Electrostatic and Lipid Anchor Contributions to the Interaction of Transducin with Membranes

Abstract

The heterotrimeric G protein transducin is a key component of the vertebrate phototransduction cascade. Transducin is peripherally attached to membranes of the rod outer segment, where it interacts with other proteins at the membrane-cytosol interface. However, upon sustained activation by light, the dissociated G t ␣ and G␤ 1 ␥ 1 subunits of transducin translocate from the outer segment to other parts of the rod cell. Here we used a computational approach to analyze the interaction strength of transducin and its subunits with acidic lipid bilayers, as well as the range of orientations that they are allowed to occupy on the membrane surface. Our results suggest that the combined constraints of electrostatics and lipid anchors substantially limit the rotational degrees of freedom of the membrane-bound transducin heterotrimer. This may contribute to a faster transducin activation rate by accelerating transducin-rhodopsin complex formation. Notably, the membrane interactions of the dissociated transducin subunits are very different from those of the heterotrimer. As shown previously, G␤ 1 ␥ 1 experiences significant attractive interactions with negatively charged membranes, whereas our new results suggest that G t ␣ is electrostatically repelled by such membranes. We suggest that this repulsion could facilitate the membrane dissociation and intracellular translocation of G t ␣. Moreover, based on similarities in sequence and electrostatic properties, we propose that the properties described for transducin are common to its homologs within the G i subfamily. In a broader view, this work exemplifies how the activity-dependent association and dissociation of a G protein can change both the affinity for membranes and the range of allowed orientations, thereby modulating G protein function.The G protein transducin is a key molecular switch in vertebrate rod cells (1-3). Like other heterotrimeric G proteins, transducin is peripherally attached to the lipid bilayer by covalent lipid modifications of the N terminus of the ␣ subunit and the C terminus of the ␥ subunit (4, 5). During the rapid course of a photoresponse, each photoexcited rhodopsin activates tens of transducin heterotrimers (G t ) at a rate of hundreds of G t molecules per second (6, 7). This activation catalyzes GDP/GTP exchange on G t ␣, which then dissociates from G␤ 1 ␥ 1 and proceeds to activate the effector, cGMP phosphodiesterase. The fact that these protein-protein interactions take place at the membrane-cytosol interface is a key reason why the phototransduction cascade operates so rapidly and efficiently (8, 9), thus motivating a more detailed investigation of G t -membrane interactions.The attachment of transducin to the disk membranes of rod outer segments is reversible, allowing transducin to undergo activation-dependent translocation from the light-sensitive outer segment compartment to the rest of the photoreceptor cell (10). A similar phenomenon was also shown for the invertebrate visual G protein, G q (11). The efficiency of transducin transl...

Functional Comparison of Rod and Cone Gαt on the Regulation of Light Sensitivity*

“…The heterotrimeric transducin is localized to the membranous ROS in the darkadapted rods due to the synergistic effect of the lipid modifications on the ␣ and ␥ subunit (38,39). Consistent with this idea, G␤ 1 ␥ 1 localization is no longer restricted to ROS and instead is evenly dispersed in the rT␣Ϫ/Ϫ rods (40).…”

In vivo optophysiology reveals that G-protein activation triggers osmotic swelling and increased light scattering of rod photoreceptors

“…Because the G-protein heterotrimer Gα t β 1 γ 1 is present in the dark-adapted rod OS at 1:8 relative to rhodopsin (36,37) and has more than twice rhodopsin's mass per molecule, it comprises ∼20% of the total mass of transduction-associated proteins in the dark-adapted rod OS. In the dark-adapted state, inactive Gα t β 1 γ 1 is anchored to OS disc membranes by N-terminal myristoylation of Gα t and C-terminal farnesylation of Gγ 1 (38). Activation of Gα t by light exposures that isomerize large fractions of rhodopsin causes the Gα t and Gβ 1 γ 1 subunits to dissociate not only from one another, but also from the disc membrane, as established by translocation of Gα t and Gβ 1 γ 1 to the IS in vivo in prolonged bright light (39).…”

Structural and kinetic modeling of an activating helix switch in the rhodopsin-transducin interface

“…In the alternative ''sequential fit'' model, the 2 spatially distant binding sites on G t , namely the G␥ C terminus with its farnesyl anchor and the G␣ C terminus, act sequentially (42). The G␥ C terminus has a function in the initial encounter interaction, which is thought to depend on proper membrane anchoring of the G protein heterotrimer by electrostatics and lipid modifications (12,43,44). The actual catalytic interaction, which leads to nucleotide exchange, occurs between the G␣ C terminus and R*.…”