Insolubility and redistribution of GPI-anchored proteins at the cell surface after detergent treatment.

Abstract

A diverse set of cell surface eukaryotic proteins including receptors, enzymes, and adhesion molecules have a glycosylphosphoinositol-lipid (GPI) modification at the carboxy-terminal end that serves as their sole means of membrane anchoring. These GPIanchored proteins are poorly solubilized in nonionic detergent such as Triton X-100. In addition these detergent-insoluble complexes from plasma membranes are significantly enriched in several cytoplasmic proteins including nonreceptor-type tyrosine kinases and caveolin/VIP-21, a component of the striated coat of caveolae. These observations have suggested that the detergent-insoluble complexes represent purified caveolar membrane preparations. However, we have recently shown by immunofluorescence and electron microscopy that GPI-anchored proteins are diffusely distributed at the cell surface but may be enriched in caveolae only after cross-linking. Although caveolae occupy only a small fraction of the cell surface (< 4%), almost all of the GPI-anchored protein at the cell surface becomes incorporated into detergent-insoluble low-density complexes. In this paper we show that upon detergent treatment the GPI-anchored proteins are redistributed into a significantly more clustered distribution in the remaining membranous structures. These results show that GPI-anchored proteins are intrinsically detergent-insoluble in the milieu of the plasma membrane, and their co-purification with caveolin is not reflective of their native distribution. These results also indicate that the association of caveolae, GPI-anchored proteins, and signalling proteins must be critically re-examined.

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

“…However, most data indicate that antibody binding and cross-linking do not disrupt (our unpublished results) (Mayor and Maxfield, 1995;Kenworthy and Edidin, 1998) and indeed may stabilize (Harder et al, 1998) the association of proteins with lipid rafts. The great exception is the observation that clustering of folate receptor is disrupted by binding of the antibody MOv19 (Smart et al, 1996).…”

“…We confirmed this in HeLa cells by incubating labeled cells in PBS containing 1% Triton X-100 for 30 min on ice and then fixing the cells and visualizing the detergent-insoluble membrane fraction by fluorescence microscopy (Mayor and Maxfield, 1995;Kenworthy and Edidin, 1998). As expected, CTXB, folate receptor, and CD59 all were present after detergent extraction at levels similar to that of mock-extracted cells, whereas transferrin receptor labeled with fluorescently tagged human transferrin was completely extracted under these conditions (our unpublished results).…”

Resistance of cell membranes to different detergents

“…Hence, separate detergent-resistant domains exist in those cells, but they are indistinguishable by the DRM criterion. Detergent treatment leads to coalescence of detergent-resistant cholesterol-sphingolipid micodomains, which come together to form aggregates or fuse into continuous membrane sheets (27,28). It has also been demonstrated that detergent intercalation into model membranes can promote the formation of DRMs (29).…”

Markers for Detergent-resistant Lipid Rafts Occupy Distinct and Dynamic Domains in Native Membranes

“…The interpretation of gradient centrifugation experiments remains controversial. There is evidence that detergents may force associations between components that are not colocalized in intact cells (Mayor and Maxfield, 1995), and fractionation results are known to be dramatically altered by varying the concentration of Triton X-100 (Field et al, 1999;Parolini et al, 1999), by use of alternative detergents (Montixi et al, 1998;Surviladze et al, 1998) or by omission of detergent altogether (Ilangumaran et al., 1999;Harder and Kuhn, 2000;Surviladze et al, 2001). Methods to observe membrane segregation in situ have also generated controversy.…”