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

Extracellular Ca2+ regulates the stimulation of Na+ transport in A6 renal epithelia

1968 — Ein Blick auf die Protestbewegungen 40 Jahre danach aus globaler Perspektive Ein Bericht von der 44. Konferenz der International Conference of Labour and Social History (ITH) in Linz, 11.-14. September 2008 Knapp 100 TeilnehmerInnen waren der Einladung zur 44. Linzer Konferenz gefolgt, die von der Internationalen Tagung der HistorikerInnen der Arbeiterbewegung und anderer sozialer Bewegungen (ITH) und der Kammer für Arbeiter und Angestellte Oberösterreich veranstaltet wurde. Marcel van..

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