Regulatory interaction of phosducin-like protein with the cytosolic chaperonin complex

Abstract

Phosducin and phosducin-like protein (PhLP) bind G protein ␤␥ subunits and regulate their activity. This report describes a previously uncharacterized binding partner unique to PhLP that was discovered by coimmunoprecipitation coupled with mass spectrometric identification. Chaperonin containing tailless complex polypeptide 1 (CCT), a cytosolic chaperone responsible for the folding of many cellular proteins, binds PhLP with a stoichiometry of one PhLP per CCT complex. Unlike protein-folding substrates of CCT, which interact only in their nonnative conformations, PhLP binds in its native state. Native PhLP competes directly for binding of protein substrates of CCT and thereby inhibits CCT activity. Overexpression of PhLP inhibited the ability of CCT to fold newly synthesized ␤-actin by 80%. These results suggest that the interaction between PhLP and CCT may be a means to regulate CCT-dependent protein folding or alternatively, to control the availability of PhLP to modulate G protein signaling.

Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

“…This interaction was later confirmed in yeast protein-protein interaction screens [68,69]. Interestingly, Pdc did not share the ability to binding CCT with PhLP [67]. CCT is an essential chaperone of protein folding found in the cytosol of eukaryotic cells [70,71].…”

“…Among the known substrates of CCT are Gα [77] and several proteins with seven β-propeller WD40 structures similar to that of Gβ [71,78,79]. PhLP1 did not bind CCT as a folding substrate, but rather it interacted in its native form, suggesting a regulatory role for PhLP1 in CCT-dependent folding [67].…”

“…Clues about other functions of PhLP1 came from a proteomics screen for PhLP1 binding partners in which a high affinity interaction of PhLP1 with the cytosolic chaperonin complex (CCT) was discovered [67]. This interaction was later confirmed in yeast protein-protein interaction screens [68,69].…”

“…PhLPs 1-3 all contain an acidic sequence in the loop between Helix 2 and 3 of the Pdc structure that is not well-conserved in Pdc [20]. This acidic region has been shown to play an important role in the binding of PhLP1 to CCT [18,80], and accordingly PhLPs1-3 all bind CCT while Pdc does not [30,67,86]. Apart from this loop and the Helix 3 region that follows, there is very little homology in the N-terminal domain between Pdc subfamily members [20].…”

“…Apart from this loop and the Helix 3 region that follows, there is very little homology in the N-terminal domain between Pdc subfamily members [20]. PhLP2 and PhLP3 are believed to bind CCT in a manner analogous to PhLP1, as native binding partners and regulators of CCT and not as nascent folding substrates [30,67,86]. These findings suggest that PhLP2 and PhLP3 might function like PhLP1 as cochaperones with CCT in protein folding.…”