FUSION BETWEEN-RETINAL ROD OUTER SEGMENT MEMBRANES AND MODEL MEMBRANES - A ROLE FOR PHOTORECEPTOR PERIPHERIN RDS/

Peripherin/rds plays an essential role in the maintenance of photorece ptor rod cell disk membrane structure. The purification of this protei n to homogeneity [Boesze-Battaglia, K., et al. (1997) Biochemistry 36, 6835-6846] has allowed us to characterize the functional role of peri pherin/rds in the maintenance of rod outer segment (ROS) membrane fusi on processes. Utilizing a cell-free fusion assay system, we report tha t the fusion of R-18-labeled ROS plasma membrane (R-18-PM) with disk m embranes 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 syntheti c 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 necess ary to promote membrane fusion. PP-5 was able to inhibit R-18-PM disk membrane fusion and promoted ANTS/DPX contents mixing in a pure vesicl e system. This peptide (PP-5) promoted calcium-induced vesicle aggrega tion of phosphatidylethanolamine:phosphatidylserine LUVs. FTIR analysi s confirmed the structural prediction of this peptide as alpha-helical . When modeled as an alpha-helix, this peptide is amphiphilic with a h ydrophobicity index of 0.75 and a hydrophobic moment of 0.59. PP-5 has substantial biochemical and functional homology with other well-chara cterized membrane fusion proteins. These results demonstrate the neces sity for peripherin/rds in ROS membrane fusion, specifically the requi rement for an intact C-terminal region of this protein.