Figure 1
Yeast G protein/MAP kinase reporter cascade. In S. cerevisiae strains BY1142 and BY1173, activation of an episomally expressed mammalian GPCR stimulates nucleotide exchange on a chimeric Gα subunit. The newly free βγ moiety activates a MAP kinase cascade that leads to transcription of the HIS3 reporter gene, allowing growth on histidine-deficient medium.
Table 1
G protein activation by E2F8
| ADE2 plasmid | Signaling | Fluorescence |
| E2F8 | +++++++ | - |
| E2F8-YFP | +++++++ | + |
| C5aR NQ | ++++ | - |
| Vector | - | - |
Yeast BY1142, which expresses a chimeric subunit, was transformed with the indicated ADE2 plasmids along with empty URA3 vector. Transformants were replica-plated onto Ura- His- Ade1 mM plates containing varying concentrations of 3-AT. Yeast were assessed for growth after 3 days. The maximal concentration of 3-AT tolerated by the transformants is indicated: 100 mM (+++++++), 50 mM (++++++), 20 mM (+++++), 10 mM (++++), 5 mM (+++), 2 mM (++), 1 mM (+), or 0 mM (-). Fluorescence was determined by examining yeast colonies on a dissecting microscope and is reported as either + (fluorescent colony) or – (non-fluorescent colony). C5aR NQ, a constitutively active mutant of the C5a receptor, was used as a positive control.
Figure 2
E2F8 effect on receptor-mediated signaling. Yeast BY1142 was transformed with a PFUS1-LacZ reporter construct (TRP1 selection), then transformed with plasmids encoding E2F8 or empty vector (ADE2 selection) and C5aR or empty vector (URA3 selection). The agonist W5Cha was used to activate the receptor. After four hours of ligand treatment, cells were lysed and β-galactosidase activity was measured by colorimetry. This experiment is representative of three independent trials, each of which examined three transformants per condition.
Table 2
Epistasis mapping and G protein specificity of E2F8 signaling
| Yeast strain | Gα subunit | Reporter cascade mutations | ADE2 plasmid | Signaling |
| BY1142 | Gαi3,Gpa1(1–41) | Intact reporter cascade | E2F8 | +++++++ |
| Vector | - | |||
| BY1187 | Gαi3,Gpa1(1–41) | ΔSte4Gβ | E2F8 | - |
| Vector | - | |||
| BY1206 | Gαi3,Gpa1(1–41) | ΔSte11MAPKKK | E2F8 | - |
| Vector | - | |||
| BY1207 | Gαi3,Gpa1(1–41) | ΔSte12 (MAPK substrate) | E2F8 | - |
| Vector | - | |||
| BY1173 | Gpa1DCGLF | Intact reporter cascade | C5aR NQ | ++++ |
| E2F8 | - | |||
| Vector | - |
A plasmid encoding E2F8 or empty vector (ADE2 selection) and empty URA3 vector were transformed into the indicated yeast strains. Transformants were replicated onto selective plates containing 3-AT and assessed for growth after 3 days. The strength of signaling is reported as in Table 1.
Table 3
Effect of RGS4 on E2F8 signaling
| ADE2 plasmid | URA3 plasmid | Signaling | Fluorescence |
| C5aR NQ | Vector | ++++ | - |
| RGS4-GFP | + | + | |
| E2F8 | Vector | +++++++ | - |
| RGS4-GFP | + | + | |
| Vector | Vector | - | - |
| RGS4-GFP | - | + |
Yeast BY1142 was transformed with the indicated plasmids. Three transformants per condition were replicated onto selective plates containing 3-AT and assessed for growth after 3 days. The strength of signaling and fluorescence is reported as in Table 1.
Figure 3
Domain structure of E2F8. E2F8 includes two E2F homology domains, both of which contain the RRXYD consensus motif that interacts directly with DNA. The region 400–630 has 16% amino acid identity with GAP-43, a known activator of heterotrimeric G proteins. Approximate boundaries of the homology regions are indicated. The percentage of amino acid identity to human E2F1 and E2F7 is indicated for the regions 1–200 and 610–867.
Figure 4
Structure/function analysis of E2F8. (A) Yeast BY1142 was transformed with the indicated E2F8 mutants (ADE2 selection) along with empty URA3 vector. Transformants were replicated onto selective plates containing 3-AT and assessed for growth after 3 days. The strength of signaling and fluorescence is reported as in Table 1. (B) Yeast cells expressing E2F8 mutants were grown in liquid culture, lysed, and examined by immunoblotting using an anti-GFP (top) antibody. The membrane was stripped and reprobed using an anti-β-actin antibody (bottom) as a loading control. The 5' UTR-E2F8-YFP construct contained the wild-type 5' UTR in addition to E2F8; the other full-length construct lacked the UTR.