Purification and light-dependent phosphorylation of a candidate fusion protein, the photoreceptor cell peripherin/rds.
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.
Boesze-Battaglia
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Department of Molecular Biology, University of Medicine and Dentistry of New Jersey-SOM, Stratford 08084, USA. Battagli@umdnj.edu
Kong
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Lamba
O P
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Stefano
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Williams
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DS
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002845:Chromatography; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007381:Intermediate Filament Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007526:Isoelectric Point; D008027:Light; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009410:Nerve Degeneration; D009419:Nerve Tissue Proteins; D064531:Peripherins; D010766:Phosphorylation; D010786:Photoreceptor Cells / Q000737:chemistry*; D012374:Rod Cell Outer Segment / Q000737:chemistry
D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D064531:Peripherins; D017886:Durapatite