Biochemical Journal

Research article

Membrane and raft association of reggie-1/flotillin-2: role of myristoylation, palmitoylation and oligomerization and induction of filopodia by overexpression

Carolin NEUMANN-GIESEN, Bianca FALKENBACH, Peter BEICHT, Stephanie CLAASEN, Georg LÜERS, Claudia A. O. STUERMER, Volker HERZOG, Ritva TIKKANEN

Abstract

The reggie protein family consists of two proteins, reggie-1 and -2, also called flotillins, which are highly ubiquitous and evolutionarily conserved. Both reggies have been shown to be associated with membrane rafts and are involved in various cellular processes such as T-cell activation, phagocytosis and insulin signalling. However, the exact molecular function of these proteins remains to be determined. In addition, the mechanism of membrane association of reggie-1, which does not contain any transmembrane domain, is not known. In this study, we have produced a fusion protein of reggie-1 with enhanced green fluorescent protein and generated targeted substitutions for the inactivation of putative palmitoylation and myristoylation sites. We were able to show that reggie-1 is myristoylated and multiply palmitoylated and that lipid modifications are necessary for membrane association of reggie-1. Overexpression of reggie-1 resulted in the induction of numerous filopodia-like protrusions in various cell lines, suggesting a role for reggie-1 as a signalling protein in actin-dependent processes.

  • actin cytoskeleton
  • filopodia
  • flotillin
  • protein acylation
  • rafts
  • reggie

Footnotes

  • Abbreviations used: DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; FCS, foetal calf serum; FL, full-length; FRT, Fischer rat thyroid; SH, short; SPFH, stomatin, prohibitin, flotillin homology; WT, wild-type; X-α-Gal, 5-bromo-4-chloroindol-3-yl α-d-galactopyranoside; YFP, yellow fluorescent protein.