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 f usion domain located within the C-terminus (Boesze-Battaglia et al., Bioche mistry 37 (1998) 9477-9487). A peptide analogue to this region, PP-5, compe titively inhibits peripherin/rds mediated fusion in a cell free assay syste m. To characterize how this region is involved in the fusion process we inv estigated two of the individual steps in membrane fusion, membrane adhesion and membrane destabilization inferred from depolarization studies. Membran e depolarization was measured as the collapse of a valinomycin induced K+ d iffusion potential in model membranes, using a potential sensitive fluoresc ent probe, diS-C-2-5. PP-5 induced membrane depolarization in a concentrati on dependent manner. PP-5 has been shown by Fourier transform infrared spec troscopy to be an amphiphilic alpha-helix. Therefore, the requirement for a n amphiphilic alpha-helix to promote depolarization was tested using two mu tant 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 induce d depolarization when added in an equimolar ratio to PP-5. Neither mutant p eptide alone or in combination with PP-5 had any effect on calcium dependen t vesicle aggregation. Using non-denaturing gel electrophoresis and size ex clusion chromatography techniques PP-5 was shown to form a tetrameric compl ex. Equimolar mixtures of PP-5 and PP-5AB formed a heterotetramer which was unable to promote membrane depolarization. The hypothesis that PP-5 tetram ers promote membrane depolarization is consistent with the calculated Hill coefficient of 3.725, determined from a Hill analysis of the depolarization data. (C) 2000 Elsevier Science B.V. All rights reserved.