Cyclodextrins but not compactin inhibit the lateral diffusion of membrane proteins independent of cholesterol

Cholesterol and glycosphingolipid-enriched membrane domains, termed lipid rafts, were proposed to play important roles in trafficking and signaling events. These functions are inhibited following putative disruption of rafts by cholesterol depletion, commonly induced by treatment with methyl-β-cyclodextrin (MβCD). However, several studies showed that the lateral diffusion of membrane proteins is inhibited by MβCD, suggesting that it may have additional effects on membrane organization unrelated to cholesterol removal. Here, we investigated this possibility by comparison of the effects of cholesterol depletion by MβCD and by metabolic inhibition (compactin), and of treatment with α-CD, which does not bind cholesterol. The studies employed two series of proteins (Ras and influenza hemagglutinin), each containing as internal controls related mutants that differ in raft association. Mild MβCD treatment retarded the lateral diffusion of both raft and non-raft mutants, whereas similar cholesterol reduction (30-33%) by metabolic inhibition enhanced selectively the diffusion of the raft-associated mutants. Moreover, α-CD also inhibited the diffusion of raft and non-raft mutants, despite its lack of effect on cholesterol content. These findings suggest that the widely used treatment with CD to reduce cholesterol has additional, cholesterol-independent effects on membrane protein mobility, which do not necessarily distinguish between raft and non-raft proteins.