Resistance of cell membranes to different detergents

Abstract

Biological membranes are composed of a puzzling variety of lipids. Such diversity would be unnecessary if lipid bilayers served only as hydrophobic barriers and homogeneous two-dimensional solvents for membrane proteins. As is now increasingly appreciated, membranes show extensive lipid-driven compartmentalization, giving rise to distinct membrane domains. These domains differ in their composition, physical properties, and biological functions.

Membranes typically exist in a fluid state characterized by unconfined diffusion of its loosely packed lipids. This state is therefore also called the liquid-disordered (l_d) phase. However, studies of liposomes (1, 2) as well as model membranes (3) have shown that certain lipids have the propensity to associate with each other, thus segregating from the l_d phase. These lipids are cholesterol, sphingolipids with their usually saturated fatty acids, and saturated glycerophospholipids. The structure of their hydrophobic moieties allows them to pack more tightly than the kinked unsaturated glycerophospholipids. As a result, they can establish a more ordered state, called the liquid-ordered (l_o) phase (4). Most likely, l_o phases also exist in cell membranes with a sufficient proportion of cholesterol and sphingolipids (5). The l_o phase is thought to form discrete microdomains interspersed in the continuous l_d phase. These microdomains have been termed “lipid rafts” (6) and are believed to play important roles in signal transduction and protein sorting (7–9).

A common biochemical method to analyze the domain organization of membranes is extraction with mild detergents like Triton X-100 (Triton). Although detergent treatment disrupts most lipid–lipid interactions, a minor fraction of cell membranes is preserved and can be isolated as detergent-resistant membranes (DRMs). DRMs prepared with Triton probably originate from the cholesterol- and sphingolipid-rich l_o phase, which resists extraction due to its tight lipid packing (5). Detergent extraction also disrupts lipid–protein interactions, so that most membrane proteins are solubilized. Only few proteins retain their association with lipids and are recovered in DRMs. Thus, DRM association of a protein is indicative of a strong interaction with highly ordered domains in the l_o phase. However, DRMs may only imperfectly reflect the distribution of membrane components between the l_o and l_d phases (10), and it is unknown how well the composition of DRMs correlates with the components of native lipid rafts in cell membranes.

DRMs have mainly been prepared with Triton (11) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (12), but other detergents such as Brij 58 (13), Brij 96 (14), Lubrol WX (15), and Brij 98 (16) have also been used. Yet, it is unclear whether DRMs prepared with different detergents similarly reflect the same aspect of membrane organization, i.e., segregation of cholesterol- and sphingolipid-rich domains in the l_o phase from the l_d phase rich in unsaturated glycerophospholipids.

To facilitate comparison of results obtained with different detergents, we analyzed the protein and lipid content of DRMs isolated with various mild detergents. We used two cell lines, epithelial Madin–Darby canine kidney (MDCK) cells and human T cell leukemia cells (Jurkat cells), to test whether the characteristics of DRMs and the effectiveness of particular detergents vary between cell types. In addition, we evaluated tools to manipulate DRM association of proteins. We found that DRMs obtained with different detergents differ considerably in their protein and lipid content, and that these differences are cell type-dependent. Our results caution against equating DRMs prepared with different detergents and from different cell types.

Acknowledgments

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