Abstract
G proteins (Galphabetagamma) are essential signaling molecules, which dissociate into Galpha and Gbetagamma upon activation by heptahelical membrane receptors. We have identified the betagamma subunit complex of the photoreceptor-specific G protein, transducin (T), as a target of the ubiquitin-proteasome pathway. Ubiquitylated species of the transducin gamma-subunit (Tgamma) but not the alpha- or beta-subunits were assembled de novo in bovine photoreceptor preparations. In addition, Tgamma was exclusively ubiquitylated when Tbetagamma was dissociated from Talpha. Ubiquitylation of Tbetagamma on Tgamma was selectively catalyzed by human ubiquitin-conjugating enzymes UbcH5 and UbcH7 and was coincident with degradation of the entire Tbetagamma subunit complex in vitro by a mechanism requiring ATP and the proteasome. We also show that Tbetagamma association with phosducin, a photoreceptor-specific protein of unknown physiological function, blocks Tbetagamma ubiquitylation and subsequent degradation. Phosphorylation of phosducin by Ca(2+)/calmodulin-dependent protein kinase II, which inhibits phosducin-Tbetagamma complex formation, completely restored Tbetagamma ubiquitylation and degradation. We conclude that Tbetagamma is a substrate of the ubiquitin-proteasome pathway and suggest that phosducin serves to protect Tbetagamma following the light-dependent dissociation of Talphabetagamma.
Highlights
G proteins (G␣␥) are essential signaling molecules, which dissociate into G␣ and G␥ upon activation by heptahelical membrane receptors
Identification of Transducin Subunits That Are Ubiquitylated in Vitro—To identify subunits of transducin that are ubiquitylated in vitro, ubiquitylation assays were conducted with supernatant from gradient-purified, dark-adapted bovine rod outer segments (ROS)
The inner segment is rich in 26 S proteasomes and phosducin, both implicated in interacting with T␥ (7–15, 18, 28 –31)
Summary
G proteins (G␣␥) are essential signaling molecules, which dissociate into G␣ and G␥ upon activation by heptahelical membrane receptors. Dark-dependent phosducin phosphorylation by protein kinase A (PKA) or by Ca2ϩ/calmodulin-dependent protein kinase II (CaMKII) reduces phosducin binding to T␥ by 3- and 300-fold, respectively [11,12,13,14,15] These observations suggested a mechanism in which the cycle of phosducin phosphorylation/ dephosphorylation regulates the light sensitivity of the photoreceptor by controlling the amount of transducin available for activation. Multiple ubiquitin molecules can be subsequently attached to a substrate as an isopeptide-linked polyubiquitin chain Such chains preferentially target the substrate moiety of the Ub-protein conjugate for degradation by the 26 S proteasome, a multicatalytic, ATP-dependent protease (reviewed in Ref. 25). The best recognized function of ubiquitylation is selective targeting of proteins for rapid degradation [22, 23], ubiquitylation per se can regulate protein trafficking, phosphorylation, and other nonproteolytic fates (reviewed in Ref. 26)
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