Purification and Light-Dependent Phosphorylation of a Candidate Fusion Protein, the Photoreceptor Cell Peripherin/rds

• Published in: Biochemistry (Easton) Vol. 36; no. 22; pp. 6835 - 6846
• Main Authors: Boesze-Battaglia, Kathleen, Kong, Fansheng, Lamba, Om P, Stefano, Frank P, Williams, David S
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 03.06.1997
• Subjects: Amino Acid Sequence; Animals; Cattle; Chromatography; Durapatite; Electrophoresis, Polyacrylamide Gel; Eye Proteins - chemistry; Eye Proteins - isolation & purification; Eye Proteins - metabolism; Hydrogen-Ion Concentration; Intermediate Filament Proteins - chemistry; Intermediate Filament Proteins - isolation & purification; Intermediate Filament Proteins - metabolism; Isoelectric Point; Light; Membrane Fusion; Membrane Glycoproteins - metabolism; Membrane Proteins - isolation & purification; Molecular Sequence Data; Nerve Degeneration; Nerve Tissue Proteins; Peripherins; Phosphorylation; Photoreceptor Cells - chemistry; Rod Cell Outer Segment - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9627370

The proteins peripherin/rds and rom-1 form a protein complex in the rims of photoreceptor outer segment disk membranes. Peripherin/rds plays an essential role in the morphogenesis and maintenance of disk membrane structure, with peripherin/rds gene mutations resulting in photoreceptor cell degeneration. We report two different chromatographic procedures for the purification of native peripherin/rds from bovine photoreceptor cell outer segments and show that the protein is a phosphoprotein that promotes membrane fusion in vitro. During one procedure, peripherin/rds was copurified in association with rom-1 by hyroxylapatite and Mono Q FPLC. During the other, it was purified free from rom-1 by concanavalin-A affinity chromatography and chromatofocusing. Analysis of homogeneous peripherin/rds from the second procedure showed that exposure of photoreceptor outer segments to light resulted in the incorporation of nearly 2 mol of phosphate per mole of peripherin/rds and a concomitant shift in the isoelectric point of the protein. In addition, we found that recombination of purified peripherin/rds into lipid vesicles increased membrane fusion, with more rapid fusion detected with phosphorylated peripherin/rds. In conclusion, studies with purified peripherin/rds reveal that the protein undergoes light-dependent phosphorylation and that it may function in membrane fusion.