title: Molecular chaperoning function of Ric-8 is to fold nascent heterotrimeric G protein α subunits.
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-AlF4(-)-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-AlF4(-)-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.
authors: Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA.
authors: Puiyee
authors: P
authors: Chan
authors: Celestine J
authors: CJ
authors: Thomas
authors: Stephen R
authors: SR
authors: Sprang
authors: Gregory G
authors: GG
authors: Tall
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived; D002417:Cattle; D002474:Cell-Free System; D056404:Chaperonin Containing TCP-1 / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018832:Molecular Chaperones / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D014908:Triticum / Q000378:metabolism
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D044385:GTP-Binding Protein alpha Subunits; D020662:Guanine Nucleotide Exchange Factors; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D056404:Chaperonin Containing TCP-1