The ClC-3 chloride transport protein traffics through the plasma membrane via interaction of an N-terminal dileucine cluster with clathrin.
ClC-3 is a ubiquitously expressed chloride transport protein that is present in synaptic vesicles and endosome/lysosome compartments. It is largely intracellular but has been observed at the plasma membrane as well. The aim of this study was to identify the pathways and regulation of ClC-3 trafficking to intracellular sites. At the steady state, approximately 94% of transfected ClC-3 was localized intracellularly, and only 6% was at the plasma membrane. Pulse labeling with [(35)S]methionine and biotinylation demonstrated that about 25% of newly synthesized ClC-3 traffics through the plasma membrane. We used both immunofluorescence microscopy and biotinylation assays to assess the trafficking of ClC-3. Plasma membrane ClC-3 was rapidly endocytosed (t((1/2)) approximately 9 min); a portion entered a recycling pool that returned to the cell surface after internalization, and the remainder trafficked to more distal intracellular compartments. ClC-3 associated with clathrin at the plasma membrane. Coimmunoprecipitation and glutathione S-transferase pulldown assays demonstrated that the N terminus of ClC-3 binds to clathrin. Alanine replacement of a dileucine acidic cluster within the cytosolic N terminus (amino acids 13-19) resulted in a molecule that had decreased endocytosis and increased surface expression. This replacement also abolished interaction with clathrin as assessed both by coimmunoprecipitation and glutathione S-transferase pulldown assays. We conclude that ClC-3 is primarily an intracellular transport protein that is transiently inserted into the plasma membrane where it is rapidly endocytosed. Internalization of ClC-3 depends on the interaction between an N-terminal dileucine cluster and clathrin.
Zhao
Zhifang
Z
Departments of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555-0620.
Li
Xinhua
X
Departments of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555-0620.
Hao
Junfang
J
Departments of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555-0620.
Winston
John H
JH
Departments of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-0620.
Weinman
Steven A
SA
Departments of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555-0620; Departments of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-0620. Electronic address: sweinman@utmb.edu.
D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology
D018118:Chloride Channels; C115376:ClC-3 channel; D002966:Clathrin