Human ClC-6 Is a Late Endosomal Glycoprotein that Associates with Detergent-Resistant Lipid Domains

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. PRINCIPAL FINDINGS: 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

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Immunolocalization of overexpressed hClC-6 in SH-SY5Y cells.

<p>Double immunofluorescence confocal images of SH-SY5Y cells, transiently transfected with pcDNA3.1(−)/hClC-6a expression vector. Overexpression levels were very high, so that transfected cells could easily be distinguished from non-transfected cells. Overexpressed hClC-6 (left column) was detected using the polyclonal α-hClC-6 antibody and visualized with anti-rabbit IgG antibodies conjugated to Alexa Fluor 488 (green signal). Markers for different endosomal compartments (middle column) were (A) mouse anti-EEA-1 (an early endosome marker); (B) mouse anti-transferrin receptor (TfR, an early/recycling endosome marker); (C) mouse anti-LAMP-1 (a late endosomal/lysosomal marker). Primary antibodies were visualized using anti-mouse IgG antibodies conjugated to Alexa Fluor 594 (red signal). In the merged pictures (right column) colocalization is indicated by a yellow signal. The scale bars represent 10 µm.</p

hClC-6 resides in detergent resistant membrane fractions.

<p>(A) DRM fractions of COS-1 cells overexpressing respectively (a) hClC-6 and (b) KKGRR/AAGAA-hClC-6; were prepared and separated on a sucrose gradient. Upward flotation of the DRM's was checked by distribution of caveolin-1 (Cav-1) which migrated to the top of the gradient (fractions 2/3/4). Transferrin receptor (TfR), a non-raft membrane protein, was used as a negative control (fractions 8/9 at the bottom of the gradient). hClC-6 expression was checked by staining with the polyclonal α-hClC-6. (B) Confocal images of double transiently transfected COS-1 cells, expressing GFP-hClC-7 and wild type hClC-6 (panels a to c) or KKGRR/AAGAA-hClC-6 (panels d to f). Wild type and KKGRR/AAGAA-hClC-6 were detected with the polyclonal α-hClC-6 antibody and visualized with anti-rabbit IgG antibodies conjugated to Alexa Fluor 594 (red signal, panels a and d). ClC-7 expression is visualized by the GFP signal (green signal, panels b and e). A yellow signal indicates colocalization (panels c and f). Scale bars represent 10 µm.</p

Characterization of the polyclonal α-hClC-6 antibody.

<p>(A) Western blotting of microsomal membranes of wild-type COS-1 cells and COS-1 cells transiently transfected with pcDNA3.1(−)/hClC-6a. Incubation with the affinity-purified polyclonal α-hClC-6 resulted in a band of approximately 100 kDa in transfected COS-1 cells, but not in wild type COS-1 cells. Such a band was not observed in transfected COS-1 cells when the blot was incubated with pre-immune serum or when the polyclonal α-hClC-6 was preincubated with the epitope-peptide before applying to the blot. (B) Immunofluorescence of COS-1 cells transfected with the bicistronic vector pcINeo/GFP-IRES/hClC-6a which ensures strict coupling between GFP expression (green signal in panels a and c) and ClC-6 expression. Incubation with pre-immune serum (panel b) never generated a positive signal in transfected cells. In contrast, a positive signal for ClC-6 (red in panel d) was exclusively observed in transfected cells incubated with polyclonal α-hClC-6. Nuclei were counterstained with blue TO-PRO®-3. The scale bar corresponds to 10 µm. (C) Western blot of microsomal membrane fractions from transiently transfected COS-1 cells expressing either hClC-6 or GFP-hClC-7. Incubation with α-hClC-6 generated a positive signal in the hClC-6 expressing membrane fractions, but not in the hClC-7 expressing membranes. Expression of GFP-hClC-7 was confirmed by incubation with a monoclonal α-GFP antibody.</p

Colocalization of overexpressed hClC-6 with different endosomal Rab-proteins.

<p>Confocal images of double transiently transfected HeLa cells, expressing hClC-6 and (A) GFP-Rab4, (B) GFP-Rab5, (C) GFP-Rab7, (D) GFP-Rab11. hClC-6 was detected with the polyclonal α-hClC-6 antibody and visualized with anti-rabbit IgG antibodies conjugated to Alexa Fluor 594. The Rab signals were visualized by the GFP signal. (E) Represents a confocal image of double transiently transfected COS-1 cells, expressing the glycosylation-deficient AAA-hClC-6 (red signal) and GFP-Rab4 (green signal). The scale bars represent 10 µm.</p

Immunolocalization of hClC-6 in transfected COS-1 cells.

<p>Double immunofluorescence confocal images of COS-1 cells transiently transfected with pcDNA3.1(−)/hClC-6a. hClC-6 expression (left column) was detected using the polyclonal α-hClC-6 antibody and visualized with anti-rabbit IgG antibodies conjugated to Alexa Fluor 488 (green signal). Organelles were stained with the following antibodies or markers (middle column): (A) mouse anti-BIP (an endoplasmic reticulum marker); (B) mouse anti-Golgin-97 (a Golgi marker); (C) mouse anti-LAMP-1 (a late endosomal/lysosomal marker); (D) mouse anti-EEA-1 (an early endosome marker); (E) mouse anti-transferrin receptor (TfR, an early/recycling endosome marker). The marker antibodies were visualized by anti-mouse IgG antibodies conjugated to Alexa Fluor 594 (red signal). The column on the right shows merged pictures of ClC-6 expression and marker staining with yellow indicating colocalization. The scale bars represent 10 µm.</p