Chan_2013_Proc.Natl.Acad.Sci.U.S.A_110_3794

We have shown that resistance to inhibitors of cholinesterase 8 (Ric-8) proteins regulate an early step of heterotrimeric G protein alpha (Galpha) subunit biosynthesis. Here, mammalian and plant cell-free translation systems were used to study Ric-8A action during Galpha subunit translation and protein folding. Galpha translation rates and overall produced protein amounts were equivalent in mock and Ric-8A-immunodepleted rabbit reticulocyte lysate (RRL). GDP-AlF4(-)-bound Galphai, Galphaq, Galpha13, and Galphas produced in mock-depleted RRL had characteristic resistance to limited trypsinolysis, showing that these G proteins were folded properly. Galphai, Galphaq, and Galpha13, but not Galphas 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 Galpha subunit trypsin protection. Dramatic results were obtained in wheat germ extract (WGE) that has no endogenous Ric-8 component. WGE-translated Galphaq was gel filtered and found to be an aggregate. Ric-8A supplementation of WGE allowed production of Galphaq that gel filtered as a approximately 100 kDa Ric-8A:Galphaq heterodimer. Addition of GTPgammaS to Ric-8A-supplemented WGE Galphaq translation resulted in dissociation of the Ric-8A:Galphaq heterodimer and production of functional Galphaq-GTPgammaS monomer. Excess Gbetagamma supplementation of WGE did not support functional Galphaq production. The molecular chaperoning function of Ric-8 is to participate in the folding of nascent G protein alpha subunits.