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
- 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 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 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 ClC-6 between early endosomes and its final destination in late endosomes
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. glycoprotein, , ClC-6, -, -
- 0. glycoprotein, , glycoprotein, -, -
- 3. K, , -, -, N410, N422 and N432
- 3. K, , -, -, asparagine residues
- 3. N-glycosylated, , -, -, sites
- 3. N-glycosylated, , ClC-6, -, region
- 9. glycoprotein, , ClC, -, -
- 9. glycoprotein, , glycoprotein, -, -
Output(Part-Of) (sent_index,
protein,
site):
- 3. -, tail
- 3. ClC-6, tail
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 3. ClC-6, -, N410, N422 and N432