Distribution and targeting of CLC-3

ClC-3 is a ubiquitously expressed chloride channel that is present in synaptic vesicles and endosome/lysosome compartments. The channel is largely intracellular, but has been observed at the plasma membrane as well. The aim of this study was to identify the trafficking pathway for delivery of ClC-3 to intracellular sites. When transiently transfected into COS-7 cells, approximately 6% of ClC-3 localized to the plasma membrane as assessed by surface biotinylation. Plasma membrane ClC-3 was rapidly endocytosed with t1/2 of approximately 9min. Biotinylation experiment detected a portion of ClC-3 recycled back to the cell surface. Antibody binding to an external HA epitope demonstrated that the plasma membrane is an intermediate trafficking site for ClC-3 destined for intracellular compartments. ClC-3 was associated with clathrin at the plasma membrane and in early endocytic vesicles. It also colocalized with transferrin-labelled endosomes. It dissociated from clathrin at later times to localize in large vesicles. GST pull-down assays demonstrated that the N-terminal of ClC-3 binds to both clathrin and AP-2. Deletion of dileucine cluster within the cytosolic N terminal (amino acids 13-19) resulted in a molecule that had decreased endocytosis and increased surface expression. This deletion also abolished interaction with clathrin in GST pull down experiments. We conclude that ClC-3 is primarily an intracellular channel but it is transiently inserted into the plasma membrane from where it is rapidly endocytosed. Internalization of ClC-3 depends on the interaction between an N-terminal dileucine cluster and clathrin.

Name thesis:degree_name

PhD

Level thesis:degree_level

Doctoral

Grantor

The University of Texas Medical Branch

Year dc:date.issued

2006

Author dc:creator

• Zhifang Zhao

Advisor dc:contributor.advisor

• Steven A. Weinman

Committee members dc:contributor.committeemember

• Roger G. O'Neil
• Lisa A. Elferink
• Cornelis Elferink
• Altenberg

• trafficking
• distribution
• ClC-3

Statement dc:rights

• Copyright © is held by the author. Presentation of this material on the TDL web site by The University of Texas Medical Branch at Galveston was made possible under a limited license grant from the author who has retained all copyrights in the works.

Language dc:language.iso

eng

Dc Identifier Other

etd-12012006-104431

OAI identifier oai:identifier

oai:utmb-ir.tdl.org:2152.3/276