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 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.
Boesze-Battaglia
K
K
Department of Molecular Biology, University of Medicine and Dentistry of New Jersey-SOM, Stratford 08084, USA. battagli@umdnj.edu
Lamba
O P
OP
Napoli
A A
AA
Sinha
S
S
Guo
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001666:Binding Sites, Antibody; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D002474:Cell-Free System; D005136:Eye Proteins / Q000502:physiology*; D006868:Hydrolysis; D007381:Intermediate Filament Proteins / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D008081:Liposomes; D008561:Membrane Fusion; D008562:Membrane Glycoproteins; D008954:Models, Biological; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000502:physiology; D064531:Peripherins; D012374:Rod Cell Outer Segment / Q000378:metabolism / Q000502:physiology*; D017550:Spectroscopy, Fourier Transform Infrared; D014357:Trypsin
D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D008081:Liposomes; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D064531:Peripherins; D014357:Trypsin