suggesting that Ste7 is ubiquitinated by the Skp1-Cullin-F-box (SCF) complex. Wang et al. 2003 PMID 12668671
- Deletion of Far1 (which eliminates cell cycle arrest in response to pheromone) eliminates Ste7 ubiquitination in response to pheromone, although Ste7 is still phosphorylated. Wang et al. 2003 PMID 12668671
- The authors believe that this suggests that prolonged arrest at the G1-S transition (a portion of the cell cycle in which the SCF complex is active) is necessary for Ste7 ubiquitination.
- However, expression of Ste5(V763A S861P) eliminates pheromone-dependent ubiquitination of Ste7. This Ste5 mutant nearly eliminates phosphorylation of Fus3, but barely affects the phosphorylation of Kss1, in response to pheromone (Flatauer et al. 2005 PMID 15713635). Thus we know that Ste7 is still phosphorylated in response to pheromone in this mutant, and thus phosphorylation of Ste7 is not sufficient for its increased ubiquitination (and degradation) rate.
- This suggests that Ste7 degradation is actually mediated by Far1 activity, and not just by prolonged arrest.
- Ubiquitination of Ste7 marks Ste7 for rapid degradation (with a half-life of ~25 minutes - estimated from figure 9). Wang et al. 2003 PMID 12668671
Reaction Definition
We do not have a clear model for Far1 activity, and it is unclear whether Far1 itself, or some downstream effector of Far1 is responsible for Ste7 ubiquitination. To simplify the model, instead of modeling Far1 dependent degradation of Ste7 we will model Fus3-dependent degradation of Ste7 using saturable kinetics. Because Kss1 is a much weaker activator of Far1, it is reasonable to assume that the contribution to Ste12 degradation from Kss1 is negligible.
Assumptions:
- The rate of ubiquitination is lumped into the degradation rate.
- Singly phosphorylated Ste7 is degraded at the same rate as unphosphorylated Ste7.
- The MAPK/target interaction properties apply to the kdeg's (presumably caused by Fus3 and Kss1's phosphorylation activity) and Km's (caused by Fus3 and Kss1's ability to bind their targets).
- Ste7 can't be degraded while bound to other proteins, but Ste7 degradation is independent of its phosphorylation state.
Fus3(docking_site, T180~pT, Y182~none) + Ste7(Ste5_site, MAPK_site) -> Fus3(docking_site, T180~pT, Y182~none)
- Rate law: Sat(kdeg_Fus3pT_Ste7, Km_deg_Fus3pT_Ste7) DeleteMolecules
Fus3(docking_site, T180~none, Y182~pY) + Ste7(Ste5_site, MAPK_site) -> Fus3(docking_site, T180~none, Y182~pY)
- Rate law: Sat(kdeg_Fus3pY_Ste7, Km_deg_Fus3pY_Ste7) DeleteMolecules
Fus3(docking_site, T180~pT, Y182~pY) + Ste7(Ste5_site, MAPK_site) -> Fus3(docking_site, T180~pT, Y182~pY)
- Rate law: Sat(kdeg_Fus3pTpY_Ste7, Km_deg_Fus3pTpY_Ste7) DeleteMolecules
Ste7 Synthesis
Reaction Definition
Ste7 synthesis must balance degradation in the absence of pheromone.
Cell -> Cell + Ste7(Ste5_site, MAPK_site, S359_T363~none)
- Synthesis rate constant ksynth_Ste7
