Ste2 synthesis/endocytosis/degradation

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Ste2 Synthesis

  • In the absence of pheromone constitutive internalization of Ste2 has a half-time of ~45 minutes. Hicke et al. 1998 PMID 9548714
    • This gives an internalization rate of ln(2)/45min ≅ 2.6 * 10-4 s-1.
  • Ste2 synthesis rate of 4 molecules per cell s-1 was measured or inferred. Yi et al. 2003 PMID 12960402
    • This same value was also subsequently used (referenced to Yi et al. 2003) by Kofahl and Klipp 2004 PMID 15300679
    • It is assumed that the steady state internalization rate and number of Ste2 molecules per cell were measured (probably via a Scatchard plot), and the synthesis rate inferred.
    • The steady state internalization rate of 4 * 10-4 s-1 was used. Yi et al. 2003 PMID 12960402

Reaction Definition

Synthesis of Ste2 must balance the constitutive internalization of Ste2 in the absence of pheromone, to give the steady state amount of Ste2 (Ste2_tot_conc). Since the internalization rate and concentration of Ste2 are measured independently, and then the synthesis rate inferred, we will use the measured internalization rate (kinternalization_Ste2 = 2.9 * 10-4 s-1 - not the value from Yi et al. since they don't explain its source) and Ste2 abundances (Ste2_tot_conc) to calculate the synthesis rate. Thus ksynth_Ste2 = Ste2_tot_conc * kinternalization_Ste2.

For pheromone-induced synthesis of Ste2, please see Ste12 mediated protein synthesis. When Ste12 mediated protein synthesis is included in a model, the synthesis of Ste2 through the reaction specified here together with synthesis of Ste2 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_Ste2 should be decreased by the amount of Ste2 synthesis that occurs through basal activation of Ste12. This cannot be determined a priori because the basal activation of Ste12 in unknown. 

Cell -> Cell + Ste2(Pheromone_site, Gpa1_site, Sst2_site, Yck_site, S338_S339~none)

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

Ste2 Internalization/Degradations

  • Yck protein kinases are required for the constitutive and pheromone-induced phosphorylation, ubiquitinylation and internalization of Ste2. Hicke et al. 1998 PMID 9548714
  • Endocytosis domain is residues 331-339 (SINNDAKSS). Dosil et al. 2000 PMID 10866688
  • S331, S338 and S339 are likely phosphorylation sites which, when phosphorylated, enable ubiquitination, likely at site K337. Hicke and Riezman 1996 PMID 8565073
  • Ste2 is weakly constitutively phosphorylated on the serines (S331, S338, S339) in the SYNNDAKSS sequence (residues 331-339) in the cytoplasmic tail, which results in ubiquitination and basal receptor internalization. Pheromone exposure results in hyperphosphorylation and hyper-ubiquitination, and much higher rate of internalization. Hicke et al. 1998 PMID 9548714
  • In the absence of pheromone constitutive internalization of Ste2 has a half-time of ~45 minutes. Mutation of S331, S338 and S339 to alanines increases the half-time to greater than 2 hours (see fig 4A, might be as high as 4-5 hours). Hicke et al. 1998 PMID 9548714
  • Mutation of S331, S338 and S339 to alanines in otherwise wild-type Ste2 has no appreciable effect on the Ste2 internalization rate upon exposure to pheromone, suggesting that phosphorylation of these residues is not responsible for the increased internalization rate in the presence of pheromone, despite the fact that S331, S338 and S339 are hyperphosphorylated in the presence of pheromone. Hicke et al. 1998 PMID 9548714
  • Hyperphosphorylation in the presence of pheromone of a Ste2 mutant missing residues 345-431 is abolished by the S331A S338A S339A mutations. Hicke et al. 1998 PMID 9548714
  • Ste2 internalization half-time of 5-6 min at 30°C (measuring either fraction of Ste2 remaining on the cell surface, or fraction of pheromone that is internalized). Hicke and Riezman 1996 PMID 8565073; Hicke et al. 1998 PMID 9548714
    • This is a surrogate for the pheromone-bound Ste2 internalization rate.
  • Ste2 degradation occurs via the vacuole rather than the proteasome. Hicke and Riezman 1996 PMID 8565073
  • Ste2 is internalized to background (undetectable) levels within 15 min of exposure to pheromone. Mulholland et al 1999 PMID 10069819
  • Pheromone induced endocytosis occurs through invaginations that are not part of the cortical actin patch. Mulholland et al 1999 PMID 10069819
  • Unoccupied receptors are endocytosed through oligomeric complexes with occupied receptors. Yesilaltay and Jenness 2000 PMID 10982387
  • Cells exposed to pheromone show a decrease in ability to bind pheromone for 20-30 minutes after stimulation, followed by an increase in pheromone binding capacity which after ~90 minutes surpasses the initial pheromone binding capacity. Experiments done at 22°C using 3H-labeled alpha factor. Jenness and Spatrick. 1986 PMID 3015412
    • The recovery of pheromone binding capacity is eliminated by treatment with cycloheximide.
    • Using cycloheximide to prevent new Ste2 synthesis, the half-life of receptors on the surface was estimated in the absence of pheromone (186 min at 22°C, 45 min at 34°C) and in the presence of 10 μM pheromone (18 min at 22°C, 10 min at 34°C).
  • A degradation rate (of unliganded Ste2) of 4 * 10-4 s-1 was measured or inferred. Yi et al. 2003 PMID 12960402
    • This same value was also subsequently used (referenced to Yi et al. 2003) by Kofahl and Klipp 2004 PMID 15300679
    • It is assumed that the steady state internalization rate and number of Ste2 molecules per cell were measured (probably via a Scatchard plot), and the synthesis rate inferred.
  • Bulk rate of ligand-bound receptor endocytosis was measured to be 4 * 10-3 s-1. Yi et al. 2003 PMID 12960402
    • This same value was also subsequently used (referenced to Yi et al 2003) by Kofahl and Klipp 2004 PMID 15300679
  • Either phosphorylation or ubiquitination is likely the rate limiting step in receptor endocytosis. Hick and Riezman 1996 PMID 8565073
  • 7.5 minutes after pheromone treatment, only 40% of Ste2 remains on the cell surface, 10% remains inside the cell, and the remainder has been degraded. Hicke and Riezman 1996 PMID 8565073
    • This suggests that internalized Ste2 is rapidly degraded.
  • Timecourse of Ste2 on surface in response to pheromone (no cycloheximide). Zanolari et al. 1992 PMID 1330324
  • In the presence of cycloheximide and pheromone, Ste2 is internalized with a half-time of 10 min at 34°C and 18 min at 22°C. In the absence of pheromone (but presence of cycloheximide), Ste2 is internalized with a half-time of 48 min at 34°C and 186 min at 22°C. Jenness and Spatrick. 1986 PMID 3015412
  • The half-time for Ste2 internalization after pheromone exposure is about 5 min at both 25°C and 37°C (in the absence of cycloheximide, determined by immunoEM). Mulholland et al. 1999 PMID 10069819
  • Pheromone is internalized with a half-time of 7.5 min (at 30°C). Rohrer et al. 1993 PMID 8392878
  • Receptor is removed from the cell surface with a half-time of 8 min (at 30°C) in response to pheromone treatment. Schandel and Jenness 1994 PMID 7935439
  • By 20min after pheromone treatment, the number of cell surface Ste2 molecules has dropped to background levels. Konopka et al. 1988 PMID 2842059
  • In the presence of cycloheximide and pheromone, Ste2 has a half-life of ~20 min (30°C). In the absence of pheromone (but presence of cycloheximide), Ste2 has a half-life of ~35 min (30°C). Since Ste2 levels were determined by Western blot, this includes internalized Ste2 proteins. Chen and Konopka 1996 PMID 8524302

Reaction Definition

It is unclear whether receptor phosphorylation, ubiquitination or internalization is the limiting step in pheromone induced receptor down-regulation (which occurs with a half-time of 4.6 minutes).

Assumptions:

  • Ste2 internalization is mediated solely by phosphorylation by Yck1, Yck2 and Yck3, which we will not discriminate between and refer to as Yck.
  • Yck has the same affinity for Ste2 regardless of whether Ste2 is ligand-bound or not. Yck has a faster phosphorylation rate for ligand-bound Ste2 than it does for Ste2 that is not ligand-bound.
    • If we were to instead assume that Yck has a higher affinity for ligand-bound Ste2, but same phosphorylation rate for both ligand-bound and -unbound Ste2, then reciprocally Ste2 must have a higher affinity for pheromone when Ste2 is bound by Yck. We assume that the differences in Ste2 phosphorylation arises due to a difference in phosphorylation rate rather than binding affinity because it turns out to be a simpler assumption.
  • Only one phosphorylation site (S338 or S339) on Ste2 is important for Ste2 internalization. (Assume this despite the fact that it appears that the SINNDAKSS serines are mainly involved in pheromone-independent Ste2 internalization, and the mechanism for Yck mediated pheromone-independent Ste2 internalization remains unclear.)
  • Yck is able to bind Ste2 independently of G protein binding.
  • Yck is unable to bind Ste2 when Sst2 is bound to Ste2 (since Sst2 binds the C-terminal tail of Ste2 where Yck phosphorylates it).
  • Yck's affinity for phosphorylated Ste2 is negligible.
  • Using phosphorylation rather than ubiquitination as the signal for internalization and degradation is sufficient for modeling purposes.
  • Receptor ubiquitinylation can be ignored and essentially included in either the receptor phosphorylation or internalization reactions. 


Ste2(Sst2_site, Yck_site, S338_S339~none) + Yck(Ste2_site) <-> 
   Ste2(Sst2_site, Yck_site!1, S338_S339~none).Yck(Ste2_site!1)





Ste2(Pheromone_site, Yck_site!2, S338_S339~none).Yck(Ste2_site!2) -> 
   Ste2(Pheromone_site, Yck_site, S338_S339~PO4) + Yck(Ste2_site)





Ste2(Pheromone_site!+, Yck_site!2, S338_S339~none).Yck(Ste2_site!2) -> 
   Ste2(Pheromone_site!+, Yck_site, S338_S339~PO4) + Yck(Ste2_site)





Ste2(S338_S339~PO4) + Cell -> Cell

  • In order for the degradation reaction to selectively degrade Ste2 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

Ste3 Internalization/Degradations

  • Ste3 undergoes constitutive endocyosis, with a half-life of 15min. Chen and Davis 2000 PMID 11062272
  • Constitutive endocytosis, but not ligand mediated endocytosis, requires Akr1p and Yck1p/Yck2p. Chen and Davis 2000 PMID 11062272
  • Constitutive endocytosis leads to receptor degradation. Chen and Davis 2002 PMID 11929601
  • Ligand-dependent endocytosis leads to receptor recycling. Chen and Davis 2002 PMID 11929601

Reaction Definition

For simplicity sake, we will only model a cells responding to α-factor, and not α cells responding to a-factor.

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