Deletional Analysis of the Rod Photoreceptor Cell Peripherin/RDS Carboxy-Terminal Region

Abstract

The C-terminal region of peripherin/rds contains three predicted α-helical domains. One of these domains, corresponding to amino acids 311-322, form an amphiphilic α-helix previously shown to promote membrane fusion. The present studies were conducted to determine how the additional α-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 (Δ1, Δ2, Δ3 and Δ4), corresponding to deletions of −19, −29, −39 and −59 amino acids were designed to disrupt the α-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 76 kDa 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 Δ1, Δ2 and Δ3 mutants formed homo-oligomeric complexes, while the Δ4 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 19 amino acids, a region downstream of the fusion peptide that is deleted in the Δ1 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.

Role of the Second Intradiscal Loop of Peripherin/rds in Homo and Hetero Associations

“…This region of P/rds has been found to promote membrane fusion activity as well as membrane targeting (13,14,35). Past experiments have shown that the deletion of the C-terminal tail of P/rds abolishes the fusogenic activity but does not have any negative effects on protein dimerization or complex assembly with Rom-1 (14).…”

“…This region of P/rds has been found to promote membrane fusion activity as well as membrane targeting (13,14,35). Past experiments have shown that the deletion of the C-terminal tail of P/rds abolishes the fusogenic activity but does not have any negative effects on protein dimerization or complex assembly with Rom-1 (14). In addition to the fusogenic activity and membrane targeting, the C terminus of P/rds may be involved in anchoring the protein or connecting the disk rim to the plasma membrane through associations with auxiliary proteins, cytoskeleton proteins, or plasma-membrane proteins.…”

“…Although P/rds possesses a clear role in maintaining the structure of rods and cones, insights into its molecular mechanism through protein interactions are quite limited. Currently, P/rds is conjectured to play a significant role in disk morphogenesis and stability (1,11), disk shedding (1,12), and membrane fusion (13,14 Cloning and identification of P/rds orthologs suggests that these proteins share several distinctive features, including four hydrophobic transmembrane domains, a large intradiscal (D2) loop containing seven highly conserved cysteines, and a highly charged cytosolic Cterminal segment (4,15,16). Over 70% of the disease-causing mutations in P/rds are located in the D2 loop, providing evidence of the importance that this region plays in proper protein function.…”

ROM-1 potentiates photoreceptor specific membrane fusion processes

“…ROM-1 forms a hetero-tetrameric complex with P/rds both in vivo and in heterologous cell expression systems (Goldberg, Moritz et al 1995;Muller-Weeks, Boesze-Battaglia et al 2002). Even though the domain involved in tetramerization has been mapped to Cys 165 -Asn 182 of P/rds the precise role of ROM-1 in P/rds dependent function has remained elusive.…”

“…Procedures for the isolation and cloning of bovine FLAG-tagged peripherin/rds ( FLAG-P/ rds) and hemaglutininin-tagged ROM-1, ( HA-ROM-1) have previously been described (Muller-Weeks, Boesze-Battaglia et al 2002). Primer design was based on sequences reported by (Connell and Molday 1990) and (Moritz and Molday 1996).…”