Figure 6. The Chaperone and GEF Activity of Ric-8A Requires Recognition of the Gα C Terminus.

(A) Stabilization of the nucleotide-bound state of Ga_i1 by the Ric-8A C terminus and the importance of the α5 helix-Ric-8A interaction for the GEF activity of Ric-8A. The effect of the MBP-fused unphosphorylated “U” or phosphorylated “P” C terminus of Ric-8A (423–530) on Gα GDP release was tested with wild-type Gα_i1 or Gα_i1 D354 mutant proteins that were preloaded with ³H-GDP. The amount of bound ³H-GDP after 20 min incubation with or without Ric-8A was determined using a filter binding method and scintillation counting. Error bars are the mean ±SD, n R 3.

(B) Ribbon diagram of the Ric-8A-Gα_q complex highlighting putative sites of interaction with the Gα C terminus (green), α5 helix (yellow), or Ras-like domain (blue).

(C) Knockout of RIC-8A in HEK293T cells results in a Gα_q abundance defect due to Gα misfolding (Papasergi-Scott et al., 2018). The rescue of Gα_q abundance upon stable expression of Ric-8A mutants was analyzed by quantitative immunoblotting of cell membrane samples. Representative western blot of membrane-bound Gα_q is shown with quantification of Ga_q levels as percentage of HEK293T Gα_q abundance. Error bars are the mean ± SEM, n = 3.