Purification and characterization of the ternary complex between phosducin‐like protein, the G protein β subunit and the cytosolic chaperonin ‐ an important intermediate in the assembly of the G protein βγ dimer

Abstract

Phosducin‐like protein 1 (PhLP1) has been recently shown to be an essential co‐chaperone, along with the cytosolic chaperonin containing the tailless‐complex polypeptide 1 (CCT), in the folding and assembly of the nascent G protein βγ subunit dimer (Gβγ). It has been proposed that a ternary PhLP1‐Gβ‐CCT complex forms during the folding process with nascent Gβ in the CCT folding cavity and PhLP above the cavity contacting Gβ and the apical domains of CCT. If PhLP1 is phosphorylated in a cluster of serine residues near its N‐terminus (S18–20) by the protein kinase CK2, it will release Gβ from CCT in a PhLP1‐Gβ complex that is then able to interact with Gγ and form the Gβγ dimer. To further test the proposed mechanism of Gβγ assembly, we have isolated the PhLP1‐Gβ‐CCT ternary complex in both states of PhLP1 phosphorylation from insect cells over‐expressing both PhLP and Gβ using a tandem affinity purification method. In addition, we have isolated the Gβ‐CCT complex in the absence of PhLP1. These complexes have been subjected to structural determination by cryo‐electron microscopy in an effort to determine the mechanism by which PhLP1 phosphorylation results in the release of PhLP1‐Gβ from CCT.