Sst2 synthesis/degradation

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Sst2 Degradation

  • Sst2 is phosphorylated at S539 in response to pheromone. This phosphorylation is eliminated in fus3Δ kss1Δ cells, but not in fus3Δ or kss1Δ cells, suggesting that Fus3 and Kss1 are capable of phosphorylating Sst2 at S539. Garrison et al. 1999 PMID 10593933
  • Sst2 is ubiquitinated in a pheromone dependent manner, and ubiquitinated Sst2 is more rapidly degraded. Hao et al. 2003 PMID 12968019
    • It seems likely that ubiquitination is due to phosphorylation at S539 by Fus3/Kss1 (as demonstrated in Garrison et al. 1999 PMID 10593933).
  • Sst2 has a half-life of ~30 minutes, which is not affected by pheromone exposure (data not shown). Dohlman et al. 1996 PMID 8756677
The amount of Sst2 remaining after pre-treatment (or not) with pheromone for one hour prior to addition of cycloheximide. Sst2 is expressed off a constitutive promoter. Figure is taken from Hao et al. 2003 PMID 12968019
The amount of Sst2 remaining after pre-treatment (or not) with pheromone for one hour prior to addition of cycloheximide. Sst2 is expressed off a constitutive promoter. Figure is taken from Hao et al. 2003 PMID 12968019
  • A degradation rate (in the absence of pheromone) of 5 * 10-2 min-1 is used in a model (no source given). Hao et al. 2003 PMID 12968019
    • This degradation rate corresponds to a half-life of 14 minutes. The figure to the right is from the same paper. We can see in this figure that the half-life of Sst2 in untreated cells is ~45 min, and in pheromone treated cells the half-life is ~30 min.
    • Same number is used (and referenced to Hao et al. 2003) by Yildirim et al. 2004 PMID 15313578

Reaction Definition

Sst2 is phosphorylated by the MAPKs in response to pheromone. It is also ubiquitinated, which results in more rapid degradation. The half-life of Sst2 in the absence of pheromone has been measured to be between 30 and 45 minutes (degradation rate of 2.6-3.8 * 10-4 s-1). In the presence of pheromone, Sst2's half-life was measured to be 30 minutes (degradation rate of 3.8 * 10-4 s-1), which is presumably a combination of Sst2's innate degradation rate, and the rates of phosphorylation, ubuititination and degradation of Sst2 in response to pheromone. Thus the degradation rate of phosphorylated Sst2 must be greater than 3.8 * 10-4 s-1.

We will use a degradation rate of 3 * 10-4 s-1 for unphosphorylated Sst2 (correspoding to a half-life of ~38 min), and a half-life of 5.8 * 10-4 s-1 for phosphorylated Sst2 (correspoding to a half-life of ~20 min).

Assumptions:

  • Kss1 and Fus3 are equally efficient at phosphorylating Sst2.
  • Phosphorylation of Sst2 by the MAPKs marks Sst2 for ubiquitination and degradation. We also model only phosphorylation and degradation, assuming that the rate of ubiquitination is lumped into the phosphorylation and degradation rates.
  • The MAPKs can only bind and phosphorylate Sst2 that is not bound to Ste2 (no evidence for this at all).
  • Since the degradation rate of Sst2 is much faster than the dilution rate of Sst2 due to cell growth (half time of ~90 min for exponentially growing cells), we will neglect Sst2 dilution as mechanism of Sst2 loss (and corresponding synthesis to maintain steady state levels). 
Fus3(docking_site, T180~none, Y182~PO4) + Sst2(Ste2_site, MAPK_site, S539~none) <->
   Fus3(docking_site!1, T180~none, Y182~PO4).Sst2(Ste2_site, MAPK_site!1, S539~none)




Kss1(docking_site, T183~none, Y185~PO4) + Sst2(Ste2_site, MAPK_site, S539~none) <->
    Kss1(docking_site!1, T183~none, Y185~PO4).Sst2(Ste2_site, MAPK_site!1, S539~none)



Fus3(docking_site, T180~PO4, Y182~none) + Sst2(Ste2_site, MAPK_site, S539~none) <->
   Fus3(docking_site!1, T180~PO4, Y182~none).Sst2(Ste2_site, MAPK_site!1, S539~none)




Kss1(docking_site, T183~PO4, Y185~none) + Sst2(Ste2_site, MAPK_site, S539~none) <->
    Kss1(docking_site!1, T183~PO4, Y185~none).Sst2(Ste2_site, MAPK_site!1, S539~none)



Fus3(docking_site, T180~PO4, Y182~PO4) + Sst2(Ste2_site, MAPK_site, S539~none) <->
   Fus3(docking_site!1, T180~PO4, Y182~PO4).Sst2(Ste2_site, MAPK_site!1, S539~none)




Kss1(docking_site, T183~PO4, Y185~PO4) + Sst2(Ste2_site, MAPK_site, S539~none) <->
    Kss1(docking_site!1, T183~PO4, Y185~PO4).Sst2(Ste2_site, MAPK_site!1, S539~none)



Fus3(docking_site!1, T180~none, Y182~PO4).Sst2(MAPK_site!1, S539~none) ->
   Fus3(docking_site, T180~none, Y182~PO4) + Sst2(MAPK_site, S539~PO4)




Kss1(docking_site!1, T183~none, Y185~PO4).Sst2(MAPK_site!1, S539~none) ->
    Kss1(docking_site, T183~none, Y185~PO4) + Sst2(MAPK_site, S539~PO4)



Fus3(docking_site!1, T180~PO4, Y182~none).Sst2(MAPK_site!1, S539~none) ->
   Fus3(docking_site, T180~PO4, Y182~none) + Sst2(MAPK_site, S539~PO4)




Kss1(docking_site!1, T183~PO4, Y185~none).Sst2(MAPK_site!1, S539~none) ->
    Kss1(docking_site, T183~PO4, Y185~none) + Sst2(MAPK_site, S539~PO4)



Fus3(docking_site!1, T180~PO4, Y182~PO4).Sst2(MAPK_site!1, S539~none) ->
   Fus3(docking_site, T180~PO4, Y182~PO4) + Sst2(Ste2_site, MAPK_site, S539~PO4)




Kss1(docking_site!1, T183~PO4, Y185~PO4).Sst2(MAPK_site!1, S539~none) ->
    Kss1(docking_site, T183~PO4, Y185~PO4) + Sst2(Ste2_site, MAPK_site, S539~PO4)



Sst2(S539~none) + Cell -> Cell

  • In order for the degradation reaction to selectively degrade Sst2 out of a complex, we need to include the parameter DeleteMolecules

  • Dummy species Cell used because BioNetGen requires at least one species on each side of reaction 
Sst2(S539~PO4) + Cell -> Cell

  • In order for the degradation reaction to selectively degrade Sst2 out of a complex, we need to include the parameter DeleteMolecules

  • Dummy species Cell used because BioNetGen requires at least one species on each side of reaction

Sst2 Synthesis

  • Sst2 synthesis is induced by pheromone exposure. Dohlman et al. 1996 PMID 8756677
  • Constitutive synthesis rate of Sst2 of 1.6 molec s-1 is used in a model. It's based on the degradation rate (4 * 10-3 s-1) and abundance (2000 molec/cell) used in the model. Yildirim et al. 2004 PMID 15313578

Reaction Definition

Synthesis of Sst2 must balance the constitutive degradation of Sst2 in the absence of pheromone, to give the steady state amount of Sst2 (Sst2_tot_conc). Since the degradation rate and concentration of Sst2 are measured independently, and then the synthesis rate inferred, we use measured degradation rates and Sst2 abundances to calculate the synthesis rate. Thus ksynth_Sst2 = Sst2_tot_conc * kdeg_Sst2.

For pheromone-induced synthesis of Sst2, please see Ste12 mediated protein synthesis. When Ste12 mediated protein synthesis is included in a model, the synthesis of Sst2 through the reaction specified here together with synthesis of Sst2 through basal pathway activation of Ste12 (in the absence of pheromone) should balance the degradation rate. So when Ste12 mediated protein synthesis is included in a model, ksynth_Sst2 should be decreased by the amount of Sst2 synthesis that occurs through basal activation of Ste12. This cannot be determined a priori because the basal activation of Ste12 in unknown. 

Cell -> Cell + Sst2(Ste2_site, MAPK_site, S539~none)

  • Dummy species Cell used because BioNetGen requires at least one species on each side of reaction
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