Background: Cofilin is a low-molecular weight actinmodulating protein, which binds to, severs, and depolymerizes actin filaments in vitro. Aip1, an actininteracting protein, was recently identified as a product of a gene on a multicopy plasmid which suppresses the temperature-sensitive phenotype of a cofilin mutant in Saccharomyces cerevisiae. Actin cytoskeleton plays an essential role in resistance to hyperosmotic stress in Dictyostelium discoideum. The roles of cofilin and Aip1 in this resistance are not known.

Farnesylation of the gamma-subunit of the retinal G-protein, transducin (Talpha/Tbetagamma), is indispensable for light-initiated signaling in photoreceptor cells. However, the farnesyl-mediated molecular interactions important for signaling are not well understood. To explore this issue, we created a functional Tbetagamma analogue in which the farnesyl group was replaced with a (3-azidophenoxy)geranyl (POG) group, a novel farnesyl analogue with a distal photoreactive azido group. In the presence of lipid membranes and/or Talpha-GDP, UV irradiation of POG-modified Tbetagamma (POG-Tbetagamma) invariably yielded a cross-linked product Tgamma-Tbeta, reflecting a constitutive interaction of the Tgamma C-terminal lipid with Tbeta. In addition to the Tgamma-Tbeta adduct, a Tgamma-Talpha cross-link was detected in the aqueous fraction. Reconstitution of POG-Tbetagamma with Talpha and light-activated rhodopsin (Rh) in photoreceptor membranes resulted in cross-linking of Tgamma with a glycerophospholipid, indicating molecular interaction of the farnesyl group with cellular membranes. The Tgamma-phospholipid cross-link was observed only in the presence of both Talpha-GDP and Rh, and was abolished by the addition of GTPgammaS or by replacing Rh with opsin. These findings suggest a transient farnesyl-membrane interaction occurs only in a signaling state formed in a transducin-Rh ternary complex. On the other hand, UV irradiation of POG-Tbetagamma in a soluble complex with phosducin, a negative regulator of G-protein, yielded a Tgamma-phosducin adduct in addition to the Tgamma-Tbeta cross-link. These results illustrate that, rather than being a static membrane anchor, the farnesyl moiety plays an active role in the dynamics of protein-protein and protein-membrane interactions at defined steps in the signal transduction process.

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*.