Abstract
This study reports the isolation and characterization of a Triton X-100-resistant membrane fraction from homogenates of rod outer segment (ROS) disk membranes purified free of the surrounding plasma membrane. A portion of the ROS disk membrane was found to be resistant to Triton X-100 extraction at 4 degrees C. This detergent-resistant fraction was isolated as a low buoyant density band on sucrose density gradients and exhibited an increase in light scattering detected at 600 nm. Biochemical analysis of the Triton X-100-resistant fraction showed it to be enriched in cholesterol and sphingomyelin relative to phospholipid and in phospholipid relative to protein compared with the soluble fraction. The Triton X-100-resistant membranes described herein did not arise simply from partial solubilization of the ROS disk membranes because detergent-treated low buoyant density fractions isolated from homogenates with octyl glucopyranoside had cholesterol and sphingomyelin content indistinguishable from that of solubilized ROS disk homogenates. Analysis of proteins associated with the Triton X-100-resistant fraction showed it to be enriched in the rim-specific protein ROM-1 and caveolin; surprisingly, the fusion protein peripherin/rds (where rds is retinal degeneration slow), also localized to the disk rim, was entirely absent from the membrane raft domain. The lipid profiles of the Triton X-100-resistant membranes were virtually identical in preparations homogenized in either the light or dark. Slightly more ROM-1 was recovered from samples prepared in the light (23%) than from samples prepared in the dark (13%), but peripherin/rds could not be detected in either preparation. When the Triton X-100-resistant membranes were treated with methyl-beta-cyclodextran to deplete membrane cholesterol, the resultant membranes contained slightly lower levels of ROM-1, specifically in the dimeric form. Cholesterol depletion also resulted in the collapse of the large caveolin complex to monomeric caveolae. The results presented herein characterize a pool of ROM-1, a photoreceptor tetraspanin protein, that may play a regulatory role in peripherin/rds-dependent fusion.
Highlights
Photoreceptor rod cells are responsible for vision under dim light
Characterization of the Lipid Composition of a Triton X-100resistant Membrane Species Isolated from rod outer segment (ROS) Disk Membranes—To identify and characterize ROS disk membranespecific detergent-resistant membranes, purified disk membranes were homogenized with 1% Triton X-100 at 4 °C in either the dark or light and fractionated by sucrose density gradient centrifugation
Samples prepared in the dark (TD) had two bands: a low density diffuse white-yellow band at a sucrose density of 20.1 Ϯ 2.2% and a nearly transparent red-orange band at 26.5 Ϯ 1.4%, similar to the Triton X-100-soluble band described by Seno et al (32)
Summary
Photoreceptor rod cells are responsible for vision under dim light. These unique post-mitotic cells are made up of a rod inner segment and a rod outer segment (ROS)1 region. The Triton X-100-resistant membranes described did not arise from partial solubilization of the ROS disk membranes because detergent-treated low buoyant density fractions isolated from homogenates with octyl glucopyranoside had cholesterol and sphingomyelin content indistinguishable from that of solubilized ROS disk homogenates.
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