Traffic, polarity, and detergent solubility of a glycosylphosphatidylinositol-anchored protein after LDL-deprivation of MDCK cells.

Abstract

Abstract. Glycosylphosphatidylinositol-anchored pro-teins, GPI-proteins, are selectively delivered to the apical surfaces of many types of morphologically polarized epithelial cells. It has been proposed that the unit for targeting GPI-proteins to the apical surface is a membrane lipid domain. This sorting domain or molecular cluster has been equated to detergent (Triton X-100)-insoluble membrane fractions that are enriched in enriched in GPI-proteins, glycosphingolipids, and cholesterol. To determine the role of cholesterol in the formation of sorting domains and to examine its importance in the intracellular traffic and membrane polarity of GPI-proteins, we studied the behavior of a model GPIprotein, gD1-DAF, in MDCK cells cultured for 3 or 14 d without their principal source of cholesterol, serum LDL. LDL deprivation affects the intracellular traffic of gD1-DAF. Surface expression of gD1-DAF is reduced in LDL-deprived cells; this reduction is most marked after 3 d of LDL deprivation. We also find a great reduction in the fraction of gD1-DAF that is detergent-insoluble in these cells and a change in its membrane milieu defined by susceptibility to cleavage with PI-specific phospholipase C. Despite these changes, the surface polarity of gD1-DAF is no different in LDLdeprived cells than in control cells.

Virus complement evasion strategies

“…Recent studies have led to strong indications that budding of a variety of enveloped viruses, such as HIV, Ebola virus, influenza virus and measles virus, does not happen randomly at the plasma membrane but at specific microdomains enriched in cholesterol and sphingolipids, called lipid rafts (Scheiffele et al, 1999;Vincent et al, 2000;Ono & Freed, 2001;Bavari et al, 2002). Glycosyl phosphatidyl inositol (GPI)-anchored complement control proteins such as CD55 and CD59 have been shown to associate with such lipid rafts (Hannan & Edidin, 1996). Although further research is necessary to explore this hypothesis, evidence obtained recently that lipid raft disruption decreases the amount of CD59 on the HIV envelope (Nguyen & Hildreth, 2000) makes it tempting to speculate that one of the mechanisms of incorporation of complement control (and other cellular) proteins in virion envelopes perhaps comprises the preferential budding of viruses at lipid rafts.…”

The Binding Activity of the Macrophage Lipoprotein(a)/Apolipoprotein(a) Receptor Is Induced by Cholesterol via a Post-translational Mechanism and Recognizes Distinct Kringle Domains on Apolipoprotein(a)

“…Thus, cholesterol loading may change the conformation of the Lp(a)/ apo(a) receptor to a more active form. Alternatively, cholesterol loading may affect the trafficking of the receptor to the plasma membrane, as was recently found for a glycosylphosphatidylinositol-anchored protein that is localized to cholesterol-rich domains of the plasma membrane (48). Since inhibition of protein synthesis blocks lysosomal degradation of r-apo(a) but not binding or lysosomal degradation of other receptor ligands, we conclude that there is a separate short-lived component of the Lp(a)/apo(a) receptor that is somehow necessary for internalization or delivery of ligand to lysosomes.…”

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

“…By preferentially including some proteins and excluding others, lipid rafts and related membrane microdomains such as caveolae may regulate the sorting and trafficking of certain plasma membrane proteins and lipids and compartmentalize cell-signaling events (Verkade and Simons, 1997;Anderson, 1998;Brown and London, 1998;Horejsi et al, 1999). Although lipid rafts have been inferred from functional and kinetic studies of intact cells (Mays et al, 1995;Hannan and Edidin, 1996;Sheets et al, 1997;Keller and Simons, 1998), most evidence of their existence is based on differential extraction of cells with detergent (Skibbens et al, 1989;Stefanova et al, 1991;Brown and Rose, 1992;Fiedler et al, 1993;Sargiacomo et al, 1993). These studies indicate that in addition to GSL and cholesterol, lipid rafts are enriched in GPI-anchored proteins, some transmembrane proteins, and diacylated cytoplasmic proteins including Src family kinases.…”