R9AP targeting to rod outer segments is independent of rhodopsin and is guided by the SNARE homology domain

Abstract

In vertebrate photoreceptor cells, rapid recovery from light excitation is dependent on the RGS9⋅Gβ5 GTPase-activating complex located in the light-sensitive outer segment organelle. RGS9⋅Gβ5 is tethered to the outer segment membranes by its membrane anchor, R9AP. Recent studies indicated that RGS9⋅Gβ5 possesses targeting information that excludes it from the outer segment and that this information is overridden by association with R9AP, which allows outer segment targeting of the entire complex. It was also proposed that R9AP itself does not contain specific targeting information and instead is delivered to the outer segment in the same post-Golgi vesicles as rhodopsin, because they are the most abundant transport vesicles in photoreceptor cells. In this study, we revisited this concept by analyzing R9AP targeting in rods of wild-type and rhodopsin-knockout mice. We found that the R9AP targeting mechanism does not require the presence of rhodopsin and further demonstrated that R9AP is actively targeted in rods by its SNARE homology domain.

FIGURE 1:

R9AP localization and expression in rhodopsin-knockout mice. (A) R9AP (green) immunostaining of retinal cross sections from Rho^−/− and wild-type mice (bars, 10 μm). Nuclei are counterstained with Hoechst (blue). (B) Representative Western blot showing R9AP and β-tubulin bands in wild-type (WT) and Rho^−/− (-/-) retinas. (C) The signal intensity of each R9AP Western blot band was normalized to the density of the β-tubulin band and plotted as a relative fluorescence. The data are averaged from measurements performed with six wild-type and six Rho^−/− retinal samples.

FIGURE 2:

Localization of FLAG-tagged R9AP constructs in mouse rods. (A) Cartoon diagram illustrating R9AP domain composition: Habc trihelical bundle, SNARE homology, and transmembrane (TM). The following FLAG-tagged constructs were electroporated into wild-type mouse rods: (B) full-length R9AP, (C) R9AP lacking Habc, (D) SNARE homology (SH) fused to transmembrane domain, (E) Habc fused to transmembrane domain, (F) transmembrane domain alone, and (G) GFP-tagged transmembrane segment 1 from mGluR1. Nuclei are counterstained with Hoechst (blue). IS, inner segment; OS, outer segment; N, outer nuclear layer; S, synapses. Bars, 10 μm. Each construct is illustrated schematically at the top, with the FLAG tag depicted as a green star.

FIGURE 3:

The SNARE homology domain is sufficient for normal R9AP targeting. Representative images of wild-type mouse retinas electroporated with the following CCIIL fused constructs: (A) FLAG-tagged SNARE homology (SH) domain, (B) FLAG-tagged Habc domain, and (C) GFP. Bars, 10 μm.

FIGURE 4:

Subcellular localization of various SNARE homology domains expressed in mouse rods. Wild-type mouse retinas were electroporated with three FLAG-tagged SNARE homology domains fused to the transmembrane domain of R9AP: (A) syntaxin 3, (B) R7BP, and (C) R9AP. Bars, 10 μm.

FIGURE 5:

Intracellular targeting of R9AP's SNARE homology domain is independent of endogenous R9AP. Images of R9AP^−/− mouse retinas electroporated with the following constructs: (A) CCIIL fused, FLAG-tagged SNARE homology domain, (B) FLAG-tagged full-length R9AP, and (C) GFP-tagged transmembrane segment 1 from mGluR1. Bars, 10 μm.