May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
A Role for Rom-1 in Peripherin/rds Dependent Membrane Fusion Processes
Author Affiliations & Notes
  • K. Boesze-Battaglia
    Biochemistry, University of Pennsylvania, Philadelphia, PA, United States
  • C. Gretzula
    Biochemistry, University of Pennsylvania, Philadelphia, PA, United States
  • Footnotes
    Commercial Relationships  K. Boesze-Battaglia, None; C. Gretzula, None.
  • Footnotes
    Support  Supprto:EY10420 and the E. Matilda Ziegler Fdn.
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 4259. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : 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.

Keywords: photoreceptors • protein purification and characterization 
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