TY - JOUR
T1 - Molecular chaperoning function of Ric-8 is to fold nascent heterotrimeric G protein α subunits
AU - Chan, Pui Yee
AU - Thomas, Celestine J.
AU - Sprang, Stephen R.
AU - Tall, Gregory G.
PY - 2013/3/5
Y1 - 2013/3/5
N2 - 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 theseGproteins 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 ofWGE allowed production ofGαq that gel filtered as a ∼100 kDa Ric-8A:Gαq heterodimer. Addition of GTPγS toRic-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.
AB - 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 theseGproteins 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 ofWGE allowed production ofGαq that gel filtered as a ∼100 kDa Ric-8A:Gαq heterodimer. Addition of GTPγS toRic-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.
KW - Chaperone
KW - GEF
UR - http://www.scopus.com/inward/record.url?scp=84874604106&partnerID=8YFLogxK
U2 - 10.1073/pnas.1220943110
DO - 10.1073/pnas.1220943110
M3 - Article
C2 - 23431197
AN - SCOPUS:84874604106
SN - 0027-8424
VL - 110
SP - 3794
EP - 3799
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 10
ER -