Interaction of the lipid raft-associated protein flotillin-2 with membrane cholesterol

Flotillin-2 and flotillin-1 are ubiquitously expressed proteins which are associated with sphingolipid and cholesterol-enriched membrane microdomains known as lipid rafts. Both proteins play a role in various cellular processes. Recently, an important role for flotillins in Niemann-Pick C1-like 1-mediated cholesterol uptake was unraveled, but their interaction mode with cholesterol still remains to be clarified.

We have shown that flotillin-2 is linked to the plasma membrane by means of fatty acid modifications, especially myristoylation and palmitoylation. Those post translational modifications facilitate the interaction with membrane cholesterol. In addition, sequence analysis of flotillin-2 showed the existence of four putative cholesterol recognition/interaction amino acid consensus motifs (CRAC).

To examine the interaction of flotillin-2 with membrane cholesterol, we produced fusion proteins and generated targeted tyrosine substitutions for the inactivation of putative CRAC-motifs. Labeling of the fusion proteins with the photoreactive cholesterol analog azocholestanol ([³H]6-azi-5α-cholestan-3β-ol) showed that flotillin-2 is able to bind cholesterol, whereas the inactivation of the putative CRAC motifs doesn’t abolish this interaction.

These results show for the first time that flotillin-2 binds to membrane cholesterol and that this interaction doesn’t solely rely on CRAC motifs. Due to that, flotillin-2 might play an active role in structuring cell membranes.