Interacting Targets of the Farnesyl of Transducin γ-Subunit

[Image: see text] G protein γ-subunits are isoprenylated and carboxyl methylated at the C-terminal cysteine residue, and the set of the posttranslational modifications is indispensable for the function of the photoreceptor G protein transducin (Tα/Tβγ). To explore farnesyl-mediated molecular interactions, we investigated molecular targets of a Tβγ analogue that was engineered to have a photoreactive farnesyl analogue, (3-azidophenoxy)geranyl (POG), covalently bound to the C-terminal cysteine of Tγ. POG-modified Tβγ was further subjected to modification by methylation at the C-terminal carboxyl group, which copies a complete set of the known posttranscriptional modifications of Tβγ. Photoaffinity labeling experiment with the photoreactive Tβγ analogue in its free form indicated that the POG moiety of Tγ interacted with Tβ. In the trimeric Tα/Tβγ complex, the POG moiety was cross-linked with Tα in addition to concurrent affinity labeling of Tβ. When photoreactive Tβγ was reconstituted with Tα and light-activated rhodopsin (Rh*) in rod outer segment (ROS) membranes, the POG moiety interacted with not only Tα and Tβ but also Rh* and membrane phospholipids. The cross-linked phospholipid species was analyzed by ELISA employing a variety of lipid-binding probes, which revealed phosphatidylethanolamine (PE) and phosphatidylserine (PS) as selective phospholipids for POG interaction in the ROS membranes. These results demonstrate that the modifying group of Tγ plays an active role in protein−protein and protein−membrane interactions and suggest that the farnesyl−PE/PS interaction may support the efficient formation of the signaling ternary complex between transducin and Rh*.