Formation of protein polymers in erythrocyte ghosts incubated with sonicated lipid vesicles. Effects on spectrin extractibility, permeability of ghosts to vesicles, intramembrane particle distribution and bleb formation

Abstract

The incubation of human erythrocyte white ghosts with phosphatidylcholine (PC) vesicles or cholesterol/phosphatidylcholine (C/PC) vesicles under hypotonic or isotonic conditions generated membrane protein cross-links. The latter appeared in the form of a high molecular weight polymer after SDS-polyacrylamide gel electrophoresis. The polymer started to develop within a few minutes of incubation, arising largely from spectrin, and required 24 h or more for completion. It occurred regardless of cholesterol depletion undergone by the ghosts in the presence of PC vesicles. It was not reversed upon further incubation in a hypotonic, vesicle-free medium. When initial incubation was carried out under hypotonic conditions, a number of other alterations were recorded: (i) spectrin extractibility was abolished; (ii) ghosts became gradually impermeable to vesicles within a few hours, a process referred to as slow ‘resealing’ and generating an irreversible sequestration of the vesicles; (iii) intramembrane particles aggregated and blebs free of intramembrane particles pinched off inward or outward. When initial incubation was conducted under isotonic conditions, the following was observed: (i) spectrin was unextractible, as could be expected; (ii) vesicles did not enter the ghosts, a fact indicating an immediate and complete impermeabilization of ghosts to vesicles, a process referred to as fast ‘resealing’; (iii) intramembrane particle aggregation and blebs free of intramembrane particles were also present. When initial incubation was performed under isotonic conditions, but in the absence of vesicles, the polymer failed to be associated with spectrin inextractibility. These data support the view that lipid vesicles generate a high molecular weight polymer-associated, slow resealing of erythrocyte ghosts that differs, at least in part, from the polymer-free, fast resealing induced by a vesicle-free isotonic medium. Resistance to β-mercaptoethanol of the polymer makes unlikely the sole participation of disulfide bonds. Absence of added Ca 2+ in the medium is inconsistent with the transglutaminase-catalyzed formation of amide linkages. When ghosts were separated from the vesicles by a cellophane membrane upon hypotonic incubation, spectrin remained extractible and no polymer developed. Sonication of the vesicles under nitrogen and in the presence of butylated hydroxytoluene did not prevent the formation of the polymer.