On the association of lipid rafts to the spectrin skeleton in human erythrocytes

Lipid rafts are local inhomogeneities in the composition of the plasma membrane of living cells, that are enriched in sphingolipids and cholesterol in a liquid-ordered state, and proteins involved in receptor-mediated-signalling. Interactions between lipid rafts and the cytoskeleton have been observed in various cell types. They are isolated as a fraction of the plasma membrane that resists solubilization by nonionic detergents at 4 °C (Detergent Resistant Membranes, DRMs). We have previously described that DRMs are anchored to the spectrin-based membrane-skeleton in human erythrocytes, and can be released by increasing the pH and ionic strength of the solubilization medium with sodium carbonate. It was unexplained why this carbonate treatment was necessary and why this requirement was not reported by other workers in this area. We show here that when contaminating leukocytes are present in erythrocyte preparations that are subjected to detergent treatment, the isolation of DRMs can occur without the requirement for carbonate treatment. This is due to the uncontrolled breakdown of erythrocyte membrane components by hydrolases that are released from contaminating neutrophils, that lead to proteolytic disruption of the supramolecular assembly of the membrane-skeleton. Results presented here corroborate the concept that DRMs are anchored to the membrane skeleton through electrostatic interactions, that most likely involve the spectrin molecule