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Stockert RJ, Potvin B, Tao L, Stanley P, Wolkoff AW: Human hepatoma cell mutant defective in cell surface protein trafficking. J Biol Chem. 1995 Jul 7;270(27):16107-13.
To isolate a mutant liver cell defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was devised. Ovalbumin-gelonin and asialoorosomucoid (ASOR)-gelonin were incubated with mutagenized HuH-7 cells, and a rare survivor termed trafficking mutant 1 (Trf1) was isolated. Trf1 cells were stably 3-fold more resistant than the parental HuH-7 to both toxic conjugates. The anterograde steps of intracellular endocytic processing of ASOR, including internalization, endosomal acidification, and ligand degradation, were unaltered in Trf1 cells. In contrast, retrograde diacytosis of asialoglycoprotein receptor (ASGR).ASOR complex back to the cell surface was enhanced by about 250%. Selective labeling revealed an approximately 46% reduction in cell surface-associated ASGR in Trf1 cells, although their total cellular ASGR content was essentially equivalent to that in HuH-7. Similar results were obtained with the transferrin receptor. Binding of 125I-ASOR and 125I-transferrin was reduced in Trf1 cells to 49 +/- 2.5% and 30 +/- 2%, respectively, of HuH-7 cells. The methionine transporter was also reduced in Trf1 cells, as revealed by a 2-fold reduction in Vmax with no change in apparent Km. Pretreatment with monensin, sodium azide, or colchicine reduced surface binding of 125I-ASOR in HuH-7 cells by 50% but had no effect on binding to Trf1 cells. This result is predicted for a cell that expresses only State 1 ASGRs, which are resistant to modulation by metabolic and cytoskeletal inhibitors in contrast to State 2, which are responsive to these agents (Weigel, P. H., and Oka, J. A. (1984) J. Biol. Chem. 259, 1150-1154). The Trf1 mutant, having lost the ability to express State 2 receptors, provides genetic evidence for the existence of these two receptor subpopulations and an approach to identifying the biochemical mechanism by which they are generated. |
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