Deletional analysis of the rod photoreceptor cell peripherin/RDS carboxy-terminal region.
The C-terminal region of peripherin/rds contains three predicted alpha-helical domains. One of these domains, corresponding to amino acids 311-322, form an amphiphilic alpha-helix previously shown to promote membrane fusion. The present studies were conducted to determine how the additional alpha-helical regions of the peripherin/rds C-terminus affect complex formation with rom-1, glycosylation, intracellular localization and membrane fusion properties. Bovine peripherin/rds and rom-1 were epitope tagged with an amino-terminal FLAG-tag or amino-terminal hemagglutinin (HA)-tag, respectively, and cloned into the pCI-neo expression vector for transient transfection into COS cells. Similarly, four C-terminal peripherin/rds truncation mutants (Delta1, Delta2, Delta3 and Delta4), corresponding to deletions of -19, -29, -39 and -59 amino acids were designed to disrupt the alpha-helical domains. Immunofluorescence microscopy and enzymatic digestions demonstrated that full-length peripherin/rds and the four C-terminal deletion mutants were localized to intracellular membranes and were all Endo-H sensitive. Western blotting and immunoprecipitation studies showed that the FLAG-tagged bovine peripherin/rds (full-length) was expressed as a 76kDa dimer, which associates with HA-tagged rom-1 to form a higher order complex. The deletion mutants were also able to associate with rom-1. However, when analyzed using non-denaturing tricine electrophoresis, full-length peripherin/rds and the Delta1, Delta2 and Delta3 mutants formed homo-oligomeric complexes, while the Delta4 mutant appeared to form only homodimers suggesting a region upstream of amino acid 300 may be involved in C-terminal interactions. Membrane fusion was then evaluated using fluorescence resonance energy transfer (RET) techniques. Intracellular COS cell membranes containing full-length peripherin/rds fused with rod outer segment plasma membrane vesicles. This fusion was inhibited with the addition of a synthetic peptide (PP-5) corresponding to the fusion domain of peripherin/rds. In contrast, fusion was negligible with any of the C-terminal truncation mutants. Collectively, these results suggest that in addition to the fusion domain, other regions of the peripherin/rds C-terminus are required for fusion. Most interesting is the observation that the last 19amino acids, a region downstream of the fusion peptide that is deleted in the Delta1 mutant, appear to be necessary for fusion. This region corresponds to the epitope for anti-peripherin/rds monoclonal antibody 2B6, which is shown to partially inhibit peripherin/rds mediated membrane fusion.
Department of Molecular Biology, University of Medicine and Dentistry of New Jersey--School of Osteopathic Medicine and UMDNJ-GSBS-Stratford Division, 2 Medical Center Drive, Stratford, NJ 08084, USA. muller@umdnj.edu
Susan
S
Muller-Weeks
Kathleen
K
Boesze-Battaglia
Catherine
C
Fitzgerald
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000235:genetics; D017353:Gene Deletion; D006031:Glycosylation; D007381:Intermediate Filament Proteins / Q000235:genetics*; D008561:Membrane Fusion / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008815:Mice, Inbred Strains; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics*; D064531:Peripherins; D011233:Precipitin Tests; D012162:Retinal Degeneration / Q000235:genetics*; D017948:Retinal Rod Photoreceptor Cells / Q000503:physiopathology*; D012374:Rod Cell Outer Segment / Q000503:physiopathology; D059470:Tetraspanins
D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D064531:Peripherins; C578996:Prph2 protein, mouse; C081477:Rom1 protein, mouse; D059470:Tetraspanins