A peptide analogue to a fusion domain within photoreceptor peripherin/rds promotes membrane adhesion and depolarization.

Photoreceptor peripherin/rds promotes membrane fusion, through a putative fusion domain located within the C-terminus (Boesze-Battaglia et al., Biochemistry 37 (1998) 9477-9487). A peptide analogue to this region, PP-5, competitively inhibits peripherin/rds mediated fusion in a cell free assay system. To characterize how this region is involved in the fusion process we investigated two of the individual steps in membrane fusion, membrane adhesion and membrane destabilization inferred from depolarization studies. Membrane depolarization was measured as the collapse of a valinomycin induced K(+) diffusion potential in model membranes, using a potential sensitive fluorescent probe, diS-C(2)-5. PP-5 induced membrane depolarization in a concentration dependent manner. PP-5 has been shown by Fourier transform infrared spectroscopy to be an amphiphilic alpha-helix. Therefore, the requirement for an amphiphilic alpha-helix to promote depolarization was tested using two mutant peptides designed to disrupt either the amphiphilic nature of PP-5 (PP-5AB) or the alpha-helical structure (PP-5HB). PP-5AB inhibited PP-5 induced depolarization when added in an equimolar ratio to PP-5. Neither mutant peptide alone or in combination with PP-5 had any effect on calcium dependent vesicle aggregation. Using non-denaturing gel electrophoresis and size exclusion chromatography techniques PP-5 was shown to form a tetrameric complex. Equimolar mixtures of PP-5 and PP-5AB formed a heterotetramer which was unable to promote membrane depolarization. The hypothesis that PP-5 tetramers promote membrane depolarization is consistent with the calculated Hill coefficient of 3.725, determined from a Hill analysis of the depolarization data.

Purification and light-dependent phosphorylation of a candidate fusion protein, the photoreceptor cell peripherin/rds.

The proteins peripherin/rds and rom-1 form a protein complex in the rims of photoreceptor outer segment disk membranes. Peripherin/rds plays an essential role in the morphogenesis and maintenance of disk membrane structure, with peripherin/rds gene mutations resulting in photoreceptor cell degeneration. We report two different chromatographic procedures for the purification of native peripherin/rds from bovine photoreceptor cell outer segments and show that the protein is a phosphoprotein that promotes membrane fusion in vitro. During one procedure, peripherin/rds was copurified in association with rom-1 by hyroxylapatite and Mono Q FPLC. During the other, it was purified free from rom-1 by concanavalin-A affinity chromatography and chromatofocusing. Analysis of homogeneous peripherin/rds from the second procedure showed that exposure of photoreceptor outer segments to light resulted in the incorporation of nearly 2 mol of phosphate per mole of peripherin/rds and a concomitant shift in the isoelectric point of the protein. In addition, we found that recombination of purified peripherin/rds into lipid vesicles increased membrane fusion, with more rapid fusion detected with phosphorylated peripherin/rds. In conclusion, studies with purified peripherin/rds reveal that the protein undergoes light-dependent phosphorylation and that it may function in membrane fusion.