RGS(Sst2)/Galpha(Gpa1)/Receptor(Ste2) interactions

From Yeast Pheromone Model

Jump to: navigation, search

Back to main model page



Contents

Sst2/Ste2 interaction

  • Mutations in either of the DEP domains of Sst2 result in a phenotype (heightened sensitivity to pheromone) similar to deletion of Sst2, demonstrating that the DEP domain is required for Sst2 function. Ballon et al. 2006 PMID 16990133
    • Coupling of Sst2's N terminal 419 residues (which contains its DEP domains) to the RGS domain from Rgs2 in sst2Δ cells greatly reduces response to pheromone (even less sensitive to pheromone than WT cells), demonstrating that the DEP domains are likely responsible for the specificity of Sst2, rather than its RGS domain.
    • There is no direct evidence that DEP mutants of Sst2 fail to bind to Gpa1, although the mutants are apparently unable to act as GAPs.
    • There is no evidence that Ste2 acts as an allosteric activator of Sst2. The RGS domain of Sst2 binds Gpa1 and has the GAP activity.
  • Sst2 binds the C-terminal tail of Ste2 (residues 297-431). Ballon et al. 2006 PMID 16990133
    • Purified Sst2-Myc pulls-down detergent-solubilized Ste2 in vitro, and this interaction is eliminated by mutations in Sst2's DEP domain.
    • No Gpa1 can be detected in the pull-down, and the interaction
    • Interaction between Sst2 and Ste2 still occurs when His6-Sst2 is expressed from baculovirus, and Ste2 is expressed in sst2Δ ste2Δ gpa1Δ ste4Δ cells, suggesting that the interaction is direct.
    • Sst2-GFP colocalizes with endocytosis-deficient Ste2(7K-to-R)-mCherry, and this interaction is eliminated when the C-terminal tail (residues 297-431) is deleted. This colocalization is detected in cells containing GPA1(G302S), a mutant that fails to interact with Sst2's RGS domain.
    • Sst2-GFP colocalizes with Ste2(297-431)-mCherry (in a GPA1(G302S) strain), suggesting that the interaction with the C-terminal tail of Ste2 is sufficient to localize Sst2 to the plasma membrane.
    • Sst2's DEP domain and Ste2's C-terminal tail are distant from the sites that each of the proteins uses to interact with Gpa1, suggesting that the three proteins may be able to bind each other cooperatively.
  • Sst2 interacts poorly (or not at all) with Ste2 that has been phosphorylated by Yck1 and Yck2. Ballon et al. 2006 PMID 16990133
    • Sst2-GFP co-localizes with endocytosis-deficient Ste2(7K-to-R)-mCherry only at the shmoo tip, but not around the rest of the periphery of the cell, suggesting that it only interacts with newly-synthesized Ste2 (since newly synthesized proteins are directed to the shmoo tip).
    • Sst2-GFP co-localizes with endocytosis-deficient Ste2(7K-to-R)-mCherry all around the cell periphery in yck1 yck2 cells. Mutation of the 19 serine and threonine residues in the C-terminal tail results in colocalization around the entire cell periphery in cells containing WT Yck1 and Yck2.
    • This may be a mechanism to prevent endocytosis of Sst2 with Ste2, and keep it localized to the shmoo tip where nascent Ste2 is deposited.

Reaction Definition

Since direct binding between Sst2 and Gpa1 has only been detected at 0°C, and does not appear to be strong enough to localize Sst2 to the membrane where Gpa1 is located, it seems likely that the main form of interaction between Gpa1 and Sst2 is through their co-binding to Ste2. Thus we will assume that Gpa1 and Sst2 do not interact directly, but Sst2 and Ste2 do bind directly.

Assumptions:

  • Sst2 cannot bind to Ste2 while Sst2 is bound to Fus3 or Kss1 (no evidence for this), but Sst2's binding to Ste2 is unaltered by Sst2 phosphorylation on S539 (since this residue is distal to the DEP domains).
Ste2(Sst2_site, Yck_site, S338_S339~none) + Sst2(Ste2_site, MAPK_site) <-> 
   Ste2(Sst2_site!1, Yck_site, S338_S339~none).Sst2(Ste2_site!1, MAPK_site)


Sst2/Gpa1 interaction

  • Sst2 and Gpa1 likely bind to each other (based on co-purification from wild-type yeast with GST-tagged Gpa1), independent of whether Gpa1 is bound to GTP or GDP. Dohlman et al. 1996 PMID 8756677
  • In pulldown experiments done at 0°C with recombinant Sst2 and Gpa1 proteins expressed in E. coli, binding was similar in the presence GDP and GTPγS and was increased with GDP-AlF4-, suggesting that Sst2 has highest affinity for transition state intermediate of Gpa1-GTP hydrolysis. Apanovitch et al. 1998 PMID 9537998
  • Sst2 binds to the C-terminal tail of Ste2, and Gpa1 binds to a different region of Ste2, so Ste2 may bridge an interaction between Sst2 and Gpa1. Ballon et al. 2006 PMID 16990133
    • There is no evidence that Sst2 is allosterically activated by its binding to Ste2, so it is likely that binding to Ste2 is essential for increasing the local concentration of Sst2 in the vicinity of Gpa1.
  • In GPA1 ste2Δ cells, Sst2-GFP is not localized to the membrane, suggesting that its interaction with Gpa1-GDP is much weaker than Sst2's interaction with Ste2 (which can localize Sst2-GFP to the membrane). Ballon et al. 2006 PMID 16990133
  • Overexpression of Sst2 is able to inhibit mating. Konopka 1993 PMID 8413281
    • Since over-expressed Sst2 does not appear to out compete Ste4:Ste18 for binding to Gpa1 (as this would result in activation of mating response rather than suppression of mating response), it seems likely that Sst2 is able to bind Gpa1 independently of Ste4:Ste18.
  • Sst2's S539 phosphorylation site is distal to its Gpa1 binding site, meaning that it is likely to be able to be phosphorylated while bound to Gpa1. Garrison et al. 1999 PMID 10593933
  • Mutation in Gpa1 of G302S makes Gpa1 insensitivie to the effects of Sst2. Dixit et al 2014 PMCID: PMC4142594

Reaction Definition

Evidence suggests that Sst2 does not compete with Ste4:Ste18 for binding to Gpa1. Since direct binding between Sst2 and Gpa1 has only been detected at 0°C, and does not appear to be strong enough to localize Sst2 to the membrane where Gpa1 is located, it seems likely that the main form of interaction between Gpa1 and Sst2 is through their co-binding to Ste2. Thus we will assume that Gpa1 and Sst2 do not interact directly.

Personal tools