The Role of Cholesterol and Glycosylphosphatidylinositol-anchored Proteins of Erythrocyte Rafts in Regulating Raft Protein Content and Malarial Infection

Abstract

Human erythrocytes are terminally differentiated, nonendocytic cells that lack all intracellular organelles. Here we show that their plasma membranes contain detergentresistant membrane rafts that constitute a small fraction (4%) of the total membrane protein, with a complex mixture of proteins that differentially associate with rafts. Depletion of raft-cholesterol abrogates association of all proteins with no significant effect on cholesterol:protein ratios in the rest of the membrane, lipid asymmetry, deformability, or transport properties of the bilayer, indicating that cholesterol is critical for protein assembly into rafts and suggesting that rafts have little influence on several erythrocyte functions. Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, which lack glycosylphosphatidylinositol-anchored proteins, show significant elevation in raft-cholesterol but no increase in raft protein association, suggesting that raft assembly does not require glycosylphosphatidylinositol-anchored proteins, raft proteins do not bind directly to cholesterol, and only threshold levels of raft-cholesterol are critical for protein recruitment. Loss of glycosylphosphatidylinositol-anchored proteins had no effect on erythrocytic infection by malarial parasite or movement of raft markers into the parasite's vacuole. However, infection is blocked following raft-cholesterol disruption, suggesting that erythrocyte rafts can be functionally exploited and providing the first evidence for the involvement of host rafts in an apicomplexan infection.

Cross-talk between Caveolae and Glycosylphosphatidylinositol-rich Domains

“…Similar observations were made on paroxysmal nocturnal hemoglobinuria erythrocytes, which were found to have 2.37 times more cholesterol than control erythrocytes (68). In contrast, we found that CHO cells stringently maintain their cholesterol levels in the absence of GPI-anchored proteins.…”

Identification of a Stomatin Orthologue in Vacuoles Induced in Human Erythrocytes by Malaria Parasites

“…Isolation of DRM Rafts-DRM rafts were isolated as previously de-scribed (4,5). Briefly, vacuolar parasites freed from the red cell membrane or uninfected erythrocytes were extracted on ice in 2 ml of 1% Triton X-100 in TBS (25 mM Tris-HCl, 150 mM NaCl pH 7.4) for 30 min in the presence of protease inhibitor mixture (Roche Applied Science) containing a broad spectrum of serine, cysteine, and metalloprotease inhibitors as well as caspain and 1 mM EDTA.…”

“…Association of Pfstomatin and SLP-2 in DRM Rafts-It has been shown that human band 7 stomatin is present in DRM rafts (5,28). However its membrane-spanning domain is not conserved in Pfstomatin or the other stomatins (Fig.…”

“…The protein flotillin-1, which is DRM-associated and forms oligomers has also been shown to induce caveolae formation in cells that do not express caveolin-1 (25). Band 7 stomatin is a homologue of flotillin-1 (26), forms large oligomers (27), but is a component of non-caveolar DRM rafts (5,28). (Note: flotillin-1 is also detected in non-caveolar DRM rafts, Ref.…”

“…Although major erythrocyte proteins are excluded from the PVM, proteins that reside in buoyant cholesterol-rich detergent-resistant membrane (DRM) rafts concentrate in the vacuole (3,4). In addition, mild depletion of erythrocyte cholesterol that has no significant effect on major erythrocyte membrane function but disrupts DRM rafts, blocks malarial infection (5). Emerging evidence suggests that DRM rafts play a role in membrane curvature (6 -8).…”