The ClC-3 chloride transport protein traffics through the plasma membrane via interaction of an N-terminal dileucine cluster with clathrin

Zhifang Zhao, Xinhua Li, Junfang Hao, John Winston, Steven A. Weinman

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

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, ∼94% of transfected ClC-3 was localized intracellularly, and only 6% was at the plasma membrane. Pulse labeling with [35S]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 ∼ 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 pull-down 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.

Original languageEnglish (US)
Pages (from-to)29022-29031
Number of pages10
JournalJournal of Biological Chemistry
Volume282
Issue number39
DOIs
StatePublished - Sep 28 2007

Fingerprint

Clathrin
Cell membranes
Chlorides
Carrier Proteins
Cell Membrane
Endocytosis
Biotinylation
Assays
Glutathione Transferase
Synaptic Vesicles
Endosomes
Recycling
Lysosomes
Fluorescence Microscopy
Alanine
Methionine
Labeling
Microscopic examination
Amino Acids
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

The ClC-3 chloride transport protein traffics through the plasma membrane via interaction of an N-terminal dileucine cluster with clathrin. / Zhao, Zhifang; Li, Xinhua; Hao, Junfang; Winston, John; Weinman, Steven A.

In: Journal of Biological Chemistry, Vol. 282, No. 39, 28.09.2007, p. 29022-29031.

Research output: Contribution to journalArticle

@article{e69b977d03ac440d983d50316b0d4406,
title = "The ClC-3 chloride transport protein traffics through the plasma membrane via interaction of an N-terminal dileucine cluster with clathrin",
abstract = "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, ∼94{\%} of transfected ClC-3 was localized intracellularly, and only 6{\%} was at the plasma membrane. Pulse labeling with [35S]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 ∼ 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 pull-down 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.",
author = "Zhifang Zhao and Xinhua Li and Junfang Hao and John Winston and Weinman, {Steven A.}",
year = "2007",
month = "9",
day = "28",
doi = "10.1074/jbc.M703506200",
language = "English (US)",
volume = "282",
pages = "29022--29031",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "39",

}

TY - JOUR

T1 - The ClC-3 chloride transport protein traffics through the plasma membrane via interaction of an N-terminal dileucine cluster with clathrin

AU - Zhao, Zhifang

AU - Li, Xinhua

AU - Hao, Junfang

AU - Winston, John

AU - Weinman, Steven A.

PY - 2007/9/28

Y1 - 2007/9/28

N2 - 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, ∼94% of transfected ClC-3 was localized intracellularly, and only 6% was at the plasma membrane. Pulse labeling with [35S]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 ∼ 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 pull-down 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.

AB - 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, ∼94% of transfected ClC-3 was localized intracellularly, and only 6% was at the plasma membrane. Pulse labeling with [35S]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 ∼ 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 pull-down 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.

UR - http://www.scopus.com/inward/record.url?scp=35348944726&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=35348944726&partnerID=8YFLogxK

U2 - 10.1074/jbc.M703506200

DO - 10.1074/jbc.M703506200

M3 - Article

VL - 282

SP - 29022

EP - 29031

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 39

ER -