Departmental Papers (Dental)

Document Type

Journal Article

Date of this Version

2-15-2000

Publication Source

Biochimica et Biophysica Acta - Biomembranes

Volume

1463

Issue

2

Start Page

343

Last Page

354

DOI

10.1016/S0005-2736(99)00226-6

Abstract

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-C2-5. PP-5 induced membrane depolarization in a concentration dependent manner. PP-5 has been shown by Fourier transform infrared spectroscopy to be an amphiphilic α-helix. Therefore, the requirement for an amphiphilic α-helix to promote depolarization was tested using two mutant peptides designed to disrupt either the amphiphilic nature of PP-5 (PP-5AB) or the α-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. Copyright (C) 2000 Elsevier Science B.V.

Keywords

Membrane fusion; Peripherin/rds and fusion peptide; Photoreceptor

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Date Posted: 08 December 2022

This document has been peer reviewed.