Characterization of structural and functional phosphoinositide domains in human erythrocyte membranes

Abstract

In the erythrocyte membrane, only a fraction (50-60%) of phosphatidylinositol 4,5-bisphosphate (PIP2) and of phosphatidylinositol 4-phosphate (PIP) is rapidly turned over by specific kinases and phosphatases and accessible to hydrolysis by the polyphosphoinositide (PPI)-specific phospholipase C (PLC). To investigate whether the metabolic segregation of PPI resulted from preferential interactions with proteins, we have measured the accessibility of PPI to bee venom phospholipase A2 (PLA2) in native erythrocyte membranes, or after treatments designed to remove peripheral proteins and cytoplasmic domains of integral proteins. In native membranes, PPI, as well as the other major phospholipids, behaved as two distinct fractions (R1 and R2) differing by their sensitivity to PLA2. Such a behavior was not observed in PIP and PIP2 containing artificial vesicles. Evidence was provided that the highly sensitive fraction of PIP and PIP2 (R1) may be identical to the PLC-sensitive and rapidly metabolized pool. Removal of peripheral proteins, followed by proteolysis of the cytoplasmic domain of integral proteins, mainly glycophorins and band 3, led to a reduction of the R1 fraction of PIP and of PIP2. It is proposed that the rapidly metabolized pool of PIP2 and PIP, involved in the regulation of major cellular functions, would be maintained in its functional state through interactions with integral proteins.

Signals determining protein tyrosine kinase and glycosyl-phosphatidylinositol-anchored protein targeting to a glycolipid-enriched membrane fraction.

“…Evidence of glycosphingolipid domains in model systems is well documented (reviewed in reference 50). Furthermore, interactions between glycosphingolipids and cholesterol (47) and the presence of phosphatidylinositol domains in biological membranes (16) have been demonstrated and further suggest a mechanism in which lipids are important for formation and stabilization of the GEM fraction.…”

“…1 For experiments examining total protein expression without gradient separation, the lysis was in 1.0 ml of a solution containing 1% Nonidet P-40-0.4% deoxycholate-66 mM EDTA-10 mM Tris (pH 7.4). The lysate was clarified by centrifugation for 5 min at 16,000 x g at 4°C.…”

Antagonist Effects of Ca2+and Spermine on Phosphatidylinositol 4,5-Bisphosphate-Mediated Transmembrane Redistribution of Phospholipids in Large Unilamellar Vesicles and in Erythrocytes

“…The lower organic phase was removed and dried under vacuum. Phospholipid separation and phosphorus assay were carried out as described before (Gascard et al, 1991(Gascard et al, , 1993b.…”

Binding of Adriamycin to Liposomes as a Probe for Membrane Lateral Organization

“…Lateral heterogeneity and domain formation in biomembranes are well established (for reviews see Knoll et al, 1991;Rodgers and Glaser, 1991;Kinnunen, 1991;Gascard et al, 1993;Tocanne et al, 1994;Welti and Glaser, 1994) and have been shown to be of importance to the membrane binding and function of proteins such as cytochrome c (Vanderkooi et al, 1973;Mustonen et al, 1987) and gelsolin (Janmey and Chaponnier, 1995), in the coupling of G-proteins to their receptors (Neubig, 1994), in the budding of viruses from the plasma membrane (Luan and Glaser, 1994), and in the activation of pancreatic lipase by colipase (Momsen et al, 1995). Considering the coupling between dynamic order and function emphasized in the modern view of biological membranes (see e.g., Kinnunen, 1991;Kinnunen and Mouritsen, 1994;; Mouritsen and , thorough understanding of the mechanisms causing lateral organization of membranes is clearly of fundamental importance.…”