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 H. Winston, Steven A. Weinman

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35 Scopus citations

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

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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