New liver cell mutants defective in the endocytic pathway.
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 alpha'' (CK2alpha'') which is missing from Trf1 cells and which corrects the Trf1 trafficking phenotype. Binding of (125)I-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 (125)I-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 Alexa(488)-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.
Stockert
Richard J
RJ
The Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA. stockert@aecom.yu.edu
Potvin
Barry
B
Nath
Sangeeta
S
Wolkoff
Allan W
AW
Stanley
Pamela
P
D017209:Apoptosis / Q000528:radiation effects; D037263:Asialoglycoprotein Receptor / Q000378:metabolism; D001212:Asialoglycoproteins / Q000378:metabolism; D001427:Bacterial Toxins / Q000494:pharmacology; D047390:Casein Kinase II / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D004167:Diphtheria Toxin / Q000494:pharmacology; D004705:Endocytosis / Q000235:genetics* / Q000528:radiation effects; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D008099:Liver / Q000166:cytology*; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D011549:Pseudomonas / Q000737:chemistry; D011990:Receptors, Transferrin / Q000378:metabolism; D012276:Ricin / Q000494:pharmacology; D014466:Ultraviolet Rays; D014909:Wheat Germ Agglutinins / Q000494:pharmacology
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D001427:Bacterial Toxins; D004167:Diphtheria Toxin; D009961:Orosomucoid; D011990:Receptors, Transferrin; D014909:Wheat Germ Agglutinins; C017030:asialoorosomucoid; D012276:Ricin; D047390:Casein Kinase II