Flotillin-1/reggie-2 traffics to surface raft domains via a novel golgi-independent pathway. Identification of a novel membrane targeting domain and a role for palmitoylation.
Flotillins are lipid raft-associated proteins, which have been implicated in neuronal regeneration and insulin signaling. We now show that newly synthesized flotillin-1 reaches the plasma membrane via a Sar1-independent and brefeldin A-resistant targeting pathway. Consistent with post-translational membrane association of flotillin, protease sensitivity experiments suggest that flotillin-1 is not a transmembrane protein but is associated with the cytoplasmic face of the plasma membrane. The N terminus of flotillin contains a prohibitin-like domain (PHB), which shows homology to a number of proteins associated with raft domains including stomatin, podocin, and prohibitin. We show that the PHB domain of flotillin can efficiently target a heterologous protein, green fluorescent protein, to the plasma membrane. Another PHB-containing protein, stomatin, traffics to the plasma membrane via the conventional secretory pathway. Plasma membrane association of both full-length flotillin and the green fluorescent protein-tagged PHB domain of flotillin is dependent on palmitoylation and requires a conserved cysteine residue, Cys-34, in the PHB domain. The results identify a novel targeting mechanism for plasma membrane association of flotillin-1 involving a Golgi-independent trafficking pathway, the PHB domain, and palmitoylation.
Morrow
Isabel C
IC
Institute for Molecular Bioscience and Centre for Functional and Applied Genomics, University of Queensland, St. Lucia, Australia.
Rea
Shane
S
Martin
Sally
S
Prior
Ian A
IA
Prohaska
Rainer
R
Hancock
John F
JF
James
David E
DE
Parton
Robert G
RG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020126:Brefeldin A / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D029941:Fish Proteins; D006056:Golgi Apparatus / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D019308:Palmitic Acid / Q000378:metabolism*; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D014709:Vero Cells
D017931:DNA Primers; D029941:Fish Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C106354:flotillins; C104436:reggie-2 protein, Carassius auratus; D020126:Brefeldin A; D019308:Palmitic Acid