Hetero-oligomerization of reggie-1/flotillin-2 and reggie-2/flotillin-1 is required for their endocytosis.

Reggie-1/flotillin-2 and reggie-2/flotillin-1 are membrane raft associated proteins which have been implicated in growth factor signaling, phagocytosis, regulation of actin cytoskeleton and membrane trafficking. Membrane and raft association of reggies is mediated by myristoylation, palmitoylation and oligomerization. We have shown that upon EGF stimulation of cells, reggie-1 is tyrosine phosphorylated by Src kinase and endocytosed into late endosomes. Here we have analyzed the mechanism of the EGF-stimulated endocytosis of reggies in more detail and show that the Src-mediated phosphorylation of reggie-1 is not the driving force for endocytosis. However, hetero-oligomerization with reggie-2 is necessary for the translocation of reggie-1, which does not take place in the absence of reggie-2. In addition, the Y163F mutant of reggie-1, which is not capable of undergoing endocytosis, oligomerizes poorly with reggie-2. EGF stimulation results in changes in the size but not in the stoichiometry of the reggie hetero-oligomers, and reggie-1 oligomer size is decreased by knockdown of reggie-2. Based on our findings, we propose a model according to which reggie hetero-oligomers are dynamic, and changes in the size of the hetero-oligomers result in endocytosis of the complex from the plasma membrane.