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Human ClC‐6 is a lipid raft associated glycoprotein that resides in late endosomes in neuronal cells but not upon overexpression in COS and HeLa cells
Author(s) -
Ignoul Sofie,
Hermans Diane,
Simaels Jeannine,
Annaert Wim,
Eggermont Jan
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a537
Subject(s) - endosome , microbiology and biotechnology , endocytic cycle , biology , glycoprotein , lipid raft , hek 293 cells , hela , intracellular , biochemistry , endocytosis , cell , signal transduction , receptor
The mammalian CLC family comprises 9 proteins (ClC‐1 to ‐7 and ClC‐Ka, ‐Kb) that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters. Recently, ClC‐6 has been described as a late endosomal/lysosomal protein in neuronal cells and a lysosomal storage disease is associated with abolition of its function. We have developed a polyclonal antibody that recognizes human ClC‐6 (hClC‐6) both upon overexpression (COS and Hela cells) and endogenous in a human neuronal cell line (SH‐SY5Y). Confocal immunofluorescence experiments showed that endogenous hClC‐6 in SH‐SY5Y cells resides in a late endosomal compartment. However, upon overexpression in COS and HeLa cells, hClC‐6 colocalized with early endosomal markers but not with different markers for late endosomes/lysosomes. This suggests that ClC‐6 is sorted to late endosomes via an intermediary early endosomal compartment and that this sorting step is rate‐limiting in COS and HeLa cells. Furthermore, hClC‐6 copurifies with detergent resistant membrane fractions suggesting partitioning in lipid rafts. Finally, hClC‐6 is N‐glycosylated upon overexpression. Three asparagines residues have been identified as acceptor sites for N‐glycosylation, but only two of three are simultaneously N‐glycosylated in the wild type protein. We therefore conclude that hClC‐6 is a lipid‐raft associated endosomal glycoprotein.