human clc-6 is a late endosomal glycoprotein that associates with detergent-resistant lipid domains人类clc-6晚期endosomal糖蛋白与detergent-resistant脂质域相关联.pdfVIP

Human ClC-6 Is a Late Endosomal Glycoprotein that Associates with Detergent-Resistant Lipid Domains 1 1 1 2. 1. Sofie Ignoul , Jeannine Simaels , Diane Hermans , Wim Annaert , Jan Eggermont * 1 Laboratory of Membrane Transport, Department of Molecular Cell Biology, University of Leuven, Leuven, Belgium, 2 Laboratory for Membrane Trafficking, Department of Human Genetics, University of Leuven and V.I.B.11, Leuven, Belgium 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 earl