N-Terminal Fatty Acylation of Transducin Profoundly Influences Its Localization and the Kinetics of Photoresponse in Rods

Abstract

N-terminal acylation of the ␣-subunits of heterotrimeric G-proteins is believed to play a major role in regulating the cellular localization and signaling of G-proteins, but physiological evidence has been lacking. To examine the functional significance of N-acylation of a well understood G-protein ␣-subunit, transducin (G␣ t ), we generated transgenic mice that expressed a mutant G␣ t lacking N-terminal acylation sequence (G␣ t G2A). Rods expressing G␣ t G2A showed a severe defect in transducin cellular localization. In contrast to native G␣ t , which resides in the outer segments of dark-adapted rods, G␣ t G2A was found predominantly in the inner compartments of the photoreceptor cells. Remarkably, transgenic rods with the outer segments containing G␣ t G2A at 5-6% of the G␣ t levels in wild-type rods showed only a sixfold reduction in sensitivity and a threefold decrease in the amplification constant. The much smaller than predicted reduction may reflect an increase in the lateral diffusion of transducin and an increased activation rate by photoexcited rhodopsin or more efficient activation of cGMP phosphodiesterase 6 by G␣ t G2A; alternatively, nonlinear relationships between concentration and the activation rate of transducin also potentially contribute to the mismatch between the amplification constant and quantitative expression analysis of G␣ t G2A rods. Furthermore, the G2A mutation reduced the GTPase activity of transducin and resulted in two to three times slower than normal recovery of flash responses of transgenic rods, indicating the role of G␣ t membrane tethering for its efficient inactivation by the regulator of G-protein signaling 9 GTPase-activating protein complex. Thus, N-acylation is critical for correct compartmentalization of transducin and controls the rate of its deactivation.

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

“…3), supporting the suggestion in Ref. 78 that association with G␤ 1 ␥ 1 is required to efficiently reattach G t ␣ to membranes.…”

Transducin γ-Subunit Sets Expression Levels of α- and β-Subunits and Is Crucial for Rod Viability

“…This supports the hypothesis that transducin heterotrimer is retained on the photoreceptor disc membranes in outer segments through the combined action of two lipid modifications, G␥ 1 isoprenylation and G␣ t acylation [for review, see Calvert et al (2006); for the most recent updates, see Kerov et al (2007) and Rosenzweig et al (2007)]. Interestingly, the distribution of G␣ t in G␥ 1 knock-out rods and G␤ 1 ␥ 1 in G␣ t knock-out rods is somewhere in between the dark-and light-adapted distributions in wild-type rods, with more transducin present in the outer segment than in lightadapted wild-type mice.…”

Trp fluorescence reveals an activation‐dependent cation‐π interaction in the Switch II region of Gαi proteins

“…Not reflected in any Ga structure is N-terminal myristoylation, which has been shown in some systems to act as a myristoyl switch, 42,43 with effects on spatial and temporal regulation of signaling 44 in the visual system. Our previous work suggested an immobile and solvent excluded environment for myristoylated N-terminal residues even in the absence of Gbc, using a series of extrinsic probes linked to specific Nterminal residues in Ga i HI proteins.…”