Proteomic Analysis of Lipid Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells.

Cell membranes are composed of a complex array of lipids and these lipids do not distribute randomly throughout the membrane but, instead, form distinct membrane domains via lipid-lipid and lipid-protein interactions.1 The lipid raft hypothesis was initially proposed in 1997 by Simons and Ikonen2 as a principle of membrane subcompartmentalization. Later in 2006 at the Keystone Symposium on Lipid Rafts and Cell Function, lipid rafts were defined as small, heterogeneous, highly dynamic membrane microdomains (10–200 nm) that are enriched in cholesterol and sphingolipids and that compartmentalize cellular processes.3 As is now increasingly appreciated, lipid rafts may play important roles in various cellular processes, such as signal transduction, membrane trafficking, cell adhesion, cytoskeletal rearrangement, and many other membrane functions.4 The concept of lipid sorting into membrane microdomains was initially introduced to explain the generation of the glycosphingolipid-rich apical membrane in epithelial cells.5 Later, this idea was expanded to include cholesterol-rich raft microdomains.2,6,7 Based on the lipid raft hypothesis,2 sphingolipids associate laterally with one another in the exoplasmic leaflet and any void space is filled with cholesterol. Cholesterol is also present in the cytoplasmic leaflet and fills the void space created by interdigitating fatty acid chains. Cholesterol is known to increase the thickness and stiffness of lipid bilayers and it is generally regarded as a key lipid component of lipid rafts.8 Plasma membranes of lens fiber cells are distinguished from other eukaryotic cell membranes by their unique lipid composition. Lens fiber cell membranes contain high concentrations of long-chain saturated fatty acids and high abundances of sphingomyelin and cholesterol.9–11 This unique lipid composition gives rise to a unique lens fiber cell membrane that is important for lens transparency.11–13 The abundance of sphingomyelin and cholesterol contributes to lens membrane rigidity and structural order. Considering this unusually high concentration of cholesterol and sphingolipid, it is expected that lipid raft domains are highly abundant and play important roles in lens fiber cell membrane. Evidence for immiscible cholesterol-rich and -poor domains in normal lens fiber cell membrane was reported,14 and cholesterol-rich domains were more pronounced and better defined in cataractous lens.15 Previously, lipid raft domains were isolated and lipids13 and caveolin-116 content was examined. Sorting of AQP0 and connexins to raft and nonraft domains was also reported.17 However, the structure and function of lipid rafts in lens fiber cells are largely unknown. Lipid rafts have been isolated from a variety of tissues relying on their insolubility in nonionic detergents at low temperature. Although detergent insolubility in itself does not accurately reflect preexisting raft domains, studying detergent-resistant membranes (DRMs) has produced results consistent with other methods, such as direct imaging.18,19 Thus, detergent resistance remains an interesting and useful tool for assigning potential membrane raft association. In this report, we isolated DRM from bovine lens fiber cells and performed quantitative proteomic studies of DRM and detergent-soluble membrane (DSM) fractions. Our results demonstrated that a significant percentage of AQP0, MP20, AQP5, and a small fraction of connexin 50 and connexin 46 reside in lipid raft domains.