Article

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

Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 03/2013; 110(10):3794-9. DOI: 10.1073/pnas.1220943110
Source: PubMed

ABSTRACT We have shown that resistance to inhibitors of cholinesterase 8 (Ric-8) proteins regulate an early step of heterotrimeric G protein α (Gα) subunit biosynthesis. Here, mammalian and plant cell-free translation systems were used to study Ric-8A action during Gα subunit translation and protein folding. Gα translation rates and overall produced protein amounts were equivalent in mock and Ric-8A-immunodepleted rabbit reticulocyte lysate (RRL). GDP-AlF-bound Gαi, Gαq, Gα13, and Gαs produced in mock-depleted RRL had characteristic resistance to limited trypsinolysis, showing that these G proteins were folded properly. Gαi, Gαq, and Gα13, but not Gαs 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-AlF-bound Gα subunit trypsin protection. Dramatic results were obtained in wheat germ extract (WGE) that has no endogenous Ric-8 component. WGE-translated Gαq was gel filtered and found to be an aggregate. Ric-8A supplementation of WGE allowed production of Gαq that gel filtered as a ∼100 kDa Ric-8A:Gαq heterodimer. Addition of GTPγS to Ric-8A-supplemented WGE Gαq translation resulted in dissociation of the Ric-8A:Gαq heterodimer and production of functional Gαq-GTPγS monomer. Excess Gβγ supplementation of WGE did not support functional Gαq production. The molecular chaperoning function of Ric-8 is to participate in the folding of nascent G protein α subunits.

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    ABSTRACT: Abstract Resistance to inhibitors of cholinesterase 8 proteins (Ric-8A and Ric-8B) collectively bind the four classes of heterotrimeric G protein α subunits. Ric-8A and Ric-8B act as non-receptor guanine nucleotide exchange factors (GEFs) toward the Gα subunits that each binds in vitro and seemingly regulate diverse G protein signaling systems in cells. Combined evidence from worm, fly and mammalian systems has shown that Ric-8 proteins are required to maintain proper cellular abundances of G proteins. Ric-8 proteins support G protein levels by serving as molecular chaperones that promote Gα subunit biosynthesis. In this review, the evidence that Ric-8 proteins act as non-receptor GEF activators of G proteins in signal transduction contexts will be weighed against the evidence supporting the molecular chaperoning function of Ric-8 in promoting G protein abundance. I will conclude by suggesting that Ric-8 proteins may act in either capacity in specific contexts. The field awaits additional experimentation to delineate the putative multi-functionality of Ric-8 towards G proteins in cells.
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