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K. Boesze-Battaglia, C. Gretzula; A Role for Rom-1 in Peripherin/rds Dependent Membrane Fusion Processes . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4259.
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Purpose: Rom-1, the non-glycosylated homologue, of peripherin/rds forms non-covalently linked heterotetramer with peripherin/rds in the disk rim. Moreover a portion of ROS rom-1 is associated with membrane rafts devoid of peripherin/rds. In these studies we sought to investigate the relationship between raft-associated rom-1 and peripherin/rds associated rom–1 with membrane fusion processes in the ROS. Methods: Using fluorescence based membrane fusion assays, fusion between purified ROS plasma membrane vesicles and a variety of target membranes was measured. These target membranes include large unilammelar vesicles containing either rom-1 alone or the peripherin/rds-rom-1, isolated cells membrane from COS cells co-transfected with peripherin/rds and rom-1 or rim specific vesicles isolated by Concanavalin A chromatography. The interaction of raft associated rom-1 and rom-1 complexed with peripherin/rds with plasma membrane specific ricin binding protein was determined using cross-linking and immunoprecipitation studies. Results: Recombinant membranes containing rom-1 alone were unable to fuse with ROS-PM vesicles. When the vesicles contained both rom-1 and peripherin/rds, fusion was restored. When these two proteins were both expressed in COS cell membranes, fusion was enhanced relative to membranes containing only peripherin/rds. Moreover the addition of a synthetic peptides corresponding to residues 298 to 312 of rom-1 to fusion assays resulted in a decrease in lag-time and no change in the initial rate of fusion. Purified rom-1 was found to associate with a plasma membrane specific ricin binding protein using affinity chromatography and cross-linking studies. Conclusions: Rom-1 is unable to promote membrane fusion but enhances peripherin/rds fusion activity. This enhancement is most likely through the formation of a fusion competent form of peripherin/rds, since addition of rom-1 peptide decreased lag-time; a parameter used to define fusion competency.
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