Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains

TLDR

Reggie‑1 and ‑2 proteins form non‑caveolar membrane microdomains that participate in T‑cell activation, phagocytosis, and signaling, yet their precise organization remains unclear. Chemical cross‑linking and deletion‑construct analyses revealed that reggie‑1 and ‑2 assemble into homo‑ and hetero‑tetramers, with tetramerization dependent on specific C‑terminal coiled‑coil domains. The tetramers are highly stable, require the C‑terminal halves of reggie proteins, depend on coiled‑coil 2 (and partly coiled‑coil 1), and the stability of reggie‑2 relies on reggie‑1, establishing tetramers as the fundamental units of reggie microdomains.

Abstract

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 signalling mediated by the cellular prion protein and insulin; this is consistent with the notion that reggie microdomains promote protein assemblies and signalling. 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 have demonstrated that reggie complexes are composed of homo- and hetero-tetramers of reggie-1 and -2. Moreover, native reggie oligomers are indeed quite stable, since non-cross-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 analysed the functional relevance of the three predicted coiled-coil stretches present in the C-terminus of reggie-1. We confirmed experimentally 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 (small interfering RNA) 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.