New liver cell mutants defective in the endocytic pathway

Richard J. Stockert, Barry Potvin, Sangeeta Nath, Allan W. Wolkoff, Pamela Stanley

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

To isolate mutant liver cells defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was utilized. Rare survivors termed trafficking mutants (Trf2-Trf7) were stable and more resistant than the parental HuH-7 cells to both toxin conjugates. They differed from the previously isolated Trf1 HuH-7 mutant as they expressed casein kinase 2 α″ (CK2α″) which is missing from Trf1 cells and which corrects the Trf1 trafficking phenotype. Binding of 125I-asialoorosomucoid (ASOR) and cell surface expression of asialoglycoprotein receptor (ASGPR) were reduced approximately 20%-60% in Trf2-Trf7 cells compared to parental HuH-7, without a reduction in total cellular ASGPR. Based on 125I-transferrin binding, cell surface transferrin receptor activity was reduced between 13% and 88% in the various mutant cell lines. Distinctive phenotypic traits were identified in the differential resistance of Trf2-Trf7 to a panel of lectins and toxins and to UV light-induced cell death. By following the endocytic uptake and trafficking of Alexa488-ASOR, significant differences in endosomal fusion between parental HuH-7 and the Trf mutants became apparent. Unlike parental HuH-7 cells in which the fusion of endosomes into larger vesicles was evident as early as 20 min, ASOR endocytosed into the Trf mutants remained within small vesicles for up to 60 min. Identifying the biochemical and genetic mechanisms underlying these phenotypes should uncover novel and unpredicted protein-protein or protein-lipid interactions that orchestrate specific steps in membrane protein trafficking.

Original languageEnglish (US)
Pages (from-to)1741-1749
Number of pages9
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1768
Issue number7
DOIs
StatePublished - Jul 1 2007

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Keywords

  • Apoptosis
  • Asialoglycoprotein receptor
  • Endocytosis
  • Glycoprotein-toxin selection
  • HuH-7 cells, casein kinase 2 α″
  • Trafficking mutant

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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