Phosducin interacts with the G-protein beta gamma-dimer of ciliate protozoan Blepharisma japonicum upon illumination

摘要

Immunological techniques and high-resolution FRET analysis were employed to investigate the in vivo colocalization and interaction of phosducin (Pdc) with the beta gamma-subunits of G-protein (G beta gamma) in the ciliate Blepharisma japonicum. Immunological techniques revealed that illumination of cells resulted in a decrease in phosphorylation levels of Pdc and its colocalization with G beta gamma. The observed light-induced Pdc dephosphorylation was also accompanied by significant enhancement of G beta gamma binding by this molecule. Possible formation of the Pdc-G beta gamma complex in cells exposed to light was corroborated by FRET between these proteins. Treatment of cells with okadaic acid, an inhibitor of phosphatase activity, entirely prevented Pdc dephosphorylation by light, colocalization of this phosphoprotein with G beta gamma and generation of the Pdc-G beta gamma complex. Cell fractionation and immunoblotting revealed that in cells exposed to light, the formation of Pdc-G beta gamma complex and its translocation into the cytoplasm occur simultaneously with a change in the gel migration of G beta. Moreover, a 33 kDa immunoanalog of 14-3-3 protein was identified and we showed that this protein is bound by phosphorylated Pdc in a cell adapted to darkness. The results of this study provide additional detailed characterization of the functional properties of the ciliate Pdc. The likely functional role of Pdc in Blepharisma is discussed.