- Gpa1 is N-myristoylated, which is required for proper localization to the membrane and Gpa1's interaction with Ste4:Ste18 Dohlman et al. 1993 PMID 8415763
- During normal growth, about half of Gpa1 is myristoylated
- Upon pheromone exposure, a greater fraction of Gpa1 becomes myristoylated.
- There is minimal conversion from unmyristoylated population to myristoylated population. The majority of the myristoylated Gpa1 is formed immediately upon Gpa1 synthesis.
- Total amount of G protein per cell was measured by quantitative Western blots and fluorescent quantification to be 10000 per cell. Yi et al. 2003 PMID 12960402
- Quantitative Western blots were used to show that there are ~8000 Gpa1 molecules per cell prior to pheromone stimulation, and about 50% more than that after pheromone stimulation. Hao et al. 2003 PMID 12968019
- They showed that only about 5500 molecules per cell of Gpa1 are myristoylated.
- Quantitative Western blots were used to show that there are ~2390 Gpa1 molecules per cell prior to pheromone stimulation. Thomson et al. In preparation
- Total Gpa1 concentration of 0.2 nM (which equates to ~7800 molecules per cell for a 5 μm cell) was used in a model but not referenced. Hao et al. 2003 PMID 12968019
- Gpa1(Q323L) lacks nucleotide hydrolysis activity and results in a constitutively active phenotype. Wu et al. 2004 PMID 15197187
- Gpa1 contains a MAPK-binding sequence at residues 21-33. Metodiev et al. 2002 PMID 12029138
Gpa1-mediated signal transmission
- When overexpressed in WT cells (with WT abundance of WT Gpa1), the GTPase-deficient Gpa1(Q323L) mutant greatly increases Fus1-lacZ gene expression both in the absence and presence of pheromone. Guo et al. 2003 PMID 14536090
- In contrast, overexpression of WT Gpa1 suppresses signaling.
- Co-expression of Gpa1(Q323L) does not induce cell cycle arrest, as halo size is unaffected, and cells develop shmoos but continue to divide. This distinguishes Gpa1(Q323L)-mediated signaling from typical signaling resulting from free Ste4 (which causes both gene expression and cell cycle arrest).
- The activity of Gpa1(Q323L) does not require Ste2, but requires Ste4, Ste11 and Ste7.
- Further supporting the notion that Gpa1(Q323L)-mediated signaling is distinct from free-Ste4-mediated signaling, the effects of overexpression of Gpa1(Q323L) and Ste4 are approximately additive across a range of pheromone concentrations.
- When Gpa1(Q323L) is expressed in gpa1Δ cells, it is unable to suppress constitutive response at all, suggesting that Gpa1(Q323L) is unable to sequester Gβγ.
- Scp160 may be involved in this mechanism, as Scp160 binds active Gpa1 in vitro, and deletion of Scp160 eliminates any effect of expression of Gpa1(Q323L).
- Active Fus3 appears to bind active Gpa1. Metodiev et al. 2002 PMID 12029138
- His-tagged Gpa1 expressed and purified from E. coli is able to pull Fus3-Myc out of cell lysates of pheromone treated cells.
- His-tagged Gpa1 expressed and purified from E. coli fails to pull Fus3-Myc out of cell lysates of cells that were not exposed to pheromone.
- Gpa1 contains a putative MAPK-binding sequence at residues 21-33.
- Mutations K21E and R22E disrupt the MAPK docking site but do not affect the induction profile of a reporter or prevent cell cycle arrest. They are, however, required for gradient tracking and shmoo morphogenesis Errede et al 2015 PMCID: PMC4569322
- Mutation of the putative MAPK-binding sequence in Gpa1 (K21E R22E) reduces the mating efficiency to 1/15th of WT efficiency. Metodiev et al. 2002 PMID 12029138
- Gpa1(K21E R22E) results in wild-type Fus3-GFP localization in the absence of pheromone, and higher than normal nuclear localization upon pheromone treatment. Blackwell et al. 2003 PMID 12556475
- Expression of Gpa1(Q323L) in WT cells causes cells to shmoo, but does not lead to cell cycle arrest. Slessareva et al. 2006 PMID 16839886
- Gpa1-dependent signaling appears to be dependent on the yeast PI3K (phosphatidylinositol 3-kinase). Slessareva et al. 2006 PMID 16839886
- Deletion or inhibition of either of the PI3K subunits Vps15 or Vps34 eliminates Gpa1(Q323L)-dependent gene expression.
- Deletion of Vps15 or Vps34 eliminates Gpa1(Q323L)-dependent Fus3 phosphorylation (but not Kss1 phosphorylation), reduces pheromone-mediated Fus3 phosphorylation (but not Kss1 phosphorylation), and reduces mating efficiency.
- Vps30 and either Vps38 or Atg14 also exist is a complex with Vps34, but deletion of either Vps30 or Vps38 does not affect Gpa1-mediated signaling.
- Deletion of either Vps15 or Vps34 results in a 5-7 fold increase in the pathway EC50.
- Active Gpa1 appears to activate PI3K activity in the endosome, which partially activates the mating pathways. Slessareva et al. 2006 PMID 16839886
- WT Gpa1 is primarily localized to the plasma membrane, whereas the constitutively active Gpa1(Q323L) allele is primarily localized to the endosome.
- Deletion of either Vps15 or Vps34 reduces Gpa1(Q323L) endosomal localization.
- Vps15 interacts preferentially with inactive Gpa1 (both by copurification and by in vitro binding).
- Vps34 copurifies with inactive and active Gpa1, and interacts in vivo preferably with active Gpa1. Presumably copurification with inactive Gpa1 is partially mediated by Vps34's interaction with Vps15.
- Expression of Gpa1(Q323L) causes a significant (<2-fold) increase in cellular PI3P, and relocalization of weak PI3P interacter Bem1 to the endosome.
Pheromone/Receptor/G protein interactions
G protein nucleotide hydrolysis/exchange
Ste12 mediated protein synthesis
Protein dilution/synthesis due to cell growth
<modelMoleculeType>Gpa1(Ste2_site, Ste4_site, nucleotide~GDP~GTP)</modelMoleculeType>
<modelSeedSpecies>Gpa1(Ste2_site, Ste4_site, nucleotide~GDP) Gpa1_tot_conc</modelSeedSpecies>