title: Human ClC-6 is a late endosomal glycoprotein that associates with detergent-resistant lipid domains.
abstract: BACKGROUND
  The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb) that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7.
  
  
  RESULTS
  Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432) have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y), endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4) and not with late endosomal/lysosomal markers (LAMP-1, Rab7). Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71-75: KKGRR) disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6 and ClC-7 when cotransfected in COS-1 cells.
  
  
  CONCLUSIONS
  We conclude that human ClC-6 is an endosomal glycoprotein that partitions in detergent resistant lipid domains. The differential sorting of endogenous (late endosomal) versus overexpressed (early and recycling endosomal) ClC-6 is reminiscent of that of other late endosomal/lysosomal membrane proteins (e.g. LIMP II), and is consistent with a rate-limiting sorting step for ClC-6 between early endosomes and its final destination in late endosomes.
authors.affiliation: Laboratory of Membrane Transport, Department of Molecular Cell Biology, University of Leuven, Leuven, Belgium.
authors.forename: Sofie
authors.initials: S
authors.lastname: Ignoul
authors.forename: Jeannine
authors.initials: J
authors.lastname: Simaels
authors.forename: Diane
authors.initials: D
authors.lastname: Hermans
authors.forename: Wim
authors.initials: W
authors.lastname: Annaert
authors.forename: Jan
authors.initials: J
authors.lastname: Eggermont
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D015153:Blotting, Western; D019556:COS Cells; D045744:Cell Line, Tumor; D018118:Chloride Channels / Q000276:immunology / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
chemical_list: D000906:Antibodies; C097539:CLCN6 protein, human; D018118:Chloride Channels