Towards the proteome of the rhodopsin-bearing post-Golgi compartment of retinal photoreceptor cells

Abstract

Polarized sorting of rhodopsin in retinal rod photoreceptor cells is mediated by post-Golgi carrier membranes that bud from the trans-Golgi network and fuse with the specialized domain of the plasma membrane in the rod inner segment. The identity of the majority of the resident proteins of this organelle still remains elusive, despite multifaceted approaches to study this compartment. In the present study we have taken a proteomic approach to the analysis of the post-Golgi carriers. First, we modified the previously established fractionation protocols in order to achieve greater purity of the isolated membranes. Specifically, the new fractionation scheme depleted the post-Golgi fraction of cytosolic proteins that were the most abundant contaminants complicating analysis of two-dimensional (2-D) gel profiles in our previous preparations. The isolated membranes were subjected to 2-D gel electrophoresis, immunoblotting and microsequencing. This analysis showed that the improved subcellular fractionation yielded a fraction highly enriched in rhodopsin-bearing post-Golgi carrier membranes. Two-dimensional mapping revealed 29 proteins that are preferentially found in this fraction and therefore represent candidates for post-Golgi membrane-specific proteins. This preparation of rhodopsin-bearing post-Golgi carriers is a first step towards the proteomics of this important organelle.

Calcium-Dependent Assembly of Centrin-G-Protein Complex in Photoreceptor Cells

“…The visual G protein, as an essential component of the visual transduction cascade, is abundantly present at the disk membrane of the photoreceptor outer segment. Studies by Deretic and coworkers have previously indicated that, after de novo synthesis in the proximal inner segment, G t subunits are cotransported with opsin-bearing membrane vesicles to the apical membrane at the base of the connecting cilium (11,35). To our knowledge, the present immunoelectron microscopic detection is the first subcellular localization of G t ␣-and thus presumably of the G t holoprotein--in the cilium.…”

The Arf GEF GBF1 and Arf4 synergize with the sensory receptor cargo, rhodopsin, to regulate ciliary membrane trafficking

“…PNS was separated into three fractions designated as the Golgi/TGN/ER-enriched, RTC-enriched (T/G/E and RTCs hereafter) and cytosol, as previously reported (Deretic, 2000;Deretic et al, 1995Deretic et al, , 1996Mazelova et al, 2009;Morel et al, 2000;Wang et al, 2012). In agreement with microscopy data, GBF1 was present in the T/G/E fraction, on RTCs and in the cytosol (Fig.…”

Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4)

“…Alternatively, more than one ARF may crosslink to the rhodopsin C-terminal peptide. ARF3, which is present in the photoreceptor Golgi (31,34), is a potential candidate for this interaction.…”