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First published online November 19, 2007
Journal of Experimental Biology 210, 4213-4223 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.005132
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Phosducin interacts with the G-protein β{gamma}-dimer of ciliate protozoan Blepharisma japonicum upon illumination

Katarzyna Sobierajska, Hanna Fabczak and Stanislaw Fabczak*

Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Street, PL-02-093 Warsaw, Poland

* Author for correspondence (e-mail: s.fabczak{at}nencki.gov.pl)

Accepted 18 September 2007

Immunological techniques and high-resolution FRET analysis were employed to investigate the in vivo colocalization and interaction of phosducin (Pdc) with the β{gamma}-subunits of G-protein (Gβ{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 Gβ{gamma}. The observed light-induced Pdc dephosphorylation was also accompanied by significant enhancement of Gβ{gamma} binding by this molecule. Possible formation of the Pdc–Gβ{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 Gβ{gamma} and generation of the Pdc–Gβ{gamma} complex. Cell fractionation and immunoblotting revealed that in cells exposed to light, the formation of Pdc–Gβ{gamma} complex and its translocation into the cytoplasm occur simultaneously with a change in the gel migration of Gβ. 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.

Key words: Blepharisma japonicum, β{gamma}-dimer, G-protein, phosducin, 14-3-3, protein, photophobic response, protein phosphorylation, Pdc–Gβ{gamma} interaction, translocation, confocal imaging, cell fractionation, FRET, immunological analysis






© The Company of Biologists Ltd 2007