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Slowed receptor trafficking in mutant CHO lines of the End1 and End2 complementation groups
L S Johnson1, J F Presley, J C Park
1Department of Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Abstract:
A mutant Chinese hamster ovary cell line with nonconditional kinetic defects in receptor internalization and recycling was isolated, based on selection for resistance to a transferrin-diphtheria toxin conjugate and screening for aberrant receptor trafficking. The 12-4 cell line internalizes transferrin at approximately 75% of the parental rate and recycles transferrin back to the cell surface at approximately 55% of the parental rate. Internalization of low density lipoprotein is also reduced to approximately 70% of the parental cell rate, demonstrating that the mutant phenotype affects the trafficking of multiple receptors. Characterization of somatic cell hybrids indicated that the 12-4 phenotype is recessive, and complementation analysis determined that the 12-4 cell line is a member of the End2 complementation group. End2 mutants have previously been described as defective in endosomal acidification but have not been known to be defective in receptor trafficking. We have found similar defects in another End2 mutant cell line, suggesting that slowed receptor trafficking is characteristic of End2 mutants. Interestingly, transferrin receptor recycling and internalization are also slowed in another complementation group of mutants, End1, that is also defective in endosomal acidification. This study demonstrates altered receptor trafficking in End1 and End2 cell lines, a novel aspect of the mutant phenotypes. These findings provide evidence, based on a cellular genetic approach, that proper endosome acidification is necessary for maintenance of normal receptor recycling.
Insights
Defects in endosomal acidification, previously linked to end2 mutants, also impair receptor trafficking. This study shows endosomal acidification is crucial for normal receptor recycling in cells.
Area of Science:
- Cell Biology
- Molecular Genetics
- Biochemistry
Background:
- Endosomal acidification is essential for various cellular processes, including receptor-mediated endocytosis.
- Mutant cell lines with defects in endosomal acidification (End1 and End2 complementation groups) were previously identified.
- The precise role of endosomal acidification in receptor trafficking kinetics remained unclear.
Purpose of the Study:
- To investigate the role of endosomal acidification in receptor internalization and recycling.
- To characterize the receptor trafficking defects in Chinese hamster ovary (CHO) cell mutants.
- To determine if altered endosomal acidification affects the trafficking of multiple receptors.
Main Methods:
- Isolation of a mutant CHO cell line (12-4) resistant to a transferrin-diphtheria toxin conjugate.
- Analysis of transferrin and low-density lipoprotein receptor trafficking kinetics in mutant and parental cells.
- Somatic cell hybridization and complementation analysis to determine the genetic basis of the mutation.
Main Results:
- The 12-4 cell line exhibited significantly reduced rates of transferrin receptor internalization (75%) and recycling (55%).
- Multiple receptor trafficking, including low-density lipoprotein, was impaired in the 12-4 mutant, indicating a broader defect.
- Complementation analysis placed the 12-4 mutation in the End2 complementation group, previously associated only with endosomal acidification defects.
Conclusions:
- Altered receptor trafficking is a novel characteristic of End1 and End2 endosomal acidification mutants.
- Proper endosomal acidification is necessary for maintaining normal rates of receptor recycling.
- This study provides cellular genetic evidence linking endosomal acidification to efficient receptor trafficking.