Fusion between Retinal Rod Outer Segment Membranes and Model Membranes:  A Role for Photoreceptor Peripherin/rds

Abstract

Peripherin/rds plays an essential role in the maintenance of photoreceptor rod cell disk membrane structure. The purification of this protein to homogeneity [Boesze-Battaglia, K., et al. (1997) Biochemistry 36, 6835-6846] has allowed us to characterize the functional role of peripherin/rds in the maintenance of rod outer segment (ROS) membrane fusion processes. Utilizing a cell-free fusion assay system, we report that the fusion of R18-labeled ROS plasma membrane (R18-PM) with disk membranes or peripherin/rds-enriched large unilammellar vesicles (LUVs) is inhibited upon trypsinolysis of peripherin/rds. To understand this phenomenon, we tested the ability of a series of overlapping synthetic C-terminal peripherin/rds peptides to mediate model membrane fusion. Within the 63 amino acid long region of the C-terminus, we identified a minimal 15 residue long amino acid sequence (PP-5), which is necessary to promote membrane fusion. PP-5 was able to inhibit R18-PM disk membrane fusion and promoted ANTS/DPX contents mixing in a pure vesicle system. This peptide (PP-5) promoted calcium-induced vesicle aggregation of phosphatidylethanolamine:phosphatidylserine LUVs. FTIR analysis confirmed the structural prediction of this peptide as alpha-helical. When modeled as an alpha-helix, this peptide is amphiphilic with a hydrophobicity index of 0.75 and a hydrophobic moment of 0.59. PP-5 has substantial biochemical and functional homology with other well-characterized membrane fusion proteins. These results demonstrate the necessity for peripherin/rds in ROS membrane fusion, specifically the requirement for an intact C-terminal region of this protein.

Role of the Second Intradiscal Loop of Peripherin/rds in Homo and Hetero Associations

“…This region of P/rds has been found to promote membrane fusion activity as well as membrane targeting (13,14,35). Past experiments have shown that the deletion of the C-terminal tail of P/rds abolishes the fusogenic activity but does not have any negative effects on protein dimerization or complex assembly with Rom-1 (14).…”

“…Although P/rds possesses a clear role in maintaining the structure of rods and cones, insights into its molecular mechanism through protein interactions are quite limited. Currently, P/rds is conjectured to play a significant role in disk morphogenesis and stability (1,11), disk shedding (1,12), and membrane fusion (13,14 Cloning and identification of P/rds orthologs suggests that these proteins share several distinctive features, including four hydrophobic transmembrane domains, a large intradiscal (D2) loop containing seven highly conserved cysteines, and a highly charged cytosolic Cterminal segment (4,15,16). Over 70% of the disease-causing mutations in P/rds are located in the D2 loop, providing evidence of the importance that this region plays in proper protein function.…”

Retinal Degeneration Slow (RDS) Glycosylation Plays a Role in Cone Function and in the Regulation of RDS·ROM-1 Protein Complex Formation

“…Its N-and C-terminal domains are cytosolic, whereas its two extracellular loop domains (D1 and D2) project into the enclosed discs in rods and the extracellular space between lamellae in cones (24). The C-terminal domain is known to direct the targeting of RDS complexes to the OS, engage in protein-protein interactions, and form an amphipathic helix that is thought to play a role in maintaining the unique membrane topology of the OS itself by potentially regulating membrane fusion or curvature (25)(26)(27)(28)(29).…”

ROM-1 potentiates photoreceptor specific membrane fusion processes

“…Previous studies in our laboratory have suggested that disks at the apical tip of the OS fuse spontaneously to delineate a packet of disks (Boesze-Battaglia 1997) and that the tetraspanin protein, peripherin/rds (P/rds a.k.a. Prph, Rds and peripherin-2) participates in this membrane fusion process in vitro (Boesze-Battaglia 1997;Boesze-Battaglia, Kong et al 1997;Boesze-Battaglia, Lamba et al 1998). Deletion of a region including the amphiphilic fusion peptide domain of P/rds in transgenic Xenous laevis resulted in the mis-localization of the mutated P/rds (Tam, Moritz et al 2002;Tam, Moritz et al 2004), supporting a key functional role for this domain.…”