TY - JOUR
T1 - New liver cell mutants defective in the endocytic pathway
AU - Stockert, Richard J.
AU - Potvin, Barry
AU - Nath, Sangeeta
AU - Wolkoff, Allan W.
AU - Stanley, Pamela
N1 - Funding Information:
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41918 and DK-41296.
PY - 2007/7
Y1 - 2007/7
N2 - 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.
AB - 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.
KW - Apoptosis
KW - Asialoglycoprotein receptor
KW - Endocytosis
KW - Glycoprotein-toxin selection
KW - HuH-7 cells, casein kinase 2 α″
KW - Trafficking mutant
UR - http://www.scopus.com/inward/record.url?scp=34250012569&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34250012569&partnerID=8YFLogxK
U2 - 10.1016/j.bbamem.2007.04.004
DO - 10.1016/j.bbamem.2007.04.004
M3 - Article
C2 - 17512493
AN - SCOPUS:34250012569
SN - 0005-2736
VL - 1768
SP - 1741
EP - 1749
JO - Biochimica et Biophysica Acta - Biomembranes
JF - Biochimica et Biophysica Acta - Biomembranes
IS - 7
ER -