Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains
Résumé
Reggie-1 and -2 proteins (flotillin-2 and -1, respectively) form their own type of non-caveolar membrane microdomains, which are involved in important cellular processes such as T cell activation, phagocytosis, and signaling mediated by the cellular prion protein and insulin; this is consistent with the notion that reggie-microdomains promote protein assemblies and signaling. While it is generally known that membrane-microdomains contain large multiprotein assemblies, the exact organization of reggie-microdomains remains elusive. Using chemical cross-linking approaches, we demonstrate here that reggie-complexes are composed of homo- and hetero-tetramers of reggie-1 and -2. Moreover, native reggie-oligomers are indeed quite stable, since uncross-linked tetramers are resistant to 8 M urea treatment. We also show that oligomerization requires the C-terminal but not the N-terminal halves of reggie-1 and -2. Using deletion constructs, we analyzed the functional relevance of the three predicted coiled-coil stretches present in the C-terminus of reggie-1. We experimentally confirmed that reggie-1 tetramerization is dependent on the presence of coiled-coil 2 and, partially, of coiled-coil 1. Furthermore, since depletion of reggie-1 by siRNA silencing induces proteasomal degradation of reggie-2, we conclude that the protein stability of reggie-2 depends on the presence of reggie-1. Our data indicate that the basic structural units of reggie-microdomains are reggie homo- and hetero-tetramers, which are dependent on the presence of reggie-1.
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