Ste2

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Contents 1 About Ste2 1.1 Ste2 dimerization 1.2 Ste2 Abundance 2 Reactions 3 Species Representation 3.1 Molecule Type 3.2 Model Seed

About Ste2

• G protein coupled receptor (GPCR) in MATa cells only.

• Can replace Ste2 with Candida albicans α-factor receptor, and using exogenous source of α-factor, can induce successful mating between a and α S. cerevisiae cells. Janiak et al. 2005 PMID 15749052
  

• Glycosylation at N25 and N32 are thought to be involved in the sorting of misfolded Ste2 proteins to the cell membrane. Mentesana and Konopka 2001 PMID 11583169

• Ste2 is phosphorylated at T382, T384, S385, T411, and T414l. Ficarro et al. 2003 PMID 12509440

Ste2 dimerization

• The existence of dominant negative Ste2 mutants is consistent with Ste2 dimerization. Dosil et al. 1998 PMID 9742115; Leavitt et al. 1999 PMID 10485282

• The hypersensitivity of endocytosis-defective Ste2 mutants is partially rescued by coexpression of WT Ste2. Reneke et al. 1988 PMID 2844413

• Overall Ste2 oligomerization is unaffected by ligand binding and G protein activation. Overton and Blumer 2000 PMID 10744981
  • Oligomerization detected between CFP/YFP tagged Ste2 lacking the C terminal tail.
  • FRET efficiency is unaffected by overexpression of another cell surface protein, suggesting that FRET between Ste2-YFP and Ste2-CFP.
  • Treatment of cells with pheromone does not alter the FRET efficiency.
  • FRET efficiency in purified plasma membrane fractions is unchanged by G protein activation or pheromone treatment.

• Ste2 may be endocytosed as a dimer/oligomer. Overton and Blumer 2000 PMID 10744981
  • CFP/YFP tagged Ste2 lacking the C terminal tail has an endocytosis defect (in response to pheromone), which is overcome by coepxression of WT Ste2.
  • This effect is specific because another cell surface protein, when coexpressed with WT Ste2, is not endocytosed in response to pheromone treatment.

• Ste2 may function as a dimer. Overton and Blumer 2000 PMID 10744981
  • A Ste2 mutant was isolated (M250I) that binds pheromone weakly, and increases the EC50 ~10-fold.
  • Coexpression of Ste2(M250I) with WT Ste2, both at WT abundances, causes a similar 10-fold increase in the EC50.
  • This is unlikely caused by sequestration of G proteins because when the M250I mutation was combined with a mutation that prevents G protein binding, the result is unchanged.

• Unoccupied receptors are endocytosed through oligomeric complexes with occupied receptors. Yesilaltay and Jenness 2000 PMID 10982387
  • Ste2-HA and Ste2-GFP coprecipitate when coexpressed, but not when cells expressing each tagger species are mixed prior to protein extraction.
  • Pheromone treatment does not change the coprecipitation efficiency.
  • Ste2(S184R), which does not bind pheromone, is efficiently internalized in response to pheromone treatment only when coexpressed with WT Ste2, whereas another abundant cell surface protein is not.

• Ste2 homo-oligomerizes with equal efficiency in the endoplasmic reticulum and the plasma membrane. Overton and Blumer 2002 PMID 12194975

• Ste2 is thought to oligomerize through GXXXG motif at residues 56-60 in the first transmembrane domain. Overton et al. 2003 PMID 14506226
  • Mutants with oligomerization defects, although expressed at WT levels, were not efficiently targeted to the plasma membrane.
  • Pheromone-binding was unaffected in oligomerization-defective mutants.
  • Cells defective in Ste2 oligomerization have signaling defects that are proportional to their oligomerization defects.

• Ste2 oligomerization is important for delivery to the cell surface membrane, and it appears to be important for proper signal transduction. Overton et al. 2003 PMID 14506226

• Ste2 subunits in an oligomer may cooperate to bind or activate G protein molecules. Chinault et al. 2004 PMID 14764600
  • Ste2 mutants that are defective in pheromone binding or G protein coupling, when coexpressed, do not rescue signaling, suggesting that Ste2 subunits in an oligomer are independently activated.
  • Different Ste2 mutants that are defective in G protein coupling, when coexpressed, partially rescue signaling, suggesting that Ste2 subunits in an oligomer may cooperate to bind or activate G protein molecules.

• Two models of Ste2 oligomers interacting with G protein, no strong evidence either way: Overton and Blumer 2005 PMID 16339714
  1. Each Ste2 in oligomer can bind its own G protein.
  2. Ste2 monomers in oligomer cooperate to bind a single G protein.

• Spectrally resolved FRET shows that nearly all Ste2 exist in homo-dimers (and not higher order oligomers). Raicu et al. 2005 PMID 15352875
  • The authors did not examine the oligomerization state in the presence of pheromone.
  • Because overall FRET doesn't change with pheromone treatment, it remains possible that pheromone causes a transition from dimers to high order oligomers.

• Ste2 oligomerization can also be dectected by BRET, but unlike FRET, it can be detected between full-length receptor molecules. Gehret et al. 2006 PMID 16709573

Ste2 Abundance

Varying Ste2 abundance by changing expression level over a wide range has little or no effect on pheromone sensitivity (EC50). However, decreased expression (below WT levels) decreases maximal activation. Sridharan et al. 2016 PMID: 27646004

• 8000 per cell via scatchard plot (35S, 30', 22C, # replicates unknown, 4202 strains, live cells with endocytosis and synthesis ongoing). Jenness et al. 1986 PMID 3023832

• 7600 per cell via scatchard plot (3H, 30', 22C,triplicates, RC629 strains, cells treated with N3Na and KF). David et al. 1997 PMID 9182592

• 3900 per cell via scatchard plot (35S, 30', ?C, replicates unknown, DJ211-5-3 strains, cells treated with N3Na and KF). Dosil et al. 2000 PMID 10866688

• 3420 per cell via scatchard plot. Chen and Konopka 1996 PMID 8524302

• 4900 per cell via scatchard plot (35S, 60', RT, duplicates, JE112 strains, cells treated with N3Na and KF). Weiner et al. 1993 PMID 8385135

• 5300 per cell via scatchard plot (35S, 30 min at 22°, triplicates, RK511-6B strain). Blumer et al. 1988 PMID 2839507

• 13,000 per cell via scatchard plot (3H, 30', 22C, # replicates unknown, 4202 bar1Δ strains, cells treated with N3Na and KF and p-tosyl-L-arginine methyl ester to stop energy metabolism and receptor internalization). Raths et al. 1988 PMID 2846561

• 11,000 per cell via scatchard plot (35S, unknown specifics, JKY78 bar1Δ strain with STE2 on CEN/ARS plasmid). Dube and Konopka 1998 PMID 9819407

• 1100 per cell via scatchard plot (35S, 30', RT, duplicates, FY70 strains, cells treated with N3Na and KF). Clark et al 1994 PMID 8132618
  • This is substantially lower than all the other estimates.

• 8000 per cell via Scatchard plot (35S, 24°C or 30°C, Bar1^-, binding done at 0°C to prevent receptor synthesis and endocytosis). Chvatchko et al. 1986 PMID 3524852

Reactions

Pheromone/Receptor/G protein interactions
Ste2 synthesis/endocytosis/degradation
G protein nucleotide hydrolysis/exchange
RGS(Sst2)/Galpha(Gpa1)/Receptor(Ste2) interactions
Ste12 mediated protein synthesis
Non-specific dephosphorylation
Protein dilution/synthesis due to cell growth

Species Representation

Molecule Type

Ste2(Pheromone_site, Gpa1_site, Sst2_site, Yck_site, S338_S339~none~PO4)

moleculizer-Ste2-definition

Model Seed

Ste2(Pheromone_site, Gpa1_site, Sst2_site, Yck_site, S338_S339~none) Ste2_tot_conc

moleculizer-Ste2-initial-population