Trafficking of the microdomain scaffolding protein reggie-1/flotillin-2

Abstract

The reggie/flotillin proteins oligomerize and associate into clusters which form scaffolds for membrane microdomains. Besides their localization at the plasma membrane, the reggies/flotillins reside at various intracellular compartments; however, the trafficking pathways used by reggie-1/flotillin-2 remain unclear. Here, we show that trafficking of reggie-1/flotillin-2 is BFA sensitive and that deletion mutants of reggie-1/flotillin-2 accumulate in the Golgi complex in HeLa, Jurkat and PC12 cells, suggesting Golgi-dependent trafficking of reggie-1/flotillin-2. Using total internal reflection fluorescence microscopy, we observed fast cycling of reggie-1/flotillin-2-positive vesicles at the plasma membrane, which engaged in transient interactions with the plasma membrane only. Reggie-1/flotillin-2 cycling was independent of clathrin, but was inhibited by cholesterol depletion and microtubule disruption. Cycling of reggie-1/flotillin-2 was negatively correlated with cell–cell contact formation but was stimulated by serum, epidermal growth factor and by cholesterol loading mediated by low density lipoproteins. However, reggie-1/flotillin-2 was neither involved in endocytosis of the epidermal growth factor itself nor in endocytosis of GPI-GFPs or the GPI-anchored cellular prion protein (PrP^c). Reggie-2/flotillin-1 and stomatin-1 also exhibited cycling at the plasma membrane similar to reggie-1/flotillin-2, but these vesicles and microdomains only partially co-localized with reggie-2/flotillin-1. Thus, regulated vesicular cycling might be a general feature of SPFH protein-dependent trafficking.

Introduction

Reggie-1 and reggie-2 were discovered in our lab as proteins upregulated during axon regeneration after optic nerve lesion (Schulte et al., 1997; Lang et al., 1998). They were independently described as proteins abundant in the floating, detergent-resistant membrane fraction prepared from murine lung tissue and therefore named flotillin-2 and -1, respectively (Bickel et al., 1997). The reggies/flotillins consist of an N-terminal SPFH domain containing residues for myristoylation and palmitoylation and thus membrane anchorage (Neumann-Giesen et al., 2004), and a C-terminal flotillin domain which is predicted to form coiled-coil structures (Schroeder et al., 1994; Bickel et al., 1997; Schulte et al., 1997). Via this domain, the reggies/flotillins form homo- and hetero-oligomers (Neumann-Giesen et al., 2004; Solis et al., 2007), which assemble into clusters of 50–100 nm at the plasma membrane (Stuermer et al., 2001). These clusters define specialized flat membrane microdomains and serve as stable scaffolds for the assembly of multiprotein complexes, similar to caveolin clusters in caveolae (reviewed in Langhorst et al. (2005)). Reggie/flotillin-dependent signaling complexes were shown to be involved in actin remodeling during T cell activation (Langhorst et al., 2006b) and in Glut4-translocation to the plasma membrane after insulin stimulation of adipocytes (Baumann et al., 2000; Kimura et al., 2001).

The biosynthetic pathway of the reggies/flotillins is controversial. Reggie-2/flotillin-1 was found to travel through the Golgi in NRK, CHO and HeLa cells (Gkantiragas et al., 2001). However, Morrow et al. (2002) observed Sar1- and Brefeldin A (BFA)-independent trafficking of reggie-2/flotillin-1 in BHK cells, suggesting a Golgi-independent pathway. In addition to their prominent localization at the plasma membrane, both reggies/flotillins reside at intracellular compartments. They localize to lipid droplets (Liu et al., 2004; Reuter et al., 2004) and to compartments of the endocytic pathway, like recycling endosomes (Gagescu et al., 2000; Solomon et al., 2002). Reggie-2/flotillin-1 was recently implicated in an unconventional, clathrin- and caveolin-independent endocytosis pathway used by GPI-anchored proteins (Glebov et al., 2006) and proteogylcan-binding ligands (Payne et al., 2007).

In this study, we investigated the trafficking pathways of reggie-1/flotillin-2 and its role in endocytosis of GPI-anchored proteins, especially the cellular prion protein PrP^c in detail. We provide evidence for Golgi-dependent trafficking of reggie-1/flotillin-2. At the plasma membrane, reggie-1/flotillin-2 exhibited vesicular cycling, which was regulated by epidermal growth factor stimulation, cholesterol loading and cell–cell contact formation. Reggie-2/flotillin-1 and stomatin-1 vesicles showed similar behavior but distinct localizations, suggesting that regulated protein delivery and retrieval by vesicular cycling might be a general function of the membrane microdomain-forming proteins of the SPFH family.