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K Boesze-Battaglia, M Kahoe; Isolation Of Photoreceptor Rom-1 Containing Membrane Rafts . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3740.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Membrane tetraspanin proteins have been found associated with membrane microdomains termed membrane rafts. The studies described herein addresses whether two photoreceptor specific tetraspanins; peripherin/rds and rom-1 are raft associated. Methods: ROS disk membranes were separated free from plasma membrane using sucrose gradient centrifugation techniques and homogenized in 5% Triton X-100 or 5% octylglucoside (OG) as a control. The Triton resistant membrane (a.k.a. membrane rafts) were isolated from a sucrose step gradient overlayed onto the samples, spun at 40K for 20 hours. The membrane rafts and soluble species were assayed for cholesterol, total phospholipid, individual lipid species, and protein. The protein composition of the rafts was determined using SDS-PAGE followed by silver staining and Western blots analysis. The blots were probed with monoclonal antibody anti-rom-1, mAb 1D5 , anti-peripherin/rds mAb 2B6 and anti-opsin mAb 4D2 (all generous gifts from Dr. R. Molday) or anti-calveolin antibody. Results: Membrane rafts isolated upon treatment with Triton X-100 at 16% (w/w) sucrose had cholesterol to phospholipid ratio of 0.26+/-0.03 and sphingomyelin to PC ratio of 0.25 as compared to OG treated samples and soluble fractions in which this ratio is 0.12. The membrane rafts were found to contain rom-1 and rhodopsin with no detectable levels of peripherin/rds. Conclusions: A unique ROS disk membrane microdomain containing rom-1 independent of peripherin/rds has been isolated in a membrane raft with high levels of cholesterol and sphingomyelin.
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