Abstract
We have isolated several Chinese hamster ovary cell lines with temperature-sensitive defects in the recycling of receptors after endocytosis. These cell lines were selected using fluorescence-activated cell sorting for retention of a pulse of labeled transferrin after a chase in the presence of unlabeled transferrin. One of these cell lines, TfT1.11, was selected for further characterization. In TfT1.11 the trapping of transferrin within the cells is paralleled by a loss of cell surface transferrin receptors. Within 4 h after the shift from 33 to 41 degrees C the surface binding of transferrin is reduced to 18% of parental cells at 41 degrees C. The trapping of transferrin and the loss of transferrin receptor from the cell surface are caused by a temperature-conditional 5.5-fold decrease in the initial rate of transferrin recycling. TfT1.11 cells also rapidly lose 89% of their ability to take up alpha 2-macroglobulin after the temperature shift to 41 degrees C. These data indicate that the TfT1.11 cell line has a pleiotropic defect in receptor recycling.
Highlights
We have isolated several Chinese hamster ovary cell from receptoris coupled to endocytic acidification
To avoid this problemcells were incubated at 41 "C This effect was demonstrated by examining the kinetics of for various times toallow expression of heat-sensitive lesions, expression of the trapping phenotype for the population of pulse labeled with Cy5-Tf for 30min, and chasedfor 1h with double-positive cellsobtained after the thirsdort (Fig. 2)
0.90 reduction in the amountof Tf internalized in a 30-min pulse, and themajority of the cells are clearly positive for both dextran accumulation and retention of Cy5-Tf (Fig. 30)
Summary
Recycling Defects for 6 min at 41 "C in a-MEM with 10 pg/mlCy5-asM.Toclear receptors of unlabeled a2M from the serum,the cells were incubated without serum at either 33 or 41 "C for 30 min prior to labeling. After labeling the cells were chilled quickly to 4 "C, washed twice with a-. Hortterm uptakewas measuredinstead of surface binding because very little binding could be detected at 4 "C even after a 1-h incubation.The temperature dependenceof a2Mbinding has been noted for other cell types as well (40). Measurements of cell-associated asM reflect both surface bounadnd internalized ligand because the off rate, like the on rate, is quite slow. Sample was determined using a luciferin/luciferase ATP determination kit for somatic cells (Sigma) and a Thorn EM1 PMT (800 V). The average number of cells/sample was determined.
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