Molecular chaperoning function of Ric-8 is to fold nascent heterotrimeric G protein α subunits

We have shown that resistance to inhibitors of cholinesterase 8 (Ric-8) proteins regulate an early step of heterotrimeric G protein alpha (G alpha) subunit biosynthesis. Here, mammalian and plant cell-free translation systems were used to study Ric-8A action during G alpha subunit translation and protein folding. G alpha translation rates and overall produced protein amounts were equivalent in mock and Ric-8A-immunodepleted rabbit reticulocyte lysate (RRL). GDP-AlF4--bound G alpha i, G alpha q, G alpha 13, and G alpha s produced in mock-depleted RRL had characteristic resistance to limited trypsinolysis, showing that these G proteins were folded properly. G alpha i, G alpha q, and G alpha 13, but not Gas produced from Ric-8A-depleted RRL were not protected from trypsinization and therefore not folded correctly. Addition of recombinant Ric-8A to the Ric-8A-depleted RRL enhanced GDP-AlF4--bound Ga subunit trypsin protection. Dramatic results were obtained in wheat germ extract (WGE) that has no endogenous Ric-8 component. WGE-translated G alpha q was gel filtered and found to be an aggregate. Ric-8A supplementation of WGE allowed production of G alpha q that gel filtered as a similar to 100 kDa Ric-8A:G alpha q heterodimer. Addition of GTP gamma S to Ric-8A-supplemented WGE G alpha q translation resulted in dissociation of the Ric-8A:G alpha q heterodimer and production of functional G alpha q-GTP gamma S monomer. Excess G beta gamma supplementation of WGE did not support functional G alpha q production. The molecular chaperoning function of Ric-8 is to participate in the folding of nascent G protein alpha subunits.