Trafficking of the microdomain scaffolding protein reggie-1/flotillin-2.
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.
Langhorst
Matthias F
MF
Department of Biology, University of Konstanz, Universitätsstrasse 10, D-78457 Konstanz, Germany. Matthiaslanghorst@email.de
Reuter
Alexander
A
Jaeger
Friederike A
FA
Wippich
Frank M
FM
Luxenhofer
Georg
G
Plattner
Helmut
H
Stuermer
Claudia A O
CA
D000818:Animals; D002784:Cholesterol / Q000378:metabolism; D004705:Endocytosis; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins; D016716:PC12 Cells; D011328:Prions / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011328:Prions; C117201:STOML1 protein, human; C106354:flotillins; D002784:Cholesterol