title: A soluble peripherin/Rds C-terminal polypeptide promotes membrane fusion and changes conformation upon membrane association.
abstract: Photoreceptor rod cells contain a unique tetraspanin fusion protein known as peripherin/rds. This protein is important in membrane fusion events hypothesized to be essential to disk membrane morphogenesis and disk shedding. In vivo and in vitro fusogenic activity has been mapped to the C-terminal domain of peripherin/rds. Moreover, a fusion peptide domain localized to a 15 amino acid long region (residues 311-325) is essential for mediating lipid bilayer fusion of model membranes. To address the functional and structural properties required for peripherin/rds dependent membrane fusion, constructs of the entire C-terminal domain (residues 284-346) were generated and polypeptides expressed. A wild type-peripherin/rds C-terminal GST fusion construct that included the entire C-terminus (PERCTER) or a C-terminal truncation mutant (PERCTN) were engineered with a thrombin cleavage site. Protein expression was induced in E. coli with IPTG, expressed proteins cleaved from the GST with thrombin and purified to homogeneity on a Superdex 75 column. Purity was confirmed by SDS-PAGE and Western blot analysis. The purified wt C-terminal protein resolved as a monomer under reducing conditions on SDS-PAGE (15%) and was immunoreactive with anti peripherin/rds antibody 2B6 (gift from Dr R. Molday). The purified polypeptide promoted the requisite steps of fusion, membrane destabilization, lipid mixing and aqueous contents mixing. Conversely, the truncation mutant lacking a portion of the fusion domain was unable to promote these steps. A common feature of most membrane fusion proteins is a change in conformation upon membrane association. Structural changes in the C-terminal polypeptide were investigated using far UV CD. The far UV CD spectra of the purified C-terminal polypeptide indicated substantial alpha-helical content in the wt peptide in isotonic aqueous buffer. An increase in intensity of 208 and 222 nm CD bands upon addition of DPC vesicles indicated an increase in alpha-helical content of the polypeptide. These results demonstrate that a purified soluble form of the C-terminus of peripherin/rds can interact with biological phospholipids; moreover, this interaction promotes a conformational change that is most consistent with an increase in alpha-helical content.
authors.affiliation: Department of Biochemistry, School of Dental Medicine, 4001 Spruce Street, University of Pennsylvania, Philadelphia 19104, USA. battagli@Biochem.dental.upenn.edu
authors.forename: Kathleen
authors.initials: K
authors.lastname: Boesze-Battaglia
authors.forename: Andrew F X
authors.initials: AF
authors.lastname: Goldberg
authors.forename: Janice
authors.initials: J
authors.lastname: Dispoto
authors.forename: Madan
authors.initials: M
authors.lastname: Katragadda
authors.forename: Gregory
authors.initials: G
authors.lastname: Cesarone
authors.forename: Arlene D
authors.initials: AD
authors.lastname: Albert
mesh_terms: D015153:Blotting, Western / Q000379:methods; D002942:Circular Dichroism / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005136:Eye Proteins / Q000378:metabolism*; D015693:Gene Products, nef / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D050356:Lipid Metabolism; D008561:Membrane Fusion / Q000502:physiology*; D008562:Membrane Glycoproteins; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D064531:Peripherins; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism; D012995:Solubility
chemical_list: D005136:Eye Proteins; C091445:GST-Nef protein, recombinant; D015693:Gene Products, nef; D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C578994:PRPH protein, human; C578997:PRPH2 protein, human; D010455:Peptides; D064531:Peripherins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase